key: cord- -iuo cw authors: lippé, roger title: deciphering novel host–herpesvirus interactions by virion proteomics date: - - journal: front microbiol doi: . /fmicb. . sha: doc_id: cord_uid: iuo cw over the years, a vast array of information concerning the interactions of viruses with their hosts has been collected. however, recent advances in proteomics and other system biology techniques suggest these interactions are far more complex than anticipated. one particularly interesting and novel aspect is the analysis of cellular proteins incorporated into mature virions. though sometimes considered purification contaminants in the past, their repeated detection by different laboratories suggests that a number of these proteins are bona fide viral components, some of which likely contribute to the viral life cycles. the present mini review focuses on cellular proteins detected in herpesviruses. it highlights the common cellular functions of these proteins, their potential implications for host–pathogen interactions, discusses technical limitations, the need for complementing methods and probes potential future research avenues. over the last decades, many host-pathogen interactions have been characterized using genetics, biochemical, and microscopy approaches. these discoveries relied on mutants, pharmacological reagents, immunoprecipitations, immunofluorescence, electron microscopy, cell fractionation, and western blotting to name a few of the methods employed. these approaches provided much precious information but, given the typical focus of these approaches on individual molecules, likely only revealed a small portion of the proteins involved. other methods such as high throughput two-hybrid and genetic screens, nucleic acid arrays, rna interference, and proteomics are now proving essential tools to tackle the complexity of these interactions. the main advantages of mass spectrometry, for instance, are that it is a fast, sensitive and potentially a quantitative approach to identify putative novel players, particularly when coupled to efficient purification schemes. already, proteomics revealed how viruses modulate the expression of host proteins (rassmann et al., ; sun et al., ; tong et al., ; antrobus et al., ; pastorino et al., ; thanthrige-don et al., ; zandi et al., ; zhang et al., zhang et al., , coombs et al., ; emmott et al., ; lu et al., lu et al., , munday et al., ; bartel et al., ; lietzen et al., ; ramirez-boo et al., ; chou et al., ) . a relatively new and interesting field is the characterization of host-pathogen interactions within mature purified virions. as reviewed on several occasions, several studies reported the presence of individual cellular proteins in viral particles (bernhard et al., ; maxwell and frappier, ; viswanathan and fruh, ; friedel and haas, ; zheng et al., ) . this includes vaccinia virus (krauss et al., ) , influenza virus (shaw et al., ) , hiv (gurer et al., ; cantin et al., ; ott, ) , vesicular stomatitis virus (moerdyk-schauwecker et al., ) , and several herpesviruses (see below). though these cellular components have often been considered purification contaminants, the presence of similar proteins in both related and unrelated viruses suggests that some of them may be biologically relevant. the identification of virion-associated host proteins could thus lead to the discovery of novel therapeutic tools against viruses. the present review focuses on their identification and putative roles with respect to the proteomics of herpesviruses. thus far, the protein composition of eight different herpesvirions has been studied by mass spectrometry. these studies include the alphaherpesvirinae herpes simplex virus type (hsv- ) and pseudorabies virus (prv; loret et al., ; kramer et al., ) , the betaherpesvirinae human and murine cytomegaloviruses (hcmv and mcmv, respectively; kattenhorn et al., ; varnum et al., ) and the gammaherpesvirinae kaposi sarcoma herpesvirus (kshv), gamma herpesvirus (γhv ), epstein-barr virus (ebv), and alcelaphine (bortz et al., ; johannsen et al., ; bechtel et al., ; zhu et al., ; dry et al., ) . interestingly, host proteins were detected in all herpesvirions analyzed so far, as summarized in table . for instance, our laboratory previously reported the protein composition of mature extracellular hsv- viral particles and identified as many as cellular proteins (loret et al., ) . similarly, studies focusing on prv and ebv reported up to and cellular proteins, respectively (johannsen et al., ; kramer et al., ) . meanwhile, varnum et al. ( ) found as many as different host proteins in extracellular hcmv virions. while fewer cellular proteins were reported for other viral particles, it is clear that herpesviruses can potentially incorporate many proteins from its host. moreover, of the different proteins detected in herpesvirions, nine protein groups are present in at least four distinct herpesvirions. this includes - - , actin, annexins, cofilin, translation factors, gapdh, heat shock proteins, pyruvate kinase m , and various rab gtpases. these results indicate that, first of all, it is common for herpesviruses to incorporate cellular proteins into their viral particles and, secondly, that www.frontiersin.org ? different viruses share similar host proteins. most excitingly, it also suggests that these host proteins may play common roles throughout the herpesviral family. this defines an interesting and novel set of host-pathogen interactions taking place within the virus itself, rather than the cell. it is tempting to speculate that some viruses might have a higher capacity to steal cellular proteins because of their size and symmetry. herpesviruses are indeed large viruses containing a layer called the tegument between their capsids and envelopes that could accommodate non-viral proteins. though some host proteins may randomly be incorporated into virions, others may rather be selected to insure the optimal replication of the viruses that carry them. bioinformatics databases such as the kegg, gene ontology, or david are useful tools to get an overview of the functional interplay of proteins (ashburner et al., ; huang da et al., ; kanehisa et al., ) . as pointed out by friedel and haas ( ) , complex statistical tools are available to quantitatively evaluate the implication of proteins in various processes but these are beyond the scope of the present review. here an analysis of the proteins identified in herpesvirions was instead performed with the ingenuity pathways analysis database (ingenuity ® systems), which contains all the known physical and functional links among cellular proteins and defines their most significant functions. that analysis indicates that many of the cellular proteins found in herpesvirions normally modulate trafficking, cell proliferation, cell death, cell migration, cell metabolism, or the cytoskeleton (figure , upper pie chart). though subtle differences between family members are noticeable when looking at individual viruses, similar functions are found (figure , other charts) . immune-related molecules are also important constituents for several viruses, including hsv- , kshv, γhv , alcelaphine, and mcmv. altogether, this provides an overall picture whereby herpesviruses, not surprisingly, modulate all of the important aspects of the cell but where each virus might deploy its energies slightly differently. the main surprise is that so many cellular proteins are detected within assembled viral particles, which raises an important question as to their biological significance and mode of action. the overall picture that several important cellular functions might be modulated by the host proteins incorporated into viral particles is intriguing. this clever strategy is consistent with the parasitic nature of all viruses, including herpesviruses, which would presumably gain some replication advantage from stealing cellular modulators rather than coding for them in their own genomes. the most critical question is the benefit for the viruses to incorporate these cellular proteins in their assembled particles, particularly since these proteins also exist in the cells. while this is open to discussion, one possibility is that some of the incorporated cellular proteins may be remnants of the final capsid envelopment process. alternatively, this may allow the prompt action of some of these proteins immediately upon viral entry. this could jumpstart the expression and/or duplication of the viral genome, as it is the case for the herpesviral vhs, vp , icp , and icp proteins that are present in virions (lam et al., ; everett, ; halford and schaffer, ; ellison et al., ; hancock et al., ; loret et al., ; sarma et al., ; loret and lippe, ) . other early potential sites of action are the process of viral entry itself, intracellular capsid transport, import of the viral genome through the nuclear pore or immune modulation, all common steps among herpesviruses. whatever the case might be, the question remains as to why the cellular pool of these proteins would not suffice. several options may be considered. first, it may be that the virions incorporate specific isoforms, splice variants or post-translationally modified proteins that could have properties or functions distinct than their cellular counterparts. second, the incorporation of a host protein from one cell type might permit the infection of a different cell type that does not express such protein. for example, alpha herpesviruses initially infect mucosal cells and could acquire host proteins that are beneficial to infect dormant neuronal cells. finally, the host proteins might be in complex with viral proteins and it is those complexes that are active to promote the infection. these possibilities are of course speculative at this point and need to be explored. one aspect where the incorporation of host proteins in mature virions might be beneficial is molecules involved in intracellular trafficking. work by numerous laboratories demonstrated that the transport machinery used to move cellular proteins is also employed by viruses (simons and warren, ; lodish et al., ; sollner, ; greber and way, ; mercer et al., ) . this is essential for their proteins and particles to reach their final destination, for example, the site of viral replication, assembly, and/or envelopment. along with snares proteins, rab and arf gtpases are master regulators of molecular trafficking throughout the cell (sollner and rothman, ; zerial and mcbride, ; mizuno-yamasaki et al., ) . so far,vamp , a snare, was identified in prv virions (kramer et al., ) but it may only be a matter of time until other snares are discovered in other members of the herpes family. this is relevant as another snare was reported to facilitate the envelopment of mcmv capsids (cepeda and fraile-ramos, ) . in contrast, a great number of rab proteins have been identified in herpesvirions, particularly hsv- and prv (table ) . one stimulating option is that these proteins regulate the displacement of viral capsids in the cell, which could justify their incorporation in the viral particles. as rab and arf proteins collectively modulate several intracellular transport steps within the cell, it is anticipated they may be involved in various stages of the infection. for instance, rab , which is present in hsv- extracellular virions (loret et al., ) , and rab were recently demonstrated to modulate the final envelopment of the virus (zenner et al., ) . similarly, rab , found in hsv- and prv (loret et al., ; kramer et al., ) , is also necessary for the efficient assembly of the related hcmv (indran and britt, ) . it will now be of interest to determine if the virion-associated pool of these gtpases actively participates in the viral life cycle. interestingly, several rab proteins have been implicated in autophagosome formation and maturation (chua et al., ) . while it is difficult to consider how virion-incorporated rab proteins play a role at that stage, they might rather be incorporated into the virions as mcmv figure | the proteins from table were analyzed with the ingenuity database to define their putative functions in the context of an infection. to this end, the protein accession numbers (or gi numbers) were queried from the ingenuity database. for the purpose of this figure, all known functions associated with these proteins were exported to microsoft excel and regrouped. in the top pie chart, the cellular proteins found in all the herpesvirions were analyzed collectively, while the other pie charts depict the host proteins incorporated into each virus. since each protein can be associated with multiples functions in the database, the results of those analyses are expressed as relative values instead of raw numbers, which consequently exceeds the original number of proteins analyzed. the percentages therefore represent the number of proteins falling into a given category with the total of each pie chart being %. a graphical legend of the categories is provided at the bottom right corner of the figure. a consequence of their involvement in autophagosome formation and concomitant viral envelopment. given the vast impact of rab proteins on the cell, it will be a major challenge to decipher all their roles in the life cycle of herpesviruses, particularly for the pool present in mature virions. molecular trafficking is not only dependent on snares, rab, and arf proteins, it is also intimately linked to the cytoskeleton. it is thus not surprising that herpesviruses devote some of their resources toward regulating this central cellular machinery. for instance, herpesviruses significantly reorganize both cellular and nuclear actin as well as microtubules (norrild et al., ; avitabile et al., ; sharma-walia et al., ; simpson-holley et al., ; de regge et al., ; saksena et al., ) . they also travel along microtubules during both entry and egress and interact with several cellular molecular motors (sodeik et al., ; smith et al., ; dohner et al., ; marozin et al., ; lee et al., ; wolfstein et al., ; radtke et al., ) as well as cortical and nuclear actin filaments (forest et al., ; feierbach et al., ; roberts and baines, ) . furthermore, some members incorporate in their viral particles tubulin or actin-related components ( table ; wong and chen, ; grunewald et al., ) . actin has been reported to compensate the loss of various viral tegument proteins in prv (del rio et al., ; michael et al., ) and may thus act as an abundant filling agent, so its significance in herpesviral particles remains enigmatic. similarly, the relevance of intermediate filament components vimentin and keratins in some herpes virions ( table ) is difficult to assess given these filaments are not as well characterized as other cytoskeletal elements. it may nevertheless be important for herpesviruses, particularly since they are not all associated with the common skin or hair contaminants often detected in mass spectrometry (hertel, ) . viruses tend to monopolize for their own purpose their host expression apparatus, including protein translation (bushell and sarnow, ) . for example, the prototypic hsv- icp viral protein regulates all aspect of mrnas including transcription, splicing, nuclear export, and translation for the benefit of the virus (rice and knipe, ; sekulovich et al., ; sandri-goldin and mendoza, ; smith et al., ; hardwicke and sandri-goldin, ; hardy and sandri-goldin, ; brown et al., ; soliman et al., ; chen et al., ; lindberg and kreivi, ; ellison et al., ; larralde et al., ; fontaine-rodriguez and knipe, ) . as these cellular functions are highly regulated, the inclusion of ddx x, a multifunctional rna helicase that also regulates transcription, nuclear export, and translation that is used by several viruses (schroder, (schroder, , ) may be relevant. its incorporation into mature virions could thus accelerate viral gene expression in the early stages of the infection. similarly, the presence of translation initiation or elongation factors in virions (table ) may also jumpstart gene expression in favor of the viruses. interestingly, hsv- does not require cells to be in the s-phase and even arrests the cell cycle at the g /s transition step (shadan et al., ; song et al., ) , which partly explains why it can grow in non-dividing neurons. while the precise mechanism of this arrest is unclear, it is known that the viral icp protein and the vp cellular partner hcf modulate the cell cycle (hobbs and deluca, ; lomonte and everett, ; piluso et al., ) . moreover, icp interacts with the host cyclin d (kawaguchi et al., ) . however, it was recently reported that stress, rather than the cell cycle per se, may be a critical feature (bringhurst and schaffer, ) . clearly, the interaction of herpesviruses with the cell proliferation apparatus is complex and likely involves several host and viral proteins. identifying novel players that might be incorporated into mature virions may thus be very useful to clarify this process. an interesting scenario is the possible regulation of apoptosis by host proteins loaded onto viral particles. apoptosis is regulated both negatively and positively by several viruses (teodoro and branton, ; goodkin et al., ) , presumably to insure their survival at the early stages of the infection but their efficient release later on. conceptually, the presence of anti-apoptotic proteins in herpes particles might thus provide a mean to quickly evade death upon entry, while the presence of pro-apoptotic proteins on newly assembled/enveloped viral particles may trigger or stimulate their extracellular release. only further work will resolve this open question. several factors generally contribute to variation among proteomic studies. hence, the preparation of the samples (e.g., in-gel trypsin digestion versus liquid digestion and chromatography) may lead to the detection of different populations of tryptic peptides. moreover, the sensitivity of the mass spectrometers and the abundance of the proteins in the samples also impact peptide detection. the relative abundance of a peptide is itself influenced by the complexity of the samples, where some proteins may evade identification. finally, each protein differs in its properties (ionization, resolution in sds-page gels), which will be reflected in their detection. this includes snares, which are transmembrane proteins resistant to sds extraction (yang et al., ; kubista et al., ) . it is thus likely that some of the proteins in table are present in more viral particles than reported and that additional proteins are indeed incorporated in herpesvirions. more specific aspects regarding herpesviruses includes the purification schemes employed to enrich the viral particles, which will directly influence the purity of the samples and hence the potential detection of contaminants. one important caveat is that some host proteins may simply stick to the large viral particles. another one is common contaminants such as some hair/skin associated keratins or as mentioned above actin, which may simply fill the virions. however, even potential contaminants cannot simply be discarded since actin and even some keratins may indeed participate in viral life cycles. moreover, the relative abundance of all the cellular proteins within the cell is unknown, so it is not possible to rule out potential contaminants on the sole basis of abundance. it is thus critical to orthogonally validate all proteomics hits. various tools are available to define the biological relevance of host proteins identified in viral particles, including western blotting, immuno-electron microscopy or functional screens. one powerful method is rna interference. however, given the dual presence of the host proteins within the viral particles and the cell itself, this becomes a challenging task. rna interference also has its own caveats (false positives and negatives). another common step is the expression of dominant positive or negative mutants. in all cases, one major difficulty is that the host proteins may be essential for the cells and their depletion may lead to cytotoxicity, thus proper controls are needed. in addition, the host proteins might be essential for the virus within the cells but only accessory within the virions. consequently, depletion of a protein may have limited impact on the virus since complemented by the other pool of that protein in the virus or the cell. small reduction or stimulation in viral yields may thus result. it such cases, it may be necessary to produce the virus on cells that lack these proteins to see if this makes a difference. one should also consider animal models since tissue culture based screens may miss important players, for instance modulators of the immune system or virulence factors. clearly, multiple experimental strategies are needed to ultimately insure the biological significance of the host proteins found in viral particles. the identification and functions of host proteins in viral particles is an important step toward the elucidation of novel host-pathogen interactions. in the case of herpesvirions, this is well under way with eight different family members analyzed so far. one main aspect is to sort biologically relevant cellular proteins from sticky contaminants. the orthogonal validation of the host proteins found in herpesvirions using biologically relevant assays is thus critical. as pointed out above, it will necessary to analyze all these proteins in the background of two pools, one cellular and one virion-associated, which are likely to complement one another. an interesting possibility is that some isoforms or specific posttranslationally modified host proteins may be loaded into the capsids. thus a detailed analysis of the host proteins present in viral particles will be important and a potential way to distinguish them from their cell-associated counterparts. another issue is the expected variation among cell types. in that respect, it would be most interesting to examine the cellular protein content of hsv- produced in neurons in opposition to the virions produced on other cell types. finally, the mechanisms by which all these host proteins are recruited to the viral particles will also need to be explored. thus the proteomics of viral particles is only the beginning of the adventure, which should prove most exciting yet challenging. i am indebted to the canadian institutes of health research (grant # mop ) for funding our proteomics research. i also wish to thank kerstin radtke for excellent suggestions and daniel henaff for critical reading of the manuscript. proteomic analysis of cells in the early stages of herpes simplex virus type- infection reveals widespread changes in the host cell proteome gene ontology: tool for the unification of biology redistribution of microtubules and golgi apparatus in herpes simplex virus-infected cells and their role in viral exocytosis proteome analysis of vaccinia virus ihd-w-infected hek cells with -dimensional gel electrophoresis and maldi-psd-tof ms of on solid phase support n-terminally sulfonated peptides host and viral proteins in the virion of kaposi's sarcomaassociated herpesvirus new insights into viral structure and virus-cell interactions through proteomics identification of proteins associated with murine gammaherpesvirus virions cellular stress rather than stage of the cell cycle enhances the replication and plating efficiencies of herpes 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gtpase-activating proteins indicates that rab a/b and rab are important for herpes simplex virus secondary envelopment rab proteins as membrane organizers proteomic analysis of pbmcs: characterization of potential hiv-associated proteins differential proteome analysis of host cells infected with porcine circovirus type mass spectrometry based proteomic studies on viruses and hosts -a review virion proteins of kaposi's sarcoma-associated herpesvirus the author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. key: cord- -d bk gu authors: conant, katelyn l.; kaleeba, johnan a. r. title: dangerous liaisons: molecular basis for a syndemic relationship between kaposi’s sarcoma and p. falciparum malaria date: - - journal: front microbiol doi: . /fmicb. . sha: doc_id: cord_uid: d bk gu the most severe manifestations of malaria (caused by plasmodium falciparum) occur as a direct result of parasitemia following invasion of erythrocytes by post-liver blood-stage merozoites, and during subsequent cyto-adherence of infected erythrocytes to the vascular endothelium. however, the disproportionate epidemiologic clustering of severe malaria with aggressive forms of endemic diseases such as kaposi’s sarcoma (ks), a neoplasm that is etiologically linked to infection with ks-associated herpesvirus (kshv), underscores the significance of previously unexplored co-pathogenetic interactions that have the potential to modify the overall disease burden in co-infected individuals. based on recent studies of the mechanisms that p. falciparum and kshv have evolved to interact with their mutual human host, several new perspectives are emerging that highlight a surprising convergence of biological themes potentially underlying their associated co-morbidities. against this background, ongoing studies are rapidly constructing a fascinating new paradigm in which the major host receptors that control parasite invasion (basigin/cd ) and cyto-adherence (cd ) are, surprisingly, also important targets for exploitation by kshv. in this article, we consider the major pathobiological implications of the co-option of basigin/cd and cd signaling pathways by both p. falciparum and kshv, not only as essential host factors for parasite persistence but also as important mediators of the pro-angiogenic phenotype within the virus-infected endothelial microenvironment. consequently, the triangulation of interactions between p. falciparum, kshv, and their mutual human host articulates a syndemic relationship that points to a conceptual framework for prevalence of aggressive forms of ks in malaria-endemic areas, with implications for the possibility of dual-use therapies against these debilitating infections in resource-limited parts of the world. plasmodium falciparum (pf) malaria is one of the world's leading health challenges, and at least two million people, mainly children below the age of years, die each year from clinical complications of the disease (snow et al., ) . the most severe manifestations of pf malaria occur as a direct result of parasitemia following invasion of erythrocytes by post-liver blood-stage merozoites, and during subsequent cyto-adherence of parasitized red blood cells (prbcs) to the vascular endothelium and other host cells and tissues. erythrocyte invasion is executed by a family of pf reticulocyte-binding-like homolog (pfrh) ligands displayed on the surface of merozoites. among at least five known members of this family, pfrh (rodriguez et al., ; lopaticki et al., ; tham et al., ) , which is indispensable for merozoite growth in cultures, is essential for invasion by all pf strains (baum et al., ; lopaticki et al., ) . the cognate erythrocyte receptor for pfrh was recently identified as cd [also known as basigin (bsg), extracellular matrix metalloproteinase inducer (emmprin), and leukocyte activation antigen, m (hereafter cd ); crosnier et al., ] . this discovery is notable for the fact that the cd /pfrh pair is essential for invasion of all laboratory-adapted and field strains of pf, a crossstrain dependency that reveals opportunities for new anti-malarial therapies based on targeting this receptor/ligand interaction. following invasion, extensive replication within infected human red blood cells (rbcs) results in surface expression of the multidomain pf erythrocyte membrane protein- (pfemp- ) family of genes (baruch et al., ) , including pfemp- that serves as a platform for sequestration of prbc from the blood circulation by adhering to endothelial and other host cells and tissues (berendt, ) . the cyto-adhesive property of infected red blood cells to the microvasculature and sequestration within vital organ systems is an important survival strategy that allows the parasite to escape immune-mediated destruction (urban et al., ) . cyto-adherence is mediated by an orchestrated set of interactions between specific regions within the ectodomain of pfemp- [notable among them being the cysteine-rich interdomain region (cidr α)], with a variety of host molecules on the surface of capillary endothelial cells. a well-characterized host receptor that mediates cyto-adherence of most pf isolates to the peripheral vasculature is human cd (ockenhouse et al., ) , although other cell adhesion molecules are also involved in execution of strain and tissue-specific cyto-adhesive events (ockenhouse et al., ; baruch et al., ; figure ). recent studies have revealed that while erythrocyte invasion and cyto-adherence represent essential evolutionary strategies for parasite growth, survival, and persistence, they are also invariably associated with alteration of cellular physiology, which in turn may contribute directly to the defining clinical manifestations of pf infection (trossaert et al., ; fried and duffy, ) . however, less examined is the provocative hypothesis that malarial disease may not be solely attributable to complications associated with the various stages of the pf lifecycle alone; rather, the sequelae of illnesses associated with pf malaria is likely to be the collective manifestation of a multitude of complex interactions between pf, other co-pathogenic infections, and the human host. in this article, we highlight emerging evidence supporting the proposition that the signaling pathways anchored by basigin/cd and cd , two of the known host receptors that control pf invasion and cyto-adherence, respectively, are also targets for functional subversion by kaposi's sarcoma (ks)-associated herpesvirus (kshv), an inherently persistent cancer-associated herpesvirus that is prevalent in malaria-endemic regions. we discuss a number of surprising nodes of pathogenetic figure | basic interactions of p. falciparum with human erythrocytes during invasion (a) and with blood capillary endothelium during cyto-adherence (b). upon exit from the liver, invasion of red blood cells by the blood-stage merozoites leads to replication and subsequent surface expression of the multi-domain pf emp- molecule that mediates cyto-adherence via the binding activities of dbl , cidr , and dbl domains of pf emp- with various host adhesion receptors. shown here is a region of pf emp- encoded by fcr s . -var (chen et al., ) . the cidr domain primarily binds to cd and to members of the immunoglobulin superfamily, including igm and cd /pecam (platelet endothelial cell adhesion molecule), whereas the dbl domain binds mainly to cd /pecam- . interface between pf and kshv in this context, and evaluate the major implications of the apparent co-option, by both pf and kshv, of cd and cd signaling pathways as a means to promote pf persistence on one hand, and virus-induced regulation of the angiogenic phenotype, on the other. we then provide a synthesis of how the triangulation of interactions between pf, kshv, and their mutual human host represents the basis for a venerable syndemic relationship that may explain the co-incidence of aggressive forms of ks in malaria-endemic regions. the most important pathological manifestation of kshv infection is ks, a multifocal and highly angiogenic mesenchymal neoplasm characterized by profound inflammation and angioproliferative expansion of spindle cells believed to be of endothelial origin (bubman and cesarman, ) . ks occurs in at least four epidemiological forms, each with its own distinguishing clinical disposition determined by age, sex, geographical location, socio-economic status, previous exposure to parasitic infections, co-infection with the human immunodeficiency virus (hiv), and the extent of acquired or iatrogenic immunosuppression (bubman and cesarman, ; dourmishev et al., ; haverkos, ) : thus, iatrogenic ks is mostly associated with organ (especially renal) transplantation and is mostly seen as localized skin lesions among people from areas where kshv is endemic. epidemic hiv/aids-associated ks is more commonly seen among hivinfected individuals, while classical ks (cks) manifests among older men of mediterranean origin as red to purple skin plaques or nodules primarily on the lower extremities. endemic ks (eks), which is strikingly similar to cks in its clinical disposition, is highly prevalent in east and central africa, where it affects children and young adults as a cutaneous disease invading soft tissue and bone, or as a fulminant lymphadenopathy that can rapidly disseminate to visceral organs (hengge et al., a,b) . eks is currently the most common cancer in adult east and central african men and follows only cervical and breast cancer in adult women (bassett et al., ; wabinga et al., ; casper, ). an important distinction is that whereas iatrogenic and aids-related ks are invariably associated with an immunosuppressed state, cks and eks are generally not (kestens et al., ) , implying that the development and/or propagation of the latter two types of ks (i.e., cks and eks) may be controlled by unique, geographically restricted cofactors unrelated to hiv or drug-induced immune suppression (pyakurel et al., ) . a number of non-competing hypotheses have been proposed to explain the contribution of socio-economic, behavioral, and environmental co-factors to the histogenesis of eks and cks, the two types of the lesion not strictly dependent on hiv or iatrogenic immunosuppression. for example, clinical studies have revealed that eks displays a notable predilection for the feet and legs of rural peasants and cultivators living in highland areas, thus inspiring the volcanic soil hypothesis first proposed by ziegler ( ) and subsequently supported by additional epidemiologic studies (montesu et al., ; montella et al., montella et al., , montella et al., , goedert et al., ) . this hypothesis proposes that walking barefoot allows soil-borne aluminosilicates, iron oxides, and other clay minerals to be taken up through sweat glands and abrasions by resident macrophages, dermal microvascular endothelial cells, and by the lymphatic system. the resulting chronic lymphatic irritation, inflammation, and immune suppression could in turn support primary infection through these portals, and/or reactivation of latent kshv within the epidermal aspects of skin. lin and colleagues also recently suggested that exposure to soil and water constituted an additional risk factor for ks development by promoting parasitic infections that could either reduce local immune reactivity or induce a condition of inflammation necessary for ks development as well (lin et al., ) . alternatively, the finding that some natural products of plants indigenous to the areas with eks could reactivate kshv (whitby et al., ) inspired the "oncoweed" hypothesis of ks development. subsequently, ruocco et al. ( ) noted that quinine, an anti-malarial drug used extensively as one of the mainstays for treatment of malaria in immunocompetent children, may trigger ks development via its immunosuppressive effects that could support virus reactivation. it is therefore noteworthy that although available evidence is not sufficient to explain the distinctive geographical distribution of ks, the four known types of the lesion have a multi-factorial etiology, with the unifying theme being that the potential co-factors in each case operate at the level of the molecular mechanisms that control kshv infection, dissemination, and the balance between virus replication and establishment of latency. this is a relevant link given the fact that a sustained state of low level lytic replication may be important for histogenesis and probably propagation of the ks lesion (grundhoff and ganem, ) . like burkitt's lymphoma, ks is one of the most prevalent childhood cancers in malaria-endemic areas. the incidence of ks displays a considerable degree of geographic variation that mirrors the prevalence of its causative agent, and may depend on etiologic mechanisms that are controlled by geographically restricted cofactors, including malaria endemicity (ziegler et al., (ziegler et al., , ascoli et al., ascoli et al., , a coluzzi et al., coluzzi et al., , b wakeham et al., ) . for example, ks incidence is particularly high in sub-saharan africa, a region with one of the highest rates of malaria deaths (snow et al., ) . epidemiological studies have also shown a disproportionately high incidence of ks among elderly men in greece, turkey, israel, and in italy where the greatest number of recorded cases are in the formerly malaria-endemic provinces of sardinia, sicily (vitale et al., ) , and in the po valley (ascoli et al., ) . such overlapping geographic clustering of pf malaria with eks or cks has inspired the co-pathogenesis hypothesis, which proposes that a previous or ongoing exposure to kshv in a setting of underlying parasite persistence (or vice versa) may result in molecular interactions between pf and kshv that could modify the overall disease burden exerted by both pathogens at a micro level. based on this attribution, we refer to the putative co-morbidity of ks and pf malaria (in settings for which the evidence for this linkage is strong), as representing an example of a classic syndemic relationship articulated by the display of co-incident clustering within defined geographic regions. in spite of evidence from case-control studies that show a disproportionately high ks prevalence in areas of currently or previously high malaria endemicity, a molecular link between ks pathogenesis and pf malaria has not been rigorously examined at a micro level, and the correlation is even more difficult to establish at a population level because of the indeterminate nature of the mechanisms by which malaria might influence ks pathogenesis outside the known clusters of endemic disease co-incidence. on one hand, the role of malaria as a co-factor for eks has been hypothesized based on the potential of the anopheles mosquito vector to contribute to person-to-person spread of kshv, and to establish an immunosuppressed state at the site of the mosquito bite, which would then create a local permissive environment for kshv infection (coluzzi et al., ; ascoli et al., a,b,c) . however, we propose an alternative model based on the provocative hypothesis that both the malaria parasite and kshv exert bidirectional influences upon each other that operate at a much more complex level beyond the permissive benefits of immune suppression or the modifying effects of occupational, socio-economic, or environmental co-factors. as a conceptual basis for our hypothesis, consideration of some of the known molecular controls that regulate the kshv life cycle reveals a number of insights into the potential pathobiological link between ks and malaria. like epstein-barr virus (ebv) and related herpesviruses, kshv establishes both lytic and long-term latent infections, the balance between which determines the timing and intensity of pathologic outcomes in specific organ systems. interestingly, both kshv and ebv are highly prevalent in malariaendemic areas and also share many features in their life cycles, including the manner in which they regulate the molecular switch between latency and lytic replication. for example, expression of the major kshv replication and transcription activator (rta) is essential for virus replication and dissemination, and is dependent on activation of p and extracellular signal-related kinase / (erk / ) mitogen-activated protein kinase (mapk; xie et al., ) . kshv rta is the genetic and functional homolog of ebv rta/brlf (bam hi fragment z rightward open-reading frame ) which, in concert with zebra transcriptional activator/bamhi fragment z rightward open-reading frame (zta/bzlf ), can initiate ebv lytic cycle (staudt and dittmer, ) . kshv rta positively regulates immediate and delayed early promoters as well as its own promoter by interacting with cellular transcription factors such as activator protein- (ap- ), octamer binding transcription factor (oct- ), recombination signal-binding protein jkappa (rbp-jκ), and ccaat/enhancer binding protein alpha (c/ebpα; staudt and dittmer, ) ; in this way, rta is susceptible to a variety of signal transduction pathways that are likely to activate its promoter. in fact, kshv rta expression (and therefore lytic replication) can be induced in vitro using a variety of chemical compounds such as ionomycin (a calcium ionophore), sodium nbutyrate (nab, a histone deacetylase inhibitor) and phorbol esters such as -o-tetradecanoylphorbol- -acetate (tpa), all of which can also be used to induce ebv lytic cycle (luka et al., ; renne et al., ; zhu et al., ; gao et al., ) . given the shared genetic and biological properties of ebv and kshv, it is not surprising that the two viruses can co-exist as latent episomes in certain peripheral effusion lymphoma-derived www.frontiersin.org cell lines (horenstein et al., ) . they also display similarities not only in the mechanism of induction of the lytic cycle but also in the distribution of endemic cancers associated with them in regions of high malaria endemicity, which perhaps reflects the contribution of malaria as a common co-factor in the pathogenesis of cancers associated with these viruses. for ebv, the epidemiological association between malaria and african endemic burkitt's lymphoma (ebl) is well established (moormann et al., ) , although the molecular mechanisms by which malaria modifies ebl and other pathobiological outcomes of ebv infection remain a matter of intense investigation (rochford et al., ) . interestingly, ebl is a b cell lymphoma propagated by a deregulation in the c-myc oncogene as a result of chromosomal translocation, and although ebv is a necessary etiological agent for ebl, it is clearly not sufficient in absence of other essential co-factors. it is also worth noting that whereas nearly all african children in endemic areas suffer from several malaria episodes as a result of chronic exposure to pf, only a fraction of them show signs of severe, life-threatening forms of the disease, implying that the biological processes underlying the progression of infection to disease are much more complex. interestingly, ebv is ubiquitous in the general human population, and in children living in malaria endemic areas, primary infection can occur within a few months of birth (piriou et al., ) , and may seroconvert within years after primary infection, followed by a tightly orchestrated viral latency state in memory b cells that reflects the balance between viral replication and host immune control. emerging new evidence now suggests that the exit from latency to the viraemic state that supports ebl development may be impacted at a molecular level by the replicative blood-stage form of the parasite life cycle. thus, chene et al. ( ) recently demonstrated that interactions between the cidr α domain of pfemp- on the surface of prbcs, with a cognate surface receptor(s) expressed on the surface of ebv-infected human memory b cells, stimulates b cell proliferation (as previously shown; donati et al., ) and reactivates ebv not only from the chronically infected akata cell line but also from latently infected b cells isolated from the peripheral blood and tonsils of healthy ebv carriers (chene et al., ) . the mechanism by which conjugation of pfemp- -expressing prbc with ebv-infected b cells triggers virus replication in this model remains to be elucidated, but the consequences of this interaction can be readily appreciated as providing a possible explanation for the increased ebv viral load that constitutes risk for ebl among children living in areas of high malaria endemicity. remarkably, we have also discovered that cross-linking of cd on the surface of kshv-infected cells with mc , a recombinant peptide derived from the cidr α domain of pfemp- that normally interacts with cd to mediate cyto-adherence (ockenhouse et al., (ockenhouse et al., , baruch et al., ) , not only upregulated cd expression (figure a ) but also reactivated the virus from latency through transcriptional activation of kshv rta (figure b) , and that the molecular mechanisms that control this process overlap with those that putatively regulate pfemp- dependent ebv reactivation from latently infected cells (chene et al., ) . remarkably, structural mimics of mc , including the two helical heptad repeats (hr and hr ) derived from kshv entry glycoprotein b (gb) also upregulated cd figure | (a) upregulation of cd by both the pf emp- -cidr α-derived mc peptide (lanes and ) and by kshv gb-derived heptad peptide ligands hr (lanes and ), and the less potent hr (lanes and ) are both blocked by a monoclonal anti-cd antibody. note that anti-cd did not block control upregulation of cd in response to treatment with sodium butyrate (nab; lanes and ). (b) activation of kshv rta by mc occurs via a cd -dependent mechanism that can be blocked by anti-cd antibody fa - . methodology : briefly, melanoma-derived mel cells were seeded in a six-well plate and either left untreated or pre-incubated with μg/ml of anti-cd monoclonal antibody clone fa - for min. at • c. after washing to remove excess antibody, cells were incubated with either μg/ml of kshv gb-derived hr , μg/ml hr peptide, μg/ml recombinant mc , or mm sodium butyrate (nab). forty-eight hours after treatment, total rna was isolated and used as template in semi-quantitative rt-pcr with primers to an internal fragment of human cd , viral rta, or human glyceraldehyde -phosphate dehydrogenase (gapdh) loading control. (c) specific activation of the kshv lytic switch protein, rta, by the pf emp- -cidr αderived peptide mc from pf malayan camp strain, but not by peptides derived from cidr α domains of a tres (which binds icam- ) or the vietnam oak knoll strain (fvo). expression and further induced virus reactivation in a cd dependent manner. indeed, like mc , the effect of these helical peptides could be blocked by a monoclonal antibody to cd (figures a,b) , suggesting that they bind a region on the exoplasmic face of cd that overlaps with mc . it is also insightful that the kshv lytic cycle was activated only by the pfemp- -cidr α-derived peptide from pf malayan camp strain, but not by peptides derived from the vietnam oak knoll (fvo) or a tres strain [that preferentially binds intercellular adhesion molecule (icam- ) and not cd ] (figure c) , demonstrating that reactivation of kshv by the parasite ligand displays some degree of strain specificity for pf that is associated with severe malaria in africa. since cd mediates parasite persistence by mediating cytoadherence and sequestration of parasitized erythrocytes away from immune surveillance, the ability of mc to stimulate kshv reactivation is significant, as it supports a model whereby in vivo cross-linking of cd on the surface of kshv-infected cells, either by its natural ligand(s) or upon conjugation with the cidr α domain of pfemp- expressed on the surface of parasitized erythrocytes (as illustrated in figure ) , represents a previously unrecognized mechanism by which kshv lytic replication could be induced in the context of a pf malaria co-infection. this model provides many new opportunities for experimental examination of the ability of parasite-derived antigens to reactivate kshv during cyto-adherence on the surface of infected blood endothelial cells and within tissues and organs: (i) biochemical analysis of structure/function relationships that control interactions of cidr α (and its structural analogs) with cd , should reveal potential targets for small-molecule inhibition of parasiteinduced sequestration or reactivation of kshv in co-infected individuals. (ii) elucidation of the signaling mechanisms that regulate cd -dependent rta activation will require multi-pronged approaches that employ dominant negative versions of the srclike kinases such as yes, fyn, and lyn that control down-stream signaling events initiated by cd ligation (see figure ). (iii) deletional mutagenesis, domain-swapping, functional complementation, and loss-of-function analysis of genetic mimics of the recently identified cd polymorphisms that lack the signaling motif (aitman et al., ; gelhaus et al., ; chilongola et al., ; fry et al., ) should elucidate the correlates of upstream signaling networks required for cd -dependent activation of the kshv lytic cycle. cd is a class ii glycoprotein involved in multiple physiological functions including cell adhesion, fatty acid uptake, non-opsonic phagocytosis, and angiogenesis (mcgilvray et al., ; podrez et al., ; febbraio, , ). the cd structure consists of a large extracellular loop and two short cytoplasmic tails at the n-and c-termini (silverstein and febbraio, ). the c-terminal tail is involved in signal transduction via association with src-like kinases, whereas the extracellular domain contains binding sites for thrombospondin- (tsp- ), a potent natural inhibitor of angiogenesis (bagavandoss and wilks, ; good et al., ; tolsma et al., ) , and a variety of other ligands including the cidr α domain of pfemp- (ockenhouse et al., ) . although cd has never been directly associated with kshv pathogenesis, some of its pleiotropic functions are consistent with its potential contribution to the basic pathobiology of kshv. for example, cd is often expressed in association with signaling structures such as lipid rafts that also contain host receptors for kshv entry, including integrins and the kshv fusion receptor complex xct/cd (akula et al., ; kaleeba and berger, ; veettil et al., ) . since cd -mediated signaling may culminate in activation of p and erk mapk pathways that overlap with one or more pathways necessary for kshv rta-dependent transcription of viral gene expression (yipp et al., ; cohen et al., ; xie et al., ) , our data suggests that figure | hypothetical model of cd -dependent rta activation. a motif displayed by pf emp- on the surface of parasitized erythrocytes interacts with its cognate epitope within the ectodomain of cd on the surface of microvascular endothelial cells. this interaction activates one or more of the src-like kinases, which in turn initiate a phosphorylation cascade that results in p and erk/mapk activation. this process culminates in activation of a cellular transcription factor, likely ap- (dimer of c-jun and c-fos), which translocates into the nucleus and stimulates kshv rta-dependent transcription of viral lytic cycle genes, starting with immediate early (ie), which then activate delayed early (de), followed by late structural genes involved in assembly of an infectious virus particle. cidr α-dependent rta activation may occur via by signals transduced through cd (figure ) . however, our model also raises many significant questions: (a) does cidr α-induced kshv replication involve mechanisms that overlap with, or distinct from those that support cidr α-induced ebv replication (chene et al., ) ? (b) does it require the traditional cd -regulated recruitment of src-like kinases and subsequent phosphorylation of p and erk mapk? (c) does it directly result in downstream activation of a specific cellular transcription factor such as ap- that is known to activate kshv rta (or ebv zta) promoter(s) (e.g., see figure )? (d) is it strictly dependent on interactions of cidr α with a distinct epitope on cd , or does it overlap with, and is therefore also inducible by, other cd ligands such as tsp- ? investigation of the role of tsp- in this context is relevant, given that tsp- is present in high concentrations in human saliva (crombie et al., (crombie et al., , shugars, ) that generally contains significant levels of kshv virions shed from the underlying oral zones of carriers (pauk et al., ; coluzzi et al., a; chene et al., ; hadinoto et al., ) . critical new experiments that address these derivative questions represent an exciting area of research into the molecular basis for the emerging new paradigm of co-pathogenesis. it is also anticipated that isolation of polymorphisms in cd and other human genes that control host interactions with pf, ebv and kshv may open up additional opportunities for population-level studies aimed at explaining the www.frontiersin.org overlapping distribution of ks, burkitt's lymphoma, and malaria in areas where these diseases display coincident endemicity. angiogenesis, defined as the development of new blood vessels, is necessary for growth and proliferation of vascular tumors like ks, the extent of which is controlled by the balance between pro-angiogenic and angiostatic elements of the human hemostatic system. one of the regulatory components of this system is tsp- , the angiostatic cd ligand known to inhibit endothelial cell proliferation, migration, and tube formation (iruela-arispe et al., ) . remarkably, kshv upregulates cd in melanomaderived cell lines but downregulates the protein in endothelial cells both at mrna and protein levels (figure ) . the mechanism(s) by which kshv accomplishes these dichotomous, cell type-specific effects are not fully understood, but one study recently demonstrated that kshv-encoded micrornas can directly target tsp- mrna for degradation (samols et al., ) , ostensibly to promote an angiogenic growth state of infected cells via attenuation of the angiostatic effects resulting from interactions between tsp- and cd . since the angiostatic signal is associated with viral lytic replication while the angiogenic phenotype is linked to the latency phase, virus regulation of cd expression and signaling in infected cells implies that the correlates of kshv latency are contextually linked to the angiogenic phenotype in diseaserelevant cells. they also provide a potential explanation for our recent findings that the kshv latency program is inefficient in melanoma cells from which the virus undergoes robust spontaneous replication, as opposed to endothelial cells in which the virus establishes a much tighter state of latency (fontana et al., unpublished findings) . in addition to direct targeting of tsp- , kshv blunts cd signaling in endothelial cells by upregulating the endothelin (et- ) system. et- is a pro-angiogenic peptide secreted by the vascular endothelium and its deregulation is implicated in the pathogenesis of many malignancies (nelson et al., ) . et- polypeptides and their cognate receptors are expressed in ks lesions (nelson et al., ; basilico et al., ) , and et- receptor blockade limited ks cell invasion in an in vivo tumor growth model (rosano et al., ) . the interplay between the pro-angiogenic effects of et- and the pathophysiology of ks is also encountered in patients with complicated pf malaria in which plasma concentrations of big et- , the precursor for bioactive et- , are elevated (wenisch et al., ) as a direct result of cyto-adherence of prbcs to human endothelial cells, independent of the parasite strain and regardless of the origin of endothelial cells (basilico et al., ) . there is, therefore, a significant degree of molecular crosstalk between pf and kshv at the level of et- biology. considered in a broader context, it is conceivable that by suppressing the angiostatic effects of cd signaling either by reducing cd expression, downregulation of tsp- , or via upregulation of et- , kshv could establish long-term persistence by establishing a state of limited cd -dependent viral reactivation, or reduce cd -dependent cyto-adherence and consequently limit the frequency of illnesses associated with this aspect of the parasite life cycle. unfortunately, the latter outcome might be associated with an increase in the likelihood for parasite access to the extra-peripheral organs such as the brain, which may elevate the probability of cerebral malaria. although measurement of these parameters in vivo is not trivial, a number of guiding principles can emerge from in vitro scrutiny of these molecular interactions based on experimental approaches that might predict their occurrence in vivo. it was recently discovered that many people of african origin harbor a high frequency of mutations and single-nucleotide polymorphisms (snps) that cause a deficiency in the cd gene, yet semi-quantitative rt-pcr (a) and western blot (b) analysis of cd and cd expression in uninfected (−) versus infected (+) endothelial (lymphatic) or melanoma-derived mel cells. kshv upregulates both cd and cd in melanoma-derived cells, whereas in lymphatic and telomerase-immortalized mixed dermal microvascular and brain endothelial cells, kshv upregulates cd (confirming a recent study; qin et al., ) but downregulates expression of cd (and its angiostatic ligand, tsp- ; data not shown); in vivo, these dichotomous effects are consistent with promotion of angiogenesis, invasion, and tumor metastasis in disease-relevant cell types. they still suffer from severe (particularly cerebral) malaria (aitman et al., ; gelhaus et al., ; chilongola et al., ; fry et al., ). the snps found in kenya and gambia introduce a premature stop codon that results in a truncated cd protein lacking the c-terminus and is therefore incompetent for signal transduction but can still bind its ligand(s), leading to the conclusion that mutations that cause cd deficiency may reduce cd -mediated parasite sequestration in peripheral organs but they may not protect from severe cerebral malaria (aitman et al., ) that is associated with cyto-adherence to brain endothelium via interactions of pfemp- with icam- but not cd (ockenhouse et al., ) . this level of linkage in which mutations in cd -a molecule that is important for parasite persistencecause a deficiency that does not alter malaria pathogenesis, implies existence of selection pressures that may be induced or maintained in the population by an endemic infection (other than malaria) whose persistence is linked to this genetic output. the fact that the mutations and their phenotypes occur at high frequency in malaria-endemic areas with a high prevalence of viruses associated with endemic cancers underscores a pathobiological paradigm whereby an inherently persistent tumor virus such as kshv (or ebv) could provide the selective pressure for introduction or maintenance of such a mutation into the genetic registry of populations living in regions of high malaria endemicity. given that pfemp- interactions with cd result in induction of the viral lytic cycle, such a virus-induced genetic output would conceivably be designed to promote virus escape from immune surveillance by limiting virus reactivation that might result from interactions between pfemp- and cd on latently infected cells. the impact of such a genetic influence, can only be measured against the host's ability to restrict virus replication and dissemination, and it could be achieved by interrogating viral genomic variability or stability in a given population against a profile of polymorphisms within the genetic registries at the cd locus on a population basis. availability of patient samples with known patho-status and disease severity from regions in which the distribution of malaria overlaps with the incidence of virus-associated endemic cancers would facilitate such a retrospective analysis. cd is a widely expressed, type i integral membrane receptor that belongs to the immunoglobulin (ig) superfamily (biswas et al., ) . it is over-expressed in a variety of disseminated human solid cancers and is a major contributor to the malignant phenotype in a variety of human cancers (riethdorf et al., ) . signaling events transduced through cd are associated with survival, metastasis, and invasion of a variety of cancer cells, mainly because it stimulates enhanced stromal release of multiple matrix metalloproteinases (mmps) and vascular endothelial growth factor (vegf), which are among the key mediators of angiogenesis and metastatic transition (marieb et al., ; tang et al., ; bougatef et al., ; kanekura and chen, ) . cd promotes hyaluronan synthesis (marieb et al., ; slomiany et al., a) , upregulates the wnt/β-catenin signaling pathway (sidhu et al., ) , and is also involved in epithelial-to-mesenchymal transition (emt; wu et al., ) . given the pleiotropic function of cd , the significance of cd /pfrh interactions in parasite invasion unlocks new avenues for investigating the nodes of pathogenetic intersection between pf malaria and other coinfecting agents such as kshv that have evolved mechanisms to subvert the cd signaling pathway to promote their existential success. several layers of this pathogenetic intersection are revealed by new data on the manner in which both pf and kshv have evolved to exploit cd and endothelial cell biology: a. whereas endothelial cells play a prominent role as a platform for cyto-adherence of prbcs, kshv displays profound tropism for this cell lineage that represents the basis for origination of the hyper-proliferating spindle cells characteristically found in ks lesions (bubman and cesarman, ; ganem, ) . b. recent studies demonstrated compelling evidence that de novo kshv infection of human endothelial cells as well as oral and fore-skin-derived fibroblasts results in upregulated expression of cd and that this effect is directly associated with various virological outcomes consistent with a pro-invasive, migratory, and pro-angiogenic phenotypes (qin et al., ; dai et al., a,b) . we have also found that kshv upregulates cd not only in melanoma-derived cells but also in chronically infected lymphatic, microvascular, and brain endothelial cells (e.g., see figure ). c. two recent reports showed that kshv promotes endothelialto-mesenchymal transition (endo-mt) through activation of notch-dependent signaling events that culminate in stimulation of an invasive phenotype analogous to that orchestrated by cd (cheng et al., ; gasperini et al., ) . kshv-induced endo-mt was dependent on the activity of membrane-type- mmp (mt -mmp; cheng et al., ) , which is consistent with a role for cd in endo-mt since mt -mmp is a cd -stimulated endopeptidase involved in extracellular matrix remodeling. it is therefore conceivable that one of the mechanisms underlying kshv-induced endo-mt may involve viral induction of mmp activity , likely through upregulation of cd . d. cd has been implicated in the entry processes of a number enveloped viruses including hiv- (pushkarsky et al., ) , measles virus (watanabe et al., ) , and severe acute respiratory syndrome coronavirus (sars-cov; chen et al., ) . based on our recent studies, we also have reason to believe that cd may regulate kshv entry as well, as an antihuman cd (neurothelin) antibody can potently block kshv glycoprotein-mediated fusion, consistent with existence of cd in the membrane of kshv-permissive cells as part of a molecular supercomplex (guo et al., ; yang et al., ; gallagher et al., ) that includes host molecules implicated in virus entry, such as integrins (akula et al., ) and the cystine transporter complex xct/cd hc (xu and hemler, ; kaleeba and berger, ) . it is also noteworthy that, like cd , cd also associates with the xct/cd hc complex and confers resistance to some chemotherapeutic drugs (okuno et al., ; yang et al., ; zou et al., ) . interestingly, another independent study also demonstrated that the transport activity of xct/cd hc, perhaps in association with cd , is a critical correlate of resistance to cisplatin (huang et al., ; www.frontiersin.org singh et al., ; chen et al., ; riglar et al., ) . for kshv-infected individuals, one implication of these molecular associations could be that virus-induced upregulation of cd (as we and others have shown; qin et al., ; dai et al., a,b) , could stabilize cd -containing multi-partite complexes and in turn potentiate their drug efflux functions, which could blunt the efficacy of chemotherapeutic strategies that might be used in the treatment of ks and other virusassociated malignancies. in support of this view, qin et al. ( ) recently showed that the intrinsic resistance of kshvpositive peripheral effusion lymphomas to the cytotoxic effects of paclitaxel and doxorubicin depends on orchestrated interactions of cd with lymphatic vessel endothelial receptor (lyve- ) and the homodimeric atp-binding cassette (abc)-g /bcrp (breast cancer resistance protein) drug transporter that is highly expressed on the surface of primary effusion lymphoma (pel)-derived cell lines. e. available evidence suggests that "outside-in" signaling may be required for the malaria parasite invasion (singh et al., ; chen et al., ; riglar et al., ) , but it remains to be determined if the structural framework that supports erythrocyte invasion through cd is distinct from, or overlaps with, the epitope on the cd ectodomain that senses signals transduced to endothelial and other cells that express this molecule. if they are the same, pfrh -mediated conjugation of merozoites with cd on normal or infected endothelial cell surfaces should result in so-called parasite-to-host "trans-signaling" events that may lead to untoward pathologic outcomes unrelated to parasite invasion itself. given that pfrh has neither a transmembrane domain nor a cytoplasmic tail, the probability of trans-signaling is likely to be high since a soluble form of pfrh [existing either as a monomer, as part of a bioavailable complex with another pathogen molecule such as pfripr (chen et al., ) , or as bound to a host "handler"] could initiate cd -dependent signaling outcomes that are likely to modify pf malaria or kshv infection (marieb et al., ; xu et al., ; ruiz et al., ; slomiany et al., b; sidhu et al., ; wu et al., ) . a first-line experimental testing of such a concept should seek to determine whether blood-borne merozoites or soluble forms of pfrh can indeed bind cd on kshv-infected cells and whether those interactions are directly associated with a cd -dependent alteration in cellular behavior, including extracellular remodeling and induction of a pro-angiogenic phenotype that is one of the defining features of the infectious process of kshv (qin et al., ; cheng et al., ; dai et al., a,b; gasperini et al., ) . in figure , we highlight some of the important nodes of intersection between kshv and the malaria parasite in endothelial cells and skin-derived melanoma cells, along with their potential impact on the virus life cycle (i.e., reactivation), ks tumorigenesis, and malaria disease outcomes. in melanoma-derived cells (mel ), kshv upregulates both cd (and its ligand, tsp- ) and cd (as shown in figure ) . binding of the pf emp- cidr α-derived peptide, mc , to virus-upregulated cd prevents akt phosphorylation while inducing rta-dependent kshv reactivation via the mapk/p pathway. remarkably, structural mimics of mc , such as the helical heptad repeat (hr) regions derived from kshv glycoprotein b (gb), can, like mc , also induce virus reactivation in a cd -dependent manner (as shown in figure ). (b) in lymphatic (lec) and other endothelial cells such as mixed tolemeraseimmortalized dermal microvascular and brain endothelial cells (tdmb), kshv upregulates cd but unlike in melanoma cells, the virus downregulates both cd and its angiostatic ligand, tsp- ; in vivo, these effects are likely to promote angiogenesis, invasion, and tumor metastasis. (c) as a key receptor for the merozoite invasion antigen, pf rh , kshv-induced upregulation of cd on the surface of infected endothelial cells increases the frequency of contacts between merozoite-bound or soluble pf rh and blood or dermal microvascular endothelial surfaces, resulting in induction of cd -mediated signals that could alter the microenvironment and cause pathologic outcomes in a variety of physiological sites in the co-infected host. kaposi's sarcoma is one of the most frequent neoplasia diagnosed in malaria-endemic regions of africa, yet despite recent progress (casper, ; casper and wald, ) , the landscape of effective therapeutic strategies for ks remains limited. it is clear that ks presents in different epidemiologic and clinical forms dictated by a variety of modifying risk factors, but the lack of data on the relative contributions of these co-factors in any given epidemiological setting frustrates efforts aimed at developing new approaches for clinical management of the disease. surgical removal of isolated nodular ks does not eliminate latent kshv at secondary sites, while traditional chemotherapy is generally toxic and may have a high failure rate in hiv-infected patients in whom durable post-therapy immune reconstitution is improbable. for epidemic (hiv/aids-associated) ks, intervention with the highly active anti-retroviral therapy (haart) in hiv/hhv (human herpes virus ) co-infected patients may have contributed to regression of ks lesions, but the initial success of haart-based therapy for ks has been eroded by several concerns. first, hiv is not necessary for endemic african or classical mediterranean ks. second, haart only limits the potentiating immunosuppressive effects of hiv but it does not remove the underlying etiology of ks. third, haart may reduce hiv viral load but it does not restore the entire t cell repertoire necessary for immunity against kshv. fourth, lack of access to haart, non-compliance with the treatment, failure to respond to treatment, and the development of drug-resistant strains of hiv confound the overall benefit of a strictly haartbased approach to ks management. fifth, the benefits of haart are not long-lasting and end up being more detrimental to many patients with advanced ks who may show no improvement while remaining in danger of developing post-therapy immune crisis (krown et al., ) . sixth, the probability of recrudescence of ks in patients treated with haart later in life is unpredictable. there is, therefore, an urgent need for a multi-pronged therapeutic approach aimed at developing strategies that are appropriate to the prevailing epidemiologic state of the disease, with the overall goal being improvement of the treatment outcome for ks patients in sub-saharan africa and other resource-limited parts of the world (mcallister et al., ) . the concept of "angio-therapy" designed to inhibit growth of angio-proliferative cancers like ks (tosetti et al., a,b; ferrari et al., ; pfeffer et al., ) led to the surprising observation that the anti-malarial peptide artesunate has anti-angiogenic effects on ks-derived endothelial cell lines (dell'eva et al., ) . artesunate is already well tolerated as an anti-malarial drug, and because it has direct effects against transformed cells, the promise of its dual use for ks is attractive for co-infected individuals. if adopted as such, the clinical benefits of artesunate for treatment of ks would represent a classic illustration of one of the defining attributes of a syndemic relationship in which the overall clinical impact of two linked infections (i.e., pf and kshv in this case) can be blunted by targeting the molecular underpinnings that link the parasite with the disease-modifying influence of kshv. in addition, it has been proposed that quinine and its chloroquine and hydroxychloroquine derivatives -drugs that have been used widely to treat malaria -are immunosuppressive and may, as such, serve as co-factors for ks by stimulating kshv reactivation, which would not only promote virus dissemination but could also support ks histogenesis (ruocco et al., ) . however, the "oncodrug" hypothesis for ks is likely to be more relevant for individuals with severely altered immunity since, in immunocompetent hosts, the viremic state induced by quinine and its derivative drugs would concomitantly expose hematogenously disseminating virions to immune surveillance which could in turn limit virus spread. with respect to malaria control, approaches that interrupt the parasite life cycle are ideal, yet in spite of many multi-national efforts in this regard, successful elimination of the disease remains a major challenge, as more than half of the world's population still lives in areas where there is a risk of contracting the disease. there are many reasons for this sobering report card, chief among them being persistent endemicity as a result of drug and insecticide resistance, inadequate support for malaria control programs, poor environmental management, the complex biology of the disease, as well as the regional variability not only in the parasite but also in the nature of its impact on specific populations and age groups. another major challenge remains the lack of practical and affordable animal models that can faithfully reflect mechanisms of malaria pathogenesis and immunity in humans. such platforms would be valuable for evaluating the efficacy of drug and vaccine candidates, and for predicting the benefits of drug combinations that can maximize safety and efficacy while minimizing the development of drug resistance. given that the invasive asexual blood stage of the pf lifecycle is the form associated with symptomatic malaria, recent efforts toward a malaria vaccine have primarily focused on targeting this stage. in this respect, the discovery of the essential merozoite invasion receptor increases the number of potential targets for a second-generation malaria-specific vaccine based on cd /pfrh interactions. however, the fact that deletion of other pfrh proteins also impairs invasion, albeit in a strainspecific manner, complicates vaccine design efforts, as it suggests that the pfrh /cd interaction may be only one of many ligand-receptor recognition events that must occur during execution of the invasion process (cowman and crabb, ; tanne, ; tham et al., ). an approach that targets cd may not be feasible, as it could impact many important physiological processes controlled by this molecule. on the other hand, innovations oriented toward development of vaccine and therapeutic strategies based on the invariant aspects of pfrh and other "accessible" pf antigens may result in a more meaningful outcome associated with minimal impact on the human host. such strategies may include the combined use of nanovehicledeliverable peptide mimetics and single-chain antibodies that can be administered before or during active parasitemia, or therapeutic lentiviral vectors carrying immunogenic epitopes that can harness the host's immune capacity. these pathogen-centered approaches could be used alone or in conjunction with the rts,s vaccine that is based on the most prominent surface antigen of the pre-liver sporozoite stage and which has already shown some promise in phase iii trials (tanne, ) . in using pfrh as the target, however, the primary goal of any given approach [antibody-based (douglas et al., ) , or otherwise] would be to elicit the safest, most long-lasting and most efficacious outcome that limits the availability of pfrh to mediate invasion, but it must also be guided by the recognition that this antigen is predominantly located within the rhoptries and it is liberated and or revealed to the immune system only for a short period of time when the merozoite contacts the erythrocyte prior to invasion. infectious agents have long been implicated in the etiology of a variety of illnesses, and although recent studies have examined the role of microbial co-infections in many disease settings, it is not known whether "cooperative pathogenesis" is sufficient to provide the driving force behind strategies in which co-pathogenic agents co-evolve and forge a state of forbearance with each other and with their shared host to advance mutually exclusive existential goals. clearly, pf malaria has been linked to a variety of other infections that display co-pathogenic relationships with the parasite, notable among them being ebl (thorley-lawson and allday, ), but recent advances have revealed that many other important examples of these relationships do exist, and in most cases they provide a surprisingly informative conceptual window into how co-infections can modify each other's disease course. for instance, the recent elucidation of the molecular mechanisms of erythrocyte invasion and cyto-adherence has: (a) illustrated the extent to which pf can exert its impact on human physiology; (b) exposed how alterations in the expression and function of the host receptors that support these processes could not only dramatically change the dynamics of malaria but may also influence the pathogenesis of other coinfections such as kshv that exploit these pathways for existential benefit; and (c) generated new interest in the structural dispositions of these receptor/ligand pairs as potential multi-domain vaccine targets against specific stages of the pf life cycle. although malaria is not considered a typical opportunistic infection in the same way that kshv is, recent advances have revealed a surprising node of intersection between kshv and malaria pathogenesis at the level of the molecular controls that regulate the persistence of these two highly successful infectious agents. to the extent that such interactions can be measured at a micro level, the concept of co-pathogenesis establishes grounds for the expectation that kshv and pf can bidirectionally influence the clinical course of each other at many physiological levels leading to a variety of clinical outcomes (table ; figure ). for example, cd -mediated cyto-adherence, by virtue of its ability to stimulate pathways that overlap with those required for kshv reactivation, may contribute to a transient increase in kshv viral load and dissemination, which in turn could increase the frequency of other defining correlates of kshv-associated disease that rely on a viremic state. on the other hand, the pathogenic mechanisms of a latent kshv infection, which include kshv-induced downregulation of cd , could effectively alter the overall disease burden by limiting peripheral sequestration; this could in turn increase parasitic access to the central nervous system, leading to a higher probability for cerebral malaria. ultimately, the emerging picture supports a syndemic link which, however serendipitous, reveals a co-evolutionary paradigm centered at the putative dueling role of cd as a mediator of parasite sequestration on one hand and kshv replication on the other. in this regard, more extensive molecular and genetic analysis is required in order to determine the extent to which kshv (or ebv, for that matter) might provide the driving force for altering the genetic registry at the cd locus in regions of high malaria endemicity. it might also be necessary to analyze the cd locus in hematopoietic versus peripheral b cells that may serve as the vehicle for virus dissemination in vivo, in order to determine whether infection increases the propensity for a heritable lesion at this locus. in conclusion, recent advances have revealed several nodes of pathobiological intersection between malaria and a variety of clinically significant infections, and although substantial progress has been made, we are still in the "embryonic stage" of understanding how co-infections interact with each other in their mutual host. derivative new research emphasis that is inspired by these concepts should include the important goal of developing practical in vivo platforms (animal models) that could facilitate systematic, experimental integration of population studies with reductionist multi-component molecular data. although we are still a long way from developing such a platform for studying the malaria and kshv co-infection paradigm, attempts toward this goal are an essential step in elucidating the extent to which triangular interactions between pf, kshv, and their mutual human host might articulate a syndemic relationship that underlies the co-incidence of aggressive ks in parts of the world with endemic malaria. once the correlates of co-pathogenesis are isolated, innovative research efforts oriented toward development of effective "combined" therapies can be launched. malaria susceptibility and cd mutation integrin alpha beta (cd c/ ) is a cellular receptor for kaposi's sarcomaassociated herpesvirus (kshv/hhv- ) entry into the target cells high incidence of classic kaposi's sarcoma in mantua kaposi's sarcoma, human herpesvirus infection and the 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how co-infection with epstein-barr virus leads to endemic burkitt lymphoma the endothelin axis: emerging role in cancer molecular basis of sequestration in severe and uncomplicated plasmodium falciparum malaria: differential adhesion of infected erythrocytes to cd and icam- identification of a platelet membrane glycoprotein as a falciparum malaria sequestration receptor role of cystine transport in intracellular glutathione level and cisplatin resistance in human ovarian cancer cell lines mucosal shedding of human herpesvirus in men antiangiogenic activity of chemopreventive drugs early age at time of primary epstein-barr virus infection results in poorly controlled viral infection in infants from western kenya: clues to the etiology of endemic burkitt lymphoma platelet cd links hyperlipidemia, oxidant stress and a prothrombotic phenotype cd facilitates hiv- infection by interacting with virusassociated cyclophilin a kshv/hhv- and hiv infection in kaposi's sarcoma development kaposi's 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kaposi sarcoma and quinine: a potentially overlooked triggering factor in millions of africans identification of cellular genes targeted by kshv-encoded micrornas endogenous mucosal antiviral factors of the oral cavity emmprin regulates the canonical wnt/beta-catenin signaling pathway, a potential role in accelerating lung tumorigenesis cd -tsp-hrgp interactions in the regulation of angiogenesis cd , a scavenger receptor involved in immunity, metabolism, angiogenesis, and behavior distinct external signals trigger sequential release of apical organelles during erythrocyte invasion by malaria parasites inhibition of functional hyaluronan-cd interactions in cd -positive primary human ovarian carcinoma cells by small hyaluronan oligosaccharides hyaluronan, cd , and emmprin regulate lactate efflux and membrane localization of monocarboxylate transporters in human breast carcinoma cells the global distribution of clinical episodes of plasmodium falciparum malaria the rta/orf transactivator 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in the elderly populations of mediterranean islands trends in cancer incidence in kyadondo county parasite infection is associated with kaposi's sarcoma associated herpesvirus (kshv) in ugandan women cd /emmprin acts as a functional entry receptor for measles virus on epithelial cells big endothelin in patients with complicated plasmodium falciparum malaria reactivation of kaposi's sarcoma-associated herpesvirus by natural products from kaposi's sarcoma endemic regions hab g/cd promotes epithelial-mesenchymal transition through tgf-beta signaling and is transcriptionally regulated by slug reactivation of kaposi's sarcoma-associated herpesvirus from latency requires mek/erk, jnk and p multiple mitogen-activated protein kinase pathways metabolic activation-related cd -cd complex sirna targeted against hab g/cd inhibits mmp- secretion, actin and fak expression in hepatocellular carcinoma cell line via erk / pathway bridge linkage role played by cd hc of anti-tumor drug resistance and cancer metastasis on cisplatin-resistant ovarian cancer cells src-family kinase signaling modulates the adhesion of plasmodium falciparum on human microvascular endothelium under flow identification of the immediate-early transcripts of kaposi's sarcoma-associated herpesvirus risk factors for kaposi's sarcoma: a casecontrol study of hiv-seronegative people in uganda endemic kaposi's sarcoma in africa and local volcanic soils kaposi's sarcoma: a comparison of classical, endemic, and epidemic forms inhibition of cd gene expression via rna interference reduces tumor cell invasion, tumorigenicity and increases chemosensitivity to paclitaxel in ho- pm cells the authors wish to express special thanks to dr. louis miller, dr. david narum, and dr. morris makobongo for expert advice and for providing recombinant mc proteins, and to anita marinelli and other members of the johnan a. r. kaleeba laboratory for technical assistance and constructive discussions. work in the authors' laboratory is supported by a grant from the u.s. department of defense (r ns and r rz to johnan a. r. kaleeba) through the intramural award program of the uniformed services university of the health sciences. the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. key: cord- -d iwkatn authors: henry, kevin a.; arbabi-ghahroudi, mehdi; scott, jamie k. title: beyond phage display: non-traditional applications of the filamentous bacteriophage as a vaccine carrier, therapeutic biologic, and bioconjugation scaffold date: - - journal: front microbiol doi: . /fmicb. . sha: doc_id: cord_uid: d iwkatn for the past years, phage display technology has been an invaluable tool for studies of protein–protein interactions. however, the inherent biological, biochemical, and biophysical properties of filamentous bacteriophage, as well as the ease of its genetic manipulation, also make it an attractive platform outside the traditional phage display canon. this review will focus on the unique properties of the filamentous bacteriophage and highlight its diverse applications in current research. particular emphases are placed on: (i) the advantages of the phage as a vaccine carrier, including its high immunogenicity, relative antigenic simplicity and ability to activate a range of immune responses, (ii) the phage’s potential as a prophylactic and therapeutic agent for infectious and chronic diseases, (iii) the regularity of the virion major coat protein lattice, which enables a variety of bioconjugation and surface chemistry applications, particularly in nanomaterials, and (iv) the phage’s large population sizes and fast generation times, which make it an excellent model system for directed protein evolution. despite their ubiquity in the biosphere, metagenomics work is just beginning to explore the ecology of filamentous and non-filamentous phage, and their role in the evolution of bacterial populations. thus, the filamentous phage represents a robust, inexpensive, and versatile microorganism whose bioengineering applications continue to expand in new directions, although its limitations in some spheres impose obstacles to its widespread adoption and use. the filamentous bacteriophage (genera inovirus and plectrovirus) are non-enveloped, rod-shaped viruses of escherichia coli whose long helical capsids encapsulate a single-stranded circular dna genome. subsequent to the independent discovery of bacteriophage by twort ( ) and d 'hérelle ( ) , the first filamentous phage, f , was isolated in loeb ( ) and later characterized as a member of a larger group of phage (ff, including f , m , and fd phage) specific for the e. coli conjugative f pilus (hofschneider and mueller-jensen, ; marvin and hoffmann-berling, ; zinder et al., ; salivar et al., ) . soon thereafter, filamentous phage were discovered that do not use f-pili for entry (if and ike; meynell and lawn, ; khatoon et al., ) , and over time the list of known filamentous phage has expanded to over members (fauquet et al., ) , including temperate and gram-positivetropic species. work by multiple groups over the past years has contributed to a relatively sophisticated understanding of filamentous phage structure, biology and life cycle (reviewed in marvin, ; rakonjac et al., ; rakonjac, ) . in the mid- s, the principle of modifying the filamentous phage genome to display polypeptides as fusions to coat proteins on the virion surface was invented by smith and colleagues (smith, ; parmley and smith, ) . based on the ideas described in parmley and smith ( ) , groups in california, germany, and the uk developed phage-display platforms to create and screen libraries of peptide and folded-protein variants (bass et al., ; devlin et al., ; mccafferty et al., ; scott and smith, ; breitling et al., ; kang et al., ) . this technology allowed, for the first time, the ability to seamlessly connect genetic information with protein function for a large number of protein variants simultaneously, and has been widely and productively exploited in studies of proteinprotein interactions. many excellent reviews are available on phage-display libraries and their applications (kehoe and kay, ; bratkovic, ; pande et al., ) . however, the phage also has a number of unique structural and biological properties that make it highly useful in areas of research that have received far less attention. thus, the purpose of this review is to highlight recent and current work using filamentous phage in novel and nontraditional applications. specifically, we refer to projects that rely on the filamentous phage as a key element, but whose primary purpose is not the generation or screening of phagedisplayed libraries to obtain binding polypeptide ligands. these tend to fall into four major categories of use: (i) filamentous phage as a vaccine carrier; (ii) engineered filamentous phage as a therapeutic biologic agent in infectious and chronic diseases; (iii) filamentous phage as a scaffold for bioconjugation and surface chemistry; and (iv) filamentous phage as an engine for evolving variants of displayed proteins with novel functions. a final section is dedicated to recent developments in filamentous phage ecology and phage-host interactions. common themes shared amongst all these applications include the unique biological, immunological, and physicochemical properties of the phage, its ability to display a variety of biomolecules in modular fashion, and its relative simplicity and ease of manipulation. nearly all applications of the filamentous phage depend on its ability to display polypeptides on the virion's surface as fusions to phage coat proteins ( table ) . the display mode determines the maximum tolerated size of the fused polypeptide, its copy number on the phage, and potentially, the structure of the displayed polypeptide. display may be achieved by fusing dna encoding a polypeptide of interest directly to the gene encoding a coat protein within the phage genome (type display on pviii, type display on piii, etc.), resulting in fully recombinant phage. much more commonly, however, only one copy of the coat protein is modified in the presence of a second, wild-type copy (e.g., type display if both recombinant and wild-type pviii genes are on the phage genome, type + display if the parmley and smith ( ), mcconnell et al. ( ) , rondot et al. ( ) hybrid (type and + systems) type + system < smith and scott ( ) , smith and petrenko ( ) pvi hybrid (type + system) yes < > kda hufton et al. ( ) pvii fully recombinant (type system) no ∼ > kda kwasnikowski et al. ( ) hybrid (type + system) yes < gao et al. ( ) pviii fully recombinant (landscape phage; type system) no ∼ - residues kishchenko et al. ( ) , petrenko et al. ( ) hybrid (type and + systems) type + system ∼ - > kda scott and smith ( ) , greenwood et al. ( ) , smith and fernandez ( ) pix fully recombinant (type + * system) yes ∼ > kda gao et al. ( ) hybrid (type + system) no < gao et al. ( ) , shi et al. ( ) , tornetta et al. ( ) asterisks indicate non-functional copies of the coat protein are present in the genome of the helper phage used to rescue a phagemid whose coat protein has been fused to a recombinant polypeptide. the copy number depends on polypeptide size; typically < copy per phage particle but for pviii peptide display can be up to ∼ % of pviii molecules in hybrid virions. the total number of pviii molecules depends on the phage genome size; one pviii molecule is added for every . nucleotides in the viral genome. recombinant gene is on a plasmid with a phage origin of replication) resulting in a hybrid virion bearing two different types of a given coat protein. multivalent display on some coat proteins can also be enforced using helper phage bearing nonfunctional copies of the relevant coat protein gene (e.g., type * + display). by far the most commonly used coat proteins for display are the major coat protein, pviii, and the minor coat protein, piii, with the major advantage of the former being higher copy number display (up to ∼ % of recombinant pviii molecules in a hybrid virion, at least for short peptide fusions), and of the latter being the ability to display some folded proteins at an appreciable copy number ( - per phage particle). while pviii display of folded proteins on hybrid phage is possible, it typically results in a copy number of much less than per virion (sidhu et al., ) . for the purposes of this review, we use the term "phage display" to refer to a recombinant filamentous phage displaying a single polypeptide sequence on its surface (or more rarely, bispecific display achieved via fusion of polypeptides to two different capsid proteins), and the term "phage-displayed library" to refer to a diverse pool of recombinant filamentous phage displaying an array of polypeptide variants (e.g., antibody fragments; peptides). such libraries are typically screened by iterative cycles of panning against an immobilized protein of interest (e.g., antigen for phage-displayed antibody libraries; antibody for phage-displayed peptide libraries) followed by amplification of the bound phage in e. coli cells. early work with anti-phage antisera generated for species classification purposes demonstrated that the filamentous phage virion is highly immunogenic in the absence of adjuvants (meynell and lawn, ) and that only the major coat protein, pviii, and the minor coat protein, piii, are targeted by antibodies (pratt et al., ; woolford et al., ) . thus, the idea of using the phage as carrier to elicit antibodies against poorly immunogenic haptens or polypeptide was a natural extension of the ability to display recombinant exogenous sequences on its surface, which was first demonstrated by de la cruz et al. ( ) . the phage particle's low cost of production, high stability and potential for high valency display of foreign antigen (via pviii display) also made it attractive as a vaccine carrier, especially during the early stages of development of recombinant protein technology. building upon existing peptide-carrier technology, the first filamentous phage-based vaccine immunogens displayed short amino acid sequences derived directly from proteins of interest as recombinant fusions to pviii or piii (de la cruz et al., ) . as library technology was developed and refined, phage-based antigens displaying peptide ligands of monoclonal antibodies (selected from random peptide libraries using the antibody, thus simulating with varying degrees of success the antibody's folded epitope on its cognate antigen; geysen et al., ; knittelfelder et al., ) were also generated for immunization purposes, with the goal of eliciting anti-peptide antibodies that also recognize the native protein. some of the pioneering work in this area used peptides derived from infectious disease antigens (or peptide ligands of antibodies against these antigens; table ) , including malaria and human immunodeficiency virus type (hiv- ). when displayed on phage, peptides encoding the repeat regions of the malarial circumsporozoite protein and merozoite surface protein were immunogenic in mice and rabbits (de la cruz et al., ; greenwood et al., ; willis et al., ; demangel et al., ) , and antibodies raised against the latter cross-reacted with the full-length protein. various peptide determinants (or mimics thereof) of hiv- gp , gp , gag, and reverse transcriptase were immunogenic when displayed on or conjugated to phage coat proteins (minenkova et al., ; di marzo veronese et al., ; de berardinis et al., ; scala et al., ; chen et al., ; van houten et al., van houten et al., , , and in some cases elicited antibodies that were able to weakly neutralize lab-adapted viruses (di marzo veronese et al., ; scala et al., ) . the list of animal and human infections for which phage-displayed peptide immunogens have been developed as vaccine leads continues to expand and includes bacterial, fungal, viral, and parasitic pathogens ( table ) . while in some cases the results of these studies have been promising, antibody epitope-based peptide vaccines are no longer an area of active research for several reasons: (i) in many cases, peptides incompletely or inadequately mimic epitopes on folded proteins (irving et al., ; see below); (ii) antibodies against a single epitope may be of limited utility, especially for highly variable pathogens (van regenmortel, ); and (iii) for pathogens for which protective immune responses are generated efficiently during natural infection, peptide vaccines offer few advantages over recombinant subunit and live vector vaccines, which have become easier to produce over time. more recently, peptide-displaying phage have been used in attempts to generate therapeutic antibody responses for chronic diseases, cancer, immunotherapy, and immunocontraception. immunization with phage displaying alzheimer's disease β-amyloid fibril peptides elicited anti-aggregating antibodies in mice and guinea pigs (frenkel et al., (frenkel et al., , esposito et al., ; tanaka et al., ) , possibly reduced amyloid plaque formation in mice (frenkel et al., ; solomon, ; esposito et al., ) , and may have helped maintain cognitive abilities in a transgenic mouse model of alzheimer's disease (lavie et al., ) ; however, it remains unclear how such antibodies are proposed to cross the blood-brain barrier. yip et al. ( ) found that antibodies raised in mice against an erbb /her peptide could inhibit breast-cancer cell proliferation. phage displaying peptide ligands of an anti-ige antibody elicited antibodies that bound purified ige molecules (rudolf et al., ) , which may be useful in allergy immunotherapy. several strategies for phage-based contraceptive vaccines have been proposed for control of animal populations. for example, immunization with phage displaying follicle-stimulating hormone peptides on pviii elicited antibodies that impaired the fertility of mice and ewes (abdennebi et al., ) . phage displaying or chemically rubinchik and chow ( ) conjugated to sperm antigen peptides or peptide mimics (samoylova et al., a,b) and gonadotropin-releasing hormone (samoylov et al., ) are also in development. for the most part, peptides displayed on phage elicit antibodies in experimental animals ( table ) , although this depends on characteristics of the peptide and the method of its display: piii fusions tend toward lower immunogenicity than pviii fusions (greenwood et al., ) possibly due to copy number differences (piii: - copies vs. pviii: estimated at several hundred copies; malik et al., ) . in fact, the phage is at least as immunogenic as traditional carrier proteins such as bovine serum albumin (bsa) and keyhole limpet hemocyanin (klh; melzer et al., ; su et al., ) , and has comparatively few endogenous b-cell epitopes to divert the antibody response from its intended target (henry et al., ) . excepting small epitopes that can be accurately represented by a contiguous short amino acid sequence, however, it has been extremely difficult to elicit antibody responses that cross-react with native protein epitopes using peptides. the overall picture is considerably bleaker than that painted by table , since in several studies either: (i) peptide ligands selected from phage-displayed libraries were classified by the authors as mimics of discontinuous epitopes if they bore no obvious sequence homology to the native protein, which is weak evidence of non-linearity, or (ii) the evidence for cross-reactivity of antibodies elicited by immunization with phage-displayed peptides with native protein was uncompelling. irving et al. ( ) describe at least one reason for this lack of success: it seems that peptide antigens elicit a set of topologically restricted antibodies that are largely unable to recognize discontinuous or complex epitopes on larger biomolecules. while the peptide may mimic the chemistry of a given epitope on a folded protein (allowing it to crossreact with a targeted antibody), being a smaller molecule, it cannot mimic the topology of that antibody's full epitope. despite this, the filamentous phage remains highly useful as a carrier for peptides with relatively simple secondary structures, which may be stablilized via anchoring to the coat proteins (henry et al., ) . this may be especially true of peptides with poor inherent immunogenicity, which may be increased by high-valency display and phage-associated adjuvanticity (see immunological mechanisms of vaccination with filamentous phage below). the filamentous phage has been used to a lesser extent as a carrier for t-cell peptide epitopes, primarily as fusion proteins with pviii ( table ) . early work, showing that immunization with phage elicited t-cell help (kölsch et al., ; willis et al., ) , was confirmed by several subsequent studies (de berardinis et al., ; ulivieri et al., ) . from the perspective of vaccination against infectious disease, de berardinis et al. ( ) showed that a cytotoxic t-cell (ctl) epitope from hiv- reverse transcriptase could elicit antigen-specific ctls in vitro and in vivo without addition of exogenous helper t-cell epitopes, presumably since these are already present in the phage coat proteins (mascolo et al., ) . similarly, efficient priming of ctls was observed against phage-displayed t-cell epitopes from hepatitis b virus (wan et al., ) and candida albicans (yang et al., a; wang et al., wang et al., , d , which, together with other types of immune responses, protected mice against systemic candidiasis. vaccination with a combination of phagedisplayed peptides elicited antigen-specific ctls that proved effective in reducing porcine cysticercosis in a randomized controlled trial (manoutcharian et al., ; morales et al., ) . while the correlates of vaccine-induced immune protection for infectious diseases, where they are known, are almost exclusively serum or mucosal antibodies (plotkin, ) , in certain vaccine applications, the filamentous phage has been used as a carrier for larger molecules that would be immunogenic even in isolation. initially, the major advantages to phage display of such antigens were speed, ease of purification and low cost of production (gram et al., ) . e. coli f a-g adhesin (van gerven et al., ) , hepatitis b core antigen (bahadir et al., ) , and hepatitis b surface antigen (balcioglu et al., ) all elicited antibody responses when displayed on piii, although none of these studies compared the immunogenicity of the phage-displayed proteins with that of the purified protein alone. phage displaying schistosoma mansoni glutathione s-transferase on piii elicited an antibody response that was both higher in titer and of different isotypes compared to immunization with the protein alone (rao et al., ) . two studies of antiidiotypic vaccines have used the phage as a carrier for antibody fragments bearing immunogenic idiotypes. immunization with phage displaying the e idiotype scfv (mimicking a vibrio anguillarum surface epitope) elicited antibodies that protected flounder fish from vibrio anguillarum challenge (xia et al., ) . a chemically linked phage-bcl tumor-specific idiotype vaccine was weakly immunogenic in mice but extended survival time in a b-cell lymphoma model (roehnisch et al., ) , and was welltolerated and immunogenic in patients with multiple myeloma (roehnisch et al., ) . one study of dna vaccination with an anti-laminarin scfv found that dna encoding a piii-scfv fusion protein elicited stronger humoral and cell-mediated immune responses than dna encoding the scfv alone (cuesta et al., ) , suggesting that under some circumstances, endogenous phage t-cell epitopes can enhance the immunogenicity of associated proteins. taken together, the results of these studies show that as a particulate virus-like particle, the filamentous phage likely triggers different types of immune responses than recombinant protein antigens, and provide additional t-cell help to displayed or conjugated proteins. however, the low copy number of piii-displayed proteins, as well as potentially unwanted phage-associated adjuvanticity, can make display of recombinant proteins by phage a suboptimal vaccine choice. although our understanding of the immune response against the filamentous phage pales in comparison to classical model antigens such as ovalbumin, recent work has begun to shed light on the immune mechanisms activated in response to phage vaccination (figure ) . the phage particle is immunogenic without adjuvant in all species tested to date, including mice (willis et al., ) , rats (dente et al., ) , rabbits (de la cruz et al., ) , guinea pigs (frenkel et al., ; kim et al., ) , fish (coull et al., ; xia et al., ) , non-human primates (chen et al., ) , and humans (roehnisch et al., ) . various routes of immunization have been employed, including oral administration (delmastro et al., ) as well as subcutaneous (grabowska et al., ) , intraperitoneal (van houten et al., ) , intramuscular (samoylova et al., a) , intravenous (vaks and benhar, ) , and intradermal injection (roehnisch et al., ) ; no published study has directly compared the effect of administration route on filamentous phage immunogenicity. antibodies are generated against only three major sites on the virion: (i) the surface-exposed n-terminal ∼ residues of the pviii monomer lattice (terry et al., ; kneissel et al., ) ; (ii) the n-terminal n and n domains of piii (van houten et al., ) ; and (iii) bacterial lipopolysaccharide (lps) embedded in the phage coat (henry et al., ) . in mice, serum antibody titers against the phage typically reach : - : after - immunizations, and are maintained for at least year postimmunization (frenkel et al., ) . primary antibody responses against the phage appear to be composed of a mixture of igm and igg b isotypes in c bl/ mice, while secondary antibody responses are composed primarily of igg and igg b isotypes, with a lesser contribution of igg c and igg isotypes (hashiguchi et al., ) . deletion of the surface-exposed n and n domains of piii produces a truncated form of this protein that does not elicit antibodies, but also results in a non-infective phage particle with lower overall immunogenicity (van houten et al., ) . figure | types of immune responses elicited in response to immunization with filamentous bacteriophage. as a virus-like particle, the filamentous phage engages multiple arms of the immune system, beginning with cellular effectors of innate immunity (macrophages, neutrophils, and possibly natural killer cells), which are recruited to tumor sites by phage displaying tumor-targeting moieties. the phage likely activates t-cell independent antibody responses, either via phage-associated tlr ligands or cross-linking by the pviii lattice. after processing by antigen-presenting cells, phage-derived peptides are presented on mhc class ii and cross-presented on mhc class i, resulting in activation of short-lived ctls and an array of helper t-cell types, which help prime memory ctl and high-affinity b-cell responses. frontiers in microbiology | www.frontiersin.org although serum anti-phage antibody titers appear to be at least partially t-cell dependent (kölsch et al., ; willis et al., ; de berardinis et al., ; van houten et al., ) , many circulating pviii-specific b cells in the blood are devoid of somatic mutation even after repeated biweekly immunizations, suggesting that under these conditions, the phage activates t-cell-independent b-cell responses in addition to highaffinity t-cell-dependent responses (murira, ) . filamentous phage particles can be processed by antigen-presenting cells and presented on mhc class ii molecules (gaubin et al., ; ulivieri et al., ) and can activate t h , t h , and t h helper t cells (yang et al., a; wang et al., d) . anti-phage t h responses were enhanced through display of ctla- peptides fused to piii (kajihara et al., ) . phage proteins can also be cross-presented on mhc class i molecules (wan et al., ) and can prime two waves of ctl responses, consisting first of short-lived ctls and later of long-lived memory ctls that require cd + t-cell help (del pozzo et al., ) . the latter ctls mediate a delayed-type hypersensitivity reaction (fang et al., ; del pozzo et al., ) . the phage particle is self-adjuvanting through multiple mechanisms. host cell wall-derived lps enhances the virion's immunogenicity, and its removal by polymyxin b chromatography reduces antibody titers against phage coat proteins (grabowska et al., ) . the phage's singlestranded dna genome contains cpg motifs and may also have an adjuvant effect. the antibody response against the phage is entirely dependent on myd signaling and is modulated by stimulation of several toll-like receptors (hashiguchi et al., ) , indicating that innate immunity plays an important but largely uncharacterized role in the activation of anti-phage adaptive immune responses. biodistribution studies of the phage after intravenous injection show that it is cleared from the blood within hours through the reticuloendothelial system (molenaar et al., ) , particularly of the liver and spleen, where it is retained for days (zou et al., ) , potentially activating marginal-zone b-cell responses. thus, the filamentous phage is not only a highly immunogenic carrier, but by virtue of activating a range of innate and adaptive immune responses, serves as an excellent model virus-like particle antigen. long before the identification of filamentous phage, other types of bacteriophage were already being used for antibacterial therapy in the former soviet union and eastern europe (reviewed in sulakvelidze et al., ) . the filamentous phage, with its nonlytic life cycle, has less obvious clinical uses, despite the fact that the host specificity of inovirus and plectrovirus includes many pathogens of medical importance, including salmonella, e. coli, shigella, pseudomonas, clostridium, and mycoplasma species. in an effort to enhance their bactericidal activity, genetically modified filamentous phage have been used as a "trojan horse" to introduce various antibacterial agents into cells. m and pf phage engineered to express either bglii restriction endonuclease (hagens and blasi, ; hagens et al., ) , lambda phage s holin (hagens and blasi, ) or a lethal catabolite gene activator protein (moradpour et al., ) effectively killed e. coli and pseudomonas aeruginosa cells, respectively, with no concomitant release of lps (hagens and blasi, ; hagens et al., ) . unfortunately, the rapid emergence of resistant bacteria with modified f pili represents a major and possibly insurmountable obstacle to this approach. however, there are some indications that filamentous phage can exert useful but more subtle effects upon their bacterial hosts that may not result in the development of resistance to infection. several studies have reported increased antibiotic sensitivity in bacterial populations simultaneously infected with either wild type filamentous phage (hagens et al., ) or phage engineered to repress the cellular sos response (lu and collins, ) . filamentous phage f infection inhibited early stage, but not mature, biofilm formation in e. coli (may et al., ) . thus, unmodified filamentous phage may be of future interest as elements of combination therapeutics against certain drug-resistant infections. more advanced therapeutic applications of the filamentous phage emerge when it is modified to express a targeting moiety specific for pathogenic cells and/or proteins for the treatment of infectious diseases, cancer and autoimmunity (figure ) . the first work in this area showed as proof-of-concept that phage encoding a gfp expression cassette and displaying a her specific scfv on all copies of piii were internalized into breast tumor cells, resulting in gfp expression (poul and marks, ) . m or fd phage displaying either a targeting peptide or antibody fragment and tethered to chloramphenicol by a labile crosslinker were more potent inhibitors of staphylococcus aureus growth than high-concentration free chloramphenicol (yacoby et al., ; vaks and benhar, ) . m phage loaded with doxorubicin and displaying a targeting peptide on piii specifically killed prostate cancer cells in vitro (ghosh et al., a) . tumorspecific peptide:pviii fusion proteins selected from "landscape" phage (romanov et al., ; abbineni et al., ; fagbohun et al., fagbohun et al., , lang et al., ; wang et al., a) were able to target and deliver sirna-, paclitaxel-, and doxorubicincontaining liposomes to tumor cells (jayanna et al., a; wang et al., a wang et al., ,b,c, b bedi et al., bedi et al., , bedi et al., , ; they were non-toxic and increased tumor remission rates in mouse models (jayanna et al., b; wang et al., b,c) . using the b -ova tumor model, eriksson et al. ( ) showed that phage displaying peptides and/or fabs specific for tumor antigens delayed tumor growth and improved survival, owing in large part to activation of tumor-associated macrophages and recruitment of neutrophils to the tumor site (eriksson et al., ) . phage displaying an scfv against β-amyloid fibrils showed promise as a diagnostic (frenkel and solomon, ) and therapeutic (solomon, ) reagent for alzheimer's disease and parkinson's disease due to the unanticipated ability of the phage to penetrate into brain tissue (ksendzovsky et al., ) . similarly, phage displaying an immunodominant peptide epitope derived from myelin oligodendrocyte glycoprotein depleted pathogenic demyelinating antibodies in brain tissue in the murine experimental autoimmune encephalomyelitis model of multiple sclerosis (rakover et al., ) . the advantages of the filamentous phage in this context over traditional antibody-drug or protein-peptide conjugates are (i) its ability to carry very high amounts of drug or peptide, and (ii) its ability to access anatomical compartments that cannot generally be reached by systemic administration of a protein. unlike most therapeutic biologics, the filamentous phage's production in bacteria complicates its use in humans in several ways. first and foremost, crude preparations of filamentous phage typically contain very high levels of contaminating lps, in the range of ∼ - endotoxin units (eu)/ml (boratynski et al., ; branston et al., ) , which have the potential to cause severe adverse reactions. lps is not completely removed by polyethylene glycol precipitation or cesium chloride density gradient centrifugation (smith and gingrich, ; branston et al., ) , but its levels can be reduced dramatically using additional purification steps such as size exclusion chromatography (boratynski et al., ; zakharova et al., ) , polymyxin b chromatography (grabowska et al., ) , and treatment with detergents such as triton x- or triton x- (roehnisch et al., ; branston et al., ) . these strategies routinely achieve endotoxin levels of < eu/ml as measured by the limulus amebocyte lysate (lal) assay, well below the fda limit for parenteral administration of eu/kg body weight/dose, although concerns remain regarding the presence of residual virion-associated lps which may be undetectable. a second and perhaps unavoidable consequence of the filamentous phage's bacterial production is inherent heterogeneity of particle size and the spectrum of host cellderived virion-associated and soluble contaminants, which may be cause for safety concerns and restrict its use to high-risk groups. many types of bacteriophage and engineered phage variants, including filamentous phage, have been proposed for prophylactic use ex vivo in food safety, either in the production pipeline (reviewed in dalmasso et al., ) or for detection of foodborne pathogens post-production (reviewed in schmelcher and loessner, ) . filamentous phage displaying a tetracysteine tag on piii were used to detect e. coli cells through staining with biarsenical dye . m phage functionalized with metallic silver were highly bactericidal against e. coli and staphylococcus epidermidis . biosensors based on surface plasmon resonance (nanduri et al., ) , piezoelectric transducers (olsen et al., ) , linear dichroism (pacheco-gomez et al., ) , and magnetoelastic sensor technology (lakshmanan et al., ; huang et al., ) were devised using filamentous phage displaying scfv or conjugated to whole igg against e. coli, listeria monocytogenes, salmonella typhimurium, and bacillus anthracis with limits of detection on the order of - bacterial cells/ml. proof of concept has been demonstrated for use of such phage-based biosensors to detect bacterial contamination of live produce (li et al., b) and eggs (chai et al., ) . the filamentous phage particle is enclosed by a rod-like protein capsid, ∼ nm long and nm wide, made up almost entirely of overlapping pviii monomers, each of which lies ∼ angstroms from its nearest neighbor and exposes two amine groups as well as at least three carboxyl groups (henry et al., ) . the regularity of the phage pviii lattice and its diversity of chemically addressable groups make it an ideal scaffold for bioconjugation (figure ) . the most commonly used approach is functionalization of amine groups with nhs esters (van houten et al., (van houten et al., , yacoby et al., ) , although this can result in unwanted acylation of piii and any displayed biomolecules. carboxyl groups and tyrosine residues can also be functionalized using carbodiimide coupling and diazonium coupling, respectively (li et al., a) . carrico et al. ( ) developed methods to specifically label pviii n-termini without modification of exposed lysine residues through a two-step transamination-oxime formation reaction. specific modification of phage coat proteins is even more easily accomplished using genetically modified phage displaying peptides (ng et al., ) or enzymes (chen et al., ; hess et al., ) , but this can be cumbersome and is less general in application. for more than a decade, interest in the filamentous phage as a building block for nanomaterials has been growing because of its unique physicochemical properties, with emerging applications in magnetics, optics, and electronics. it has long been known that above a certain concentration threshold, phage can form ordered crystalline suspensions (welsh et al., ) . lee et al. ( ) engineered m phage to display a zns-binding peptide on piii and showed that, in the presence of zns nanoparticles, they selfassemble into highly ordered film biomaterials that can be aligned using magnetic fields. taking advantage of the ability to display substrate-specific peptides at known locations on the phage filament hess et al., ) , this pioneering figure | chemically addressable groups of the filamentous bacteriophage major coat protein lattice. the filamentous phage virion is made up of ∼ , - , overlapping copies of the -residue major coat protein, pviii, arranged in a shingle-type lattice. each monomer has an array of chemically addressable groups available for bioorthogonal conjugation, including two primary amine groups (shown in red), three carboxyl groups (show in blue) and two hydroxyl groups (show in green). the n-terminal residues generally exposed to the immune system for antibody binding are in bold underline. figure adapted from structural data of marvin, , freely available in pdb and scope databases. work became the basis for construction of two-and threedimensional nanomaterials with more advanced architectures, including semiconducting nanowires (mao et al., (mao et al., , , nanoparticles , and nanocomposites (oh et al., ; chen et al., ) . using hybrid m phage displaying co o -and gold-binding peptides on pviii as a scaffold to assemble nanowires on polyelectrolyte multilayers, nam et al. ( ) produced a thin, flexible lithium ion battery, which could be stamped onto platinum microband current collectors (nam et al., ) . the electrochemical properties of such batteries were further improved through piii-display of single-walled carbon nanotube-binding peptides (lee et al., ) , offering an approach for sustainable production of nanostructured electrodes from poorly conductive starting materials. phagebased nanomaterials have found applications in cancer imaging (ghosh et al., b; yi et al., ) , photocatalytic water splitting (nam et al., a; neltner et al., ) , light harvesting (nam et al., b; chen et al., ) , photoresponsive technologies (murugesan et al., ) , neural electrodes (kim et al., ) , and piezoelectric energy generation (murugesan et al., ) . thus, the unique physicochemical properties of the phage, in combination with modular display of peptides and proteins with known binding specificity, have spawned wholly novel materials with diverse applications. it is worth noting that the unusual biophysical properties of the filamentous phage can also be exploited in the study of structures of other macromolecules. magnetic alignment of high-concentration filamentous phage in solution can partially order dna, rna, proteins, and other biomolecules for measurement of dipolar coupling interactions (hansen et al., (hansen et al., , dahlke ojennus et al., ) in nmr spectroscopy. because of their large population sizes, short generation times, small genome sizes and ease of manipulation, various filamentous and non-filamentous bacteriophages have been used as models of experimental evolution (reviewed in husimi, ; wichman and brown, ; kawecki et al., ; hall et al., ) . the filamentous phage has additional practical uses in protein engineering and directed protein evolution, due to its unique tolerance of genetic modifications that allow biomolecules to be displayed on the virion surface. first and foremost among these applications is in vitro affinity maturation of antibody fragments displayed on piii. libraries of variant fabs and single chain antibodies can be generated via random or sitedirected mutagenesis and selected on the basis of improved or altered binding, roughly mimicking the somatic evolution strategy of the immune system (marks et al., ; bradbury et al., ) . however, other in vitro display systems, such as yeast display, have important advantages over the filamentous phage for affinity maturation (although each display technology has complementary strengths; koide and koide, ) , and regardless of the display method, selection of "improved" variants can be slow and cumbersome. iterative methods have been developed to combine computationally designed mutations (lippow et al., ) and circumvent the screening of combinatorial libraries, but these have had limited success to date. recently, esvelt et al. ( ) developed a novel strategy for directed evolution of filamentous phage-displayed proteins, called phage-assisted continuous evolution (pace), which allows multiple rounds of evolution per day with little experimental intervention. the authors engineered m phage to encode an exogenous protein (the subject for directed evolution), whose functional activity triggers gene iii expression from an accessory plasmid; variants of the exogenous protein arise by random mutagenesis during phage replication, the rate of which can be increased by inducible expression of error-prone dna polymerases. by supplying limiting amounts of receptive e. coli cells to the engineered phage variants, esvelt et al. ( ) elegantly linked phage infectivity and production of offspring with the presence of a desired protein phenotype. carlson et al. ( ) later showed that pace selection stringency could be modulated by providing small amounts of piii independently of protein phenotype, and undesirable protein functions negatively selected by linking them to expression of a truncated piii variant that impairs infectivity in a dominant negative fashion. pace is currently limited to protein functions that can be linked in some way to the expression of a gene iii reporter, such as protein-protein interaction, recombination, dna or rna binding, and enzymatic catalysis (meyer and ellington, ) . this approach represents a promising avenue for both basic research in molecular evolution (dickinson et al., ) and synthetic biology, including antibody engineering. filamentous bacteriophage have been recovered from diverse environmental sources, including soil (murugaiyan et al., ) , coastal fresh water (xue et al., ) , alpine lakes (hofer and sommaruga, ) and deep sea bacteria (jian et al., ) , but not, perhaps surprisingly, the human gut (kim et al., ) . the environmental "phageome" in soil and water represent the largest source of replicating dna on the planet, and is estimated to contain upward of viral particles (ashelford et al., ; chibani-chennoufi et al., ; suttle, ) . the few studies attempting to investigate filamentous phage environmental ecology using classical environmental microbiology techniques (typically direct observation by electron microscopy) found that filamentous phage made up anywhere from to % of all viral particles (demuth et al., ; pina et al., ; hofer and sommaruga, ) . there was some evidence of seasonal fluctuation of filamentous phage populations in tandem with the relative abundance of free-living heterotrophic bacteria (hofer and sommaruga, ) . environmental metagenomics efforts are just beginning to unravel the composition of viral ecosystems. the existing data suggest that filamentous phage comprise minor constituents of viral communities in freshwater (roux et al., ) and reclaimed and potable water (rosario et al., ) but have much higher frequencies in wastewater and sewage (cantalupo et al., ; alhamlan et al., ) , with the caveat that biases inherent to the methodologies for ascertaining these data (purification of viral particles, sequencing biases) have not been not well validated. there are no data describing the population dynamics of filamentous phage and their host species in the natural environment. at the individual virus-bacterium level, it is clear that filamentous phage can modulate host phenotype, including the virulence of important human and crop pathogens. this can occur either through direct effects of phage replication on cell growth and physiology, or, more typically, by horizontal transfer of genetic material contained within episomes and/or chromosomally integrated prophage. temperate filamentous phage may also play a role in genome evolution (reviewed in canchaya et al., ) . perhaps the best-studied example of virulence modulation by filamentous phage is that of vibrio cholerae, whose full virulence requires lysogenic conversion by the cholera toxin-encoding ctxφ phage (waldor and mekalanos, ) . integration of ctxφ phage occurs at specific sites in the genome; these sequences are introduced through the combined action of another filamentous phage, fs φ, and a satellite filamentous phage, tlc-knφ (hassan et al., ) . thus, filamentous phage species interact and coevolve with each other in addition to their hosts. infection by filamentous phage has been implicated in the virulence of yersinia pestis (derbise et al., ) , neisseria meningitidis (bille et al., (bille et al., , , vibrio parahaemolyticus (iida et al., ) , e. coli :k :h (gonzalez et al., ) , xanthomonas campestris (kamiunten and wakimoto, ) , and p. aeruginosa (webb et al., ) , although in most of these cases, the specific mechanisms modulating virulence are unclear. phage infection can both enhance or repress virulence depending on the characteristics of the phage, the host bacterium, and the environmental milieu, as is the case for the bacterial wilt pathogen ralstonia solanacearum (yamada, ) . since infection results in downregulation of the pili used for viral entry, filamentous phage treatment has been proposed as a hypothetical means of inhibiting bacterial conjugation and horizontal gene transfer, so as to prevent the spread of antibiotic resistance genes (lin et al., ) . finally, the filamentous phage may also play a future role in the preservation of biodiversity of other organisms in at-risk ecosystems. engineered phage have been proposed for use in bioremediation, either displaying antibody fragments of desired specificity for filtration of toxins and environmental contaminants (petrenko and makowski, ) , or as biodegradable polymers displaying peptides selected for their ability to aggregate pollutants, such as oil sands tailings (curtis et al., (curtis et al., , . engineered phage displaying peptides that specifically bind inorganic materials have also been proposed for use in more advanced and less intrusive mineral separation technologies (curtis et al., ). the filamentous phage represents a highly versatile organism whose uses extend far beyond traditional phage display and affinity selection of antibodies and polypeptides of desired specificity. its high immunogenicity and ability to display a variety of surface antigens make the phage an excellent particulate vaccine carrier, although its bacterial production and preparation heterogeneity likely limits its applications in human vaccines at present, despite being apparently safe and well-tolerated in animals and people. unanticipated characteristics of the phage particle, such as crossing of the blood-brain barrier and formation of highly ordered liquid crystalline phases, have opened up entirely new avenues of research in therapeutics for chronic disease and the design of nanomaterials. our comparatively detailed understanding of the interactions of model filamentous phage with their bacterial hosts has allowed researchers to harness the phage life cycle to direct protein evolution in the lab. hopefully, deeper knowledge of phage-host interactions at an ecological level may produce novel strategies to control bacterial pathogenesis. while novel applications of the filamentous phage continue to be developed, the phage is likely to retain its position as a workhorse for therapeutic antibody discovery for many years to come, even with the advent of competing technologies. kh and js conceived and wrote the manuscript. ma-g read the manuscript and commented on the text. evolutionary selection of new breast cancer cell-targeting peptides and phages with the cell-targeting peptides fully displayed on the major coat and their effects on actin dynamics during cell internalization generating fsh antagonists and agonists through immunization against fsh receptor n-terminal decapeptides metagenomics-based analysis of viral communities in 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filamentous prophage in escherichia coli o :k :h and yersinia pestis biovar orientalis immunisation with phage displaying peptides representing single epitopes of the glycoprotein g can give rise to partial protective immunity to hsv- phage display as a rapid gene expression system: production of bioactive cytokinephage and generation of neutralizing monoclonal antibodies multiple display of foreign peptides on a filamentous bacteriophage. peptides from plasmodium falciparum circumsporozoite protein as antigens genetically modified filamentous phage as bactericidal agents: a pilot study augmentation of the antimicrobial efficacy of antibiotics by filamentous phage therapy of experimental pseudomonas infections with a nonreplicating genetically modified phage viral host-adaptation: insights from evolution experiments with phages pf filamentous phage as an alignment tool for generating local and global structural information in nucleic acids tunable alignment of macromolecules by 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immunogenic ligands selection and evolution of bacteriophages in cellstat filamentous phage associated with recent pandemic strains of vibrio parahaemolyticus exploring peptide mimics for the production of antibodies against discontinuous protein epitopes landscape phage ligands for pc prostate carcinoma cells landscape phage fusion protein-mediated targeting of nanomedicines enhances their prostate tumor cell association and cytotoxic efficiency dynamic modulation of dna replication and gene transcription in deep-sea filamentous phage sw in response to changes of host growth and temperature th -type immune response induced by a phage clone displaying a ctla -binding domain mimicmotif effect of infection with filamentous phage xf on the growth, ultrastructure and virulence of xanthomonas campestris pv. oryzae n linkage of recognition and replication functions by assembling combinatorial antibody fab libraries along phage surfaces experimental evolution filamentous phage display in the new millennium a new filamentous bacteriophage with sex-factor specificity diversity and abundance of single-stranded dna viruses in human feces genetically engineered bacteriophage delivers a tumor necrosis factor alpha antagonist coating on neural electrodes expression of a foot-and-mouth disease virus immunodominant epitope by a filamentous bacteriophage vector structure of a foreign peptide displayed on the surface of bacteriophage m epitope structures recognised by antibodies against the major coat protein (g p) of filamentous bacteriophage fd (inoviridae) mimotope vaccination-from allergy to cancer affinity maturation of single-domain antibodies by yeast surface display genetics of the immune response. i. the immune response to the phage fd in high and low responding inbred strains of mice convection-enhanced delivery of m bacteriophage to the brain multivalent display system on filamentous bacteriophage pvii minor coat protein phage immobilized magnetoelastic sensor for the detection of salmonella typhimurium specific probe selection from landscape phage display library and its application in enzyme-linked immunosorbent assay of free prostate-specific antigen efrh-phage immunization of alzheimer's disease animal model improves behavioral performance in morris water maze trials ordering of quantum dots using genetically engineered viruses fabricating genetically engineered high-power lithium-ion batteries using multiple virus genes chemical modification of m bacteriophage and its application in cancer cell imaging direct detection of salmonella typhimurium on fresh produce using phagebased magnetoelastic biosensors mimotopes selected with a neutralizing antibody against urease b from helicobacter pylori induce enzyme inhibitory antibodies in mice upon vaccination phage display for site-specific immunization and characterization of high-risk human papillomavirus specific e monoclonal antibodies inhibition of bacterial conjugation by phage m and its protein g p: quantitative analysis and model computational design of antibody-affinity improvement beyond in vivo maturation isolation of a bacteriophage specific for the f plus and hfr mating types of escherichia coli k- engineered bacteriophage targeting gene networks as adjuvants for antibiotic therapy role of capsid structure and membrane protein processing in determining the size and copy number of peptides displayed on the major coat protein of filamentous bacteriophage recombinant bacteriophage-based multiepitope vaccine against taenia solium pig cysticercosis viral assembly of oriented quantum dot nanowires virus-based toolkit for the directed synthesis of magnetic and semiconducting nanowires genetically engineered phage fibers and coatings for antibacterial applications molecular evolution of proteins on filamentous phage. mimicking the strategy of the immune system model-building studies of inovirus: genetic variations on a geometric theme filamentous phage structure, infection and assembly a fibrous dna phage (fd) and a spherical rna phage (fr) specific for male strains of e. coli. ii. physical characteristics phage display of a ctl epitope elicits a long-term in vivo cytotoxic response exposure of conjugative plasmid carrying escherichia coli biofilms to male-specific bacteriophages phage antibodies: filamentous phage displaying antibody variable domains constrained peptide libraries as a tool for finding mimotopes humoral immune response against proteophosphoglycan surface antigens of entamoeba histolytica elicited by immunization with synthetic mimotope peptides antigenicity and immunogenicity of phage library-selected peptide mimics of the major surface proteophosphoglycan antigens of entamoeba histolytica immunisation with phage-displayed variable region from meningococcal pora outer membrane protein induces bactericidal antibodies against neisseria meningitidis derivation of vaccines from mimotopes. immunologic properties of human hepatitis b virus surface 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photo-responsive nanowires formed by linking site-directed mutagenesis and chemical reaction virus-enabled synthesis and assembly of nanowires for lithium ion battery electrodes stamped microbattery electrodes based on self-assembled m viruses biologically templated photocatalytic nanostructures for sustained light-driven water oxidation virus-templated assembly of porphyrins into light-harvesting nanoantennae spr biosensor for the detection of l. monocytogenes using phage-displayed antibody production of hydrogen using nanocrystalline protein-templated catalysts on m phage quantitative synthesis of genetically encoded glycopeptide libraries displayed on m phage graphene sheets stabilized on genetically engineered m viral templates as conducting frameworks for hybrid energy-storage materials affinity-selected filamentous bacteriophage as a probe for acoustic wave biodetectors of salmonella typhimurium detection of pathogenic bacteria using a homogeneous immunoassay based on shear alignment of virus particles and linear dichroism phage display: concept, innovations, applications and future antibody-selectable filamentous fd phage vectors: affinity purification of target genes potential applications of phage display to bioremediation a library of organic landscapes on filamentous phage abundance, morphology and distribution of planktonic viruslike particles in two high-mountain lakes correlates of protection induced by vaccination targeted gene delivery to mammalian cells by filamentous bacteriophage conditional lethal mutants of the small filamentous coliphage m . ii. two genes for coat proteins selection of antigenic and immunogenic mimics of hepatitis c virus using sera from patients towards a solution for hepatitis c virus hypervariability: mimotopes of the hypervariable region can induce antibodies crossreacting with a large number of viral variants filamentous bacteriophages: biology and applications, " in els (the encyclopaedia of life sciences) filamentous bacteriophage: biology, phage display and nanotechnology applications antigen-specific therapy of eae via intranasal delivery of filamentous phage displaying a myelin immunodominant epitope expression of a -kilodalton glutathione s-transferase antigen of schistosoma mansoni on the surface of filamentous phages and evaluation of its vaccine potential structural requirements for the activity of the mirb ferrisiderophore transporter of aspergillus fumigatus chemically linked phage idiotype vaccination in the murine b cell lymphoma model phage idiotype vaccination: first phase i/ii clinical trial in patients with multiple myeloma phage display selection of peptides that affect prostate carcinoma cells attachment and invasion a helper phage to improve single-chain antibody presentation in phage display metagenomic analysis of viruses in reclaimed water assessing the diversity and specificity of two freshwater viral communities through metagenomics recombinant expression and neutralizing activity of an mhc class ii binding epitope of toxic shock syndrome toxin- epitope-specific antibody response to ige by mimotope immunization some physical-chemical and biological properties of the rod-shaped coliphage m generation and characterization of phage-gnrh chemical conjugates for potential use in cat and dog immunocontraception phage display allows identification of zona pellucida-binding peptides with species-specific properties: novel approach for development of contraceptive vaccines for wildlife infective and inactivated filamentous phage as carriers for immunogenic peptides vaccination with filamentous bacteriophages targeting dec- induces dc maturation and potent anti-tumor t-cell responses in the absence of adjuvants the use of filamentous bacteriophage fd to deliver hla-a -restricted peptides and to induce strong antitumor ctl responses selection of hiv-specific immunogenic epitopes by screening random peptide libraries with hiv- -positive sera application of bacteriophages for detection of foodborne pathogens searching for peptide ligands with an epitope library de novo selection of high-affinity antibodies from synthetic fab libraries displayed on phage as pix fusion proteins high copy display of large proteins on phage for functional selections filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface effect of dna copy number on genetic stability of phage-displayed peptides hydroxyapatite chromatography of phage-display virions phage display libraries of peptides and proteins displayed on filamentous phage generation of anti-β-amyloid antibodies via phage display technology towards alzheimer's disease vaccination filamentous bacteriophage as a novel therapeutic tool for alzheimer's disease treatment comparison of phage pviii and klh as vector in inducing the production of cytokines in c bl/ j mice bacteriophage therapy viruses in the sea a mimotope peptide of aβ fibril-specific antibodies with aβ fibrillation inhibitory activity induces anti-aβ conformer antibody response by a displayed form on an m phage in mice accessibility of peptides displayed on filamentous bacteriophage virions: susceptibility to proteinases antibody fab display and selection through fusion to the pix coat protein of filamentous phage an investigation on the nature of ultra-microscopic viruses antigenic properties of hcmv peptides displayed by filamentous bacteriophages vs. synthetic peptides in vivo characteristics of targeted drug-carrying filamentous bacteriophage nanomedicines presentation of the functional receptor-binding domain of the bacterial adhesin f a-g on bacteriophage m engineering filamentous phage carriers to improve focusing of antibody responses against peptides filamentous phage as an immunogenic carrier to elicit focused antibody responses against a synthetic peptide basic research in hiv vaccinology is hampered by reductionist thinking lysogenic conversion by a filamentous phage encoding cholera toxin induction of hepatitis b virus-specific cytotoxic t lymphocytes response in vivo by filamentous phage display vaccine crosspresentation of phage particle antigen in mhc class ii and endoplasmic reticulum marker-positive compartments bio-mimetic nanostructure self-assembled from au@ag heterogeneous nanorods and phage fusion proteins for targeted tumor optical detection and photothermal therapy enhanced tumor delivery and antitumor activity in vivo of liposomal doxorubicin modified with mcf- -specific phage fusion protein paclitaxel-loaded peg-pe-based micellar nanopreparations targeted with tumor specific landscape phage fusion protein enhance apoptosis and efficiently reduce tumors hybrid phage displaying slaqvkytsassi induces protection against candida albicans challenge in balb/c mice protective immune responses against systemic candidiasis mediated by phage-displayed specific epitope of candida albicans heat shock protein in c bl/ j mice enhanced binding and killing of target tumor cells by drug-loaded liposomes modified with tumor-specific phage fusion coat protein paclitaxel-loaded polymeric micelles modified with mcf- cell-specific phage protein: enhanced binding to target cancer cells and increased cytotoxicity cytoplasmic delivery of liposomes into mcf- breast cancer cells mediated by cell-specific phage fusion coat protein bacteriophage and phenotypic variation in pseudomonas aeruginosa biofilm development evidence for tilted smectic liquid crystalline packing of fd inovirus from x-ray fiber diffraction experimental evolution of viruses: microviridae as a model system immunological properties of foreign peptides in multiple display on a filamentous bacteriophage adsorption protein of bacteriophage fl: solubilization in deoxycholate and localization in the fl virion sensitive and selective bacterial detection using tetracysteine-tagged phages in conjunction with biarsenical dye phage display particles expressing tumor-specific antigens induce preventive and therapeutic anti-tumor immunity in murine p model development of a phage displayed disulfide-stabilized fv fragment vaccine against vibrio anguillarum high frequency of a novel filamentous phage, vcyφ, within an environmental vibrio cholerae population targeting antibacterial agents by using drug-carrying filamentous bacteriophages filamentous phages of ralstonia solanacearum: doubleedged swords for pathogenic bacteria prophylactic vaccination with phage-displayed epitope of c. albicans elicits protective immune responses against systemic candidiasis in c bl/ mice epitope mapping of mycoplasma hyopneumoniae using phage displayed peptide libraries and the immune responses of the selected phagotopes m phage-functionalized single-walled carbon nanotubes as nanoprobes for second near-infrared window fluorescence imaging of targeted tumors comparison of phage piii, pviii and gst as carrier proteins for peptide immunisation in balb/c mice spontaneous assembly of viruses on multilayered polymer surfaces characterization of murine coronavirus neutralization epitopes with phage-displayed peptides purification of filamentous bacteriophage for phage display using size-exclusion chromatography conformational mimicry of a chlamydial neutralization epitope on filamentous phage f , a rodshaped male-specific bacteriophage that contains dna biodistribution of filamentous phage peptide libraries in mice this work was supported by funding from the national research council of canada (kh, ma-g) and the canada research chair program (js). we thank jyothi kumaran and roger mackenzie for critical appraisal of the manuscript, and jasna rakonjac for inviting us to contribute it. this is national research council canada publication number . key: cord- -i qcanti authors: yang, jing; chen, hao; wang, zhenzhong; yu, xianglong; niu, xiaoyu; tang, yi; diao, youxiang title: development of a quantitative loop-mediated isothermal amplification assay for the rapid detection of novel goose parvovirus date: - - journal: front microbiol doi: . /fmicb. . sha: doc_id: cord_uid: i qcanti an infectious disease characterized with short bills and protruding tongues has attacked to meat ducks in china since march , which has caused ducks poor growth and enormous economic losses to duck industry of china. it was eventually proved to be caused by parvovirus after pathogen isolation and identification. as the genomic sequence analysis showed, this pathogen shared . – . % of nucleotide identity with goose parvovirus (gpv), and it was called duck-origin novel goose parvovirus (n-gpv). in this study, a quantitative loop-mediated isothermal amplification (qlamp) assay was developed for the rapid diagnosis of n-gpv. a set of four specific primers, two inner and two outer, were designed targeting at vp gene, which could be completed within min at °c in water bath or on a real-time pcr instrument for quantitative analysis. specificity test of lamp assay showed that there was no cross-reactivity between n-gpv and other duck pathogens, and the detection limit of qlamp assay was . × ( ) copies/μl. the repeatability of this method was confirmed by inter-assay and intra-assay tests with variability ranging from . to . %. the results have indicated that the qlamp assay was a simple, rapid, accurate, sensitive, and specific method for detecting n-gpv, especially on field detection. waterfowl parvovirus causes high morbidity and mortality in geese and muscovy ducks, and mortality rate ranges from to %. these diseases can lead to enteric symptoms, watery diarrhea, prostration, and growth retardation, thus resulting in serious economic losses to waterfowl industry (glávits et al., ) . in , a disease, which emerged in france and poland in the early s in mule and muscovy ducks was first reported in chinese mainland (chen et al., ) . it mainly caused short beak, protruding tongues, fragile tibia and pteroid, and growth retardation to commercial ducks. the morbidity rate ranges from to %, and even up to %, thereby resulting in enormous economic losses to duck industry of china. in order to identify the exact pathogen that caused this disease, pathogen isolation and identification and genomic sequence analysis were carried out. isolated pathogen was detected by polymerase chain reaction (pcr) assay, and the result showed that only gpv was positive; genomic sequence analysis showed that this new pathogen shared . - . % of nucleotide identity with goose parvovirus (gpv). these two studies above indicated that this new pathogen shared higher homology with other gpv strains. therefore, it was called duck-origin novel goose parvovirus (n-gpv) . in common with other waterfowl parvovirus, n-gpv was also a member of the parvoviridae family with a single-stranded dna genome, and has a small, nonenveloped, icosahedral capsid packaging a singlestranded dna genome of approximately , nucleotides. there are two open reading frames (orfs) encoding nonstructural proteins (ns), and capsid proteins (vp) (chen et al., ) . recently, with advances in virological diagnositic techniques, a novel nucleic acid amplification method-loop-mediated isothermal amplification (lamp) has attracted intense attention, which relies on auto-cycling strand displacement dna synthesis by the bst dna polymerase large fragment with high strand displacement activity and a set of specific primers that recognizes six distinct sequences in the target dna (notomi et al., ) . in addition, lamp has been considered as a time-saving, lowcost, highly specific and sensitive method (chotiwan et al., ) , which can be completed within min under condition of constant temperature, and it has been established to detect gpv, muscovy duck parvovirus (mdpv), porcine parvovirus (ppv), canine parvovirus (cpv), and others targeting at vp gene (cho et al., ; chen et al., ; ji et al., ; jinlong et al., ) . results of lamp can be judged by either turbidity, or end-products that can be visible to naked eyes with fluorescent reagents such as sybr green i (tomita et al., ) . lately, a novel eva green-based quantitative lamp assay has become popular (wang et al., ; lee et al., ) . compared with sybr green i-based quantitative lamp assay, eva green dye r is less inhibitory to pcr and less likely to cause nonspecific amplification (ihrig et al., ) . meanwhile, aerosol pollution of this method can be avoided due to tubes closed. after amplification, results can be analyzed by amplification curve, turbidity, or fluorescence of end-products under ultraviolet light, thus judging whether target dna is amplified (mori et al., ) . in this study, a detection method quantitative loopmediated isothermal amplification (qlamp) for amplifying vp gene of n-gpv was established. amplification curves and good linear relationship have been obtained. specificity of this method was determined by gpv and other duckorigin viruses, such as duck plague virus, duck tembusu virus, duck hepatitis virus, duck reovirus, muscovy duck parvovirus, and h n -aiv. in addition, sensitivity of qlamp was carried out with plasmid substances after tenfold series dilutions. these test results above demonstrated that this method has advantages with strong specificity, high sensitivity, easy operated, and low cost so that is valuable and suitable for clinical application in basic level production. n-gpv used in this study was isolated from morbid ducks liver in liaocheng city, shandong province, china (named sdlc strain). gpv (hn strain), duck plague virus (zb strain), duck tembusu virus (gl strain), duck hepatitis virus (lc strain), duck reovirus (hb strain), muscovy duck parvovirus (gx strain), and duck-origin h n -aiv (gt strain) were preserved by the research institute of poultry disease of shandong agricultural university. sixty clinical liver samples and cloacal swabs were collected from dead ducks and clinical healthy ducks respectively in shandong, jiangsu, and henan province of china in different large-scale duck farms. the liver samples were homogenized in dulbecco's modified eagle medium (dmem), frozen and thawed three times, then centrifuged at × g for min. the supernatant were filtered through a . µm filter, and the filtrate was inoculated into duck embryo fibroblasts (def) cells. the culture supernatant was harvested after days post-inoculated for three passages, and detected by pcr. dna was extracted by tianamp genomic dna kit (tiangen biotech, beijing, china). rna was extracted by minibest universal rna extraction kit (takara, dalian, china). vp gene of n-gpv, gpv, and mdpv were aligned by the megalign software, and phylogenetic tree was constructed with mega version . using the neighbor-joining method (figure ). the reference waterfowl parvovirus isolates are listed in table . a set of four primers and pcr primers were designed based on the vp gene of n-gpv using an online program (primerexplorer v , http://primerexplorer.jp/elamp . . /index. html) and primer . software respectively. primers including two inner (fip and bip), two outer (f and b ), and pcr primers are shown in table . outer primers were used to amplify target dna, and the amplified pcr products were purified with a gel extraction kit (takara, dalian, china), cloned into the pmd -t vector (takara, dalian, china) and transformed into e. coli dh α competent cells. clones were sequenced by bgi tech (bgi tech, shenzhen, china), and analyzed results. high fidelity clone was cultured and extracted plasmid with pureplasmid mini kit (cwbio, beijing, china). meanwhile, . × copies/µl- . × copies/µl substances were used for constructing standard curve. the qlamp assay was conducted in a µl reaction system, which containing µl fip and bip primers ( µm of each), µl f and b primers ( µm of each), . µl × isothemal amplification buffer, . µl mm mgso , . µl mm dntp mix, . µl × evagreen r dye, µl bst dna polymerase, µl target dna, . µl nuclease-free water, and performed using real time pcr system (applied biosystems, foster city, ca, united states). mixtures were incubated at • c in water bath for min, and terminated by heating at • c for min (followed by neb typical lamp protocol), or carried out on real time pcr system followed by procedure: cycles of • c s and • c s, fluorescence signals were collected at the end of • c step, and the cutoff point for tt (time threshold, a cycle indicates min) value was determined as by the previous reported method . data was analyzed by sds software program (version . ). the qlamp results were also analyzed by gel electrophoresis with % agarose gel, and observed under uv light. the pcr assay was conducted in µl reaction system, containing µl × ex taq mix, µl ddh o, µl forward primer, µl forward primer, µl reverse primer, and µl dna, and performed using pcr system (applied biosystems, foster city, ca, united states). pcr conditions were as follows: • c for min, cycles of denaturation ( • c for s), annealing ( • c for s), and extension ( • c for s), followed by a final extension at • c for min. ten-fold series dilutions ( - copies/µl) of standard substance were used as templates for qlamp amplification to confirm its sensitivity. quantitative loop-mediated isothermal amplification assay was carried out using different viruses including gpv, duck plague virus, duck tembusu virus, duck hepatitis virus, duck reovirus, muscovy duck parvovirus, and h n -aiv to validate specificity of this method. sensitivity test was performed to compare pcr and lamp assay, and plasmid with different dilutions was used as templates for pcr detection. f , b primers for pcr amplification were shown in table . pcr results were electrophoresed in % tae agarose gel and observed in the uv transilluminator. to confirm that the qlamp detection method has good reproducibility to detect n-gpv, tenfold series dilutions of plasmid substances, from . × copies/µl to . × copies/µl were used. for the intra-assay test, triplicate from each dilution were tested in the same run. for the inter-assay test, samples from each dilution were tested in three independent runs. the results were evaluated by coefficient of variation. sixty clinical liver samples and cloacal swabs from dead ducks and clinical healthy ducks respectively were collected to test reliability of qlamp, pcr assay, and virus isolation assay. to determine genetic relationship between sdlc and other gpv and mdpv strains, phylogenetic tree of vp genes was constructed. phylogenetic trees based on vp genes showed that the sdlc strains were in the same branch with european gpv and vaccine isolates, and shared . - . % identity with gpv isolates, while only shared . - . % with mdpv, which indicated that evolutionary relationship of sdlc strain is closer to gpv stains, and n-gpv is a novel variant of gpv. concentration of positive plasmid was . ng/µl, determined by ds- spectrophotometer (denovix, america), and the copy number was . × copies/µl. the standard curve was generated by tt values obtained from this study, and a good linear relationship was established between the log of the plasmid copy numbers (copies/µl) and the tt values (r = . ), with a regression line revealing an average intercept of . and an average slope of - . (figure ) . performed by ten-fold series dilutions ( . × - . × copies/µl) of standard substance, the qlamp method for n-gpv detection has the lowest limit of copies, and no amplification signals were observed in negative control (nc) (figure a) . in our strategy, the results of qlamp assay could not only be evaluated by naked eyes at the endpoint with evagreen r fluorescent dye in the uv light ( figure b ) and agarose gel electrophoresis (figure c) , it could also be quantitatively monitored using the real-time pcr system during the reaction. specificity test of qlamp method was carried out by seven duckorigin viruses and a goose-origin virus (mentioned in virus and samples). the results showed that our assay could only detect n-gpv and gpv, while no amplification signals were detected in other viruses ( figure a) . the specificity of qlamp assay could also be confirmed by green fluorescence in the uv light (figure b) , and typical ladder pattern seen in the agarose gel electrophoresis (figure c ). in addition, no positive results were obtained in negative control (nc), which indicates that this qlamp assay has a good specificity for n-gpv detection. figure | standard curve of quantitative loop-mediated isothermal amplification (qlamp) assay using tenfold series dilutions of plasmid substances in te buffer ( . × copies/µl- . × copies/µl). frontiers in microbiology | www.frontiersin.org in the intra-assay test, the coefficient of variation of the tt values varied from . to . %; in the inter-assay test, the coefficient of variation of the tt values varied from . to . % ( table ) . these results revealed that the qlamp method has a high reproducibility and excellent stability in detecting n-gpv. different dilution plasmids ( . × - . × copies/µl) were used as templates for lamp and pcr amplification. results showed that the detection limit of qlamp assay was copies, while pcr assay was copies, which indicated the sensitivity of lamp method was times higher than pcr method. loop-mediated isothermal amplification, pcr, and virus isolation assay were performed on clinical samples and cloacal swabs of ducks from different regions of china. the results were shown by statistical analysis in table . the results showed that the consistency of these three methods in every sample. the sensitivity of qlamp assay was the highest, and there was no positive amplification in negative control in qlamp assay, pcr, and virus isolation assay. currently, as a laboratory assay and conventional method, pcr is used most widely. however, not only does it require expensive equipments and much time, but also it has a high requirement for experiment conditions. meanwhile, although the taqmanbased real-time pcr assay has been established for the detection of n-gpv, it is time-consuming and costly; in addition, the detection limit ( . × copies/µl) is almost the same as qlamp assay ( . × copies/µl) (niu et al., ) , so the rapid, simple, reliable, and cost-efficient qlamp assay is definitely a good choice in detecting n-gpv. although traditional sybr green i-based lamp assay is a good method with high sensitivity and specificity, adding fluorescence dye after reaction would cause aerosol pollution, thus resulting in a false positive. however, sybr green i would inhibit pcr amplification if added before lamp reaction (eischeid, ) . so, the novel evagreen-based quantitative lamp assay has overcome these disadvantages and is more suitable for further application. this method could also be carried out in a water bath or on a real-time pcr instrument for quantitative analysis, and results can be easily observed by turbidity, fluorescence in the uv by naked eyes and agarose gel electrophoresis. this study was the first to report the evagreen-based lamp assay for detecting n-gpv. based on the data above, the detection limit of this method was as low as . × copies/µl, especially the intra and inter-assay variations, -just . - . % only. in addition, the specificity of this method was times higher than traditional pcr assay, and was almost the same as qpcr assay. novel goose parvovirus is newly discovered in recent years, which mainly causes ducks growth retardation and high infection rate to meat ducks, while gpv mainly causes serious death to goslings. according to the view of homology, the n-gpv strain is closer to european gpv and vaccine isolates, while separated from asian isolates. therefore, it is clear that n-gpv is a member of gpv-related parvovirus. n-gpv and gpv are almost indistinguishable using this method due to higher nucleotide homology, but it's also a great method for the detection of n-gpv, because these two pathogens are from different poultry. the qlamp assay has proved to be a highly sensitive, specific, and reliable diagnostic method that can detect n-gpv in a simple, rapid, and cost-efficient way. it could be undoubtedly used as a point-of-care strategy for clinical laboratories to prevent diseases from breaking out (notomi et al., ) . conceived and designed the experiments: yd, yt, and jy. performed the experiments: jy, hc, and zw. analyzed the data: jy, xy, and xn. contributed reagents/materials/analysis tools: jy and hc. wrote the paper: jy. isolation and genomic characterization of a duck-origin gpv-related parvovirus from cherry valley ducklings in china evidence for vertical transmission of novel duck-origin goose parvovirus-related parvovirus rapid detection of porcine parvovirus dna by sensitive loop-mediated isothermal amplification detection of canine parvovirus in fecal samples using loop-mediated isothermal amplification rapid and specific detection of asian-and africanlineage zika viruses syto dyes and evagreen outperform sybr green in real-time pcr comparative pathological studies on domestic geese (anser anser domestica) and muscovy ducks (cairina moschata) experimentally infected with parvovirus strains of goose and muscovy duck origin application of the dna-specific dye evagreen for the routine quantification of dna in microplates molecular detection of muscovy duck parvovirus by loop-mediated isothermal amplification assay a simple and rapid method for detection of goose parvovirus in the field by loop-mediated isothermal amplification one-pot reverse transcriptional loop-mediated isothermal amplification (rt-lamp) for detecting mers-cov real-time turbidimetry of lamp reaction for quantifying template dna development of a taqman-based real-time pcr assay for the detection of novel gpv loop-mediated isothermal amplification (lamp): principle, features, and future prospects loop-mediated isothermal amplification of dna an immunoassay-based reversetranscription loop-mediated isothermal amplification assay for the rapid detection of avian influenza h n virus viremia loop-mediated isothermal amplification (lamp) of gene sequences and simple visual detection of products dna quantification using evagreen and a real-time pcr instrument the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © yang, chen, wang, yu, niu, tang and diao. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- -e ciil authors: feng, min; dai, manman; xie, tingting; li, zhenhui; shi, meiqing; zhang, xiquan title: innate immune responses in alv-j infected chicks and chickens with hemangioma in vivo date: - - journal: front microbiol doi: . /fmicb. . sha: doc_id: cord_uid: e ciil avian leukosis virus subgroup j (alv-j) infection can cause tumors and immunosuppression. since the precise mechanism of the innate immune response induced by alv-j is unknown, we investigated the antiviral innate immune responses induced by alv-j in chicks and chickens that had developed tumors. spleen levels of interleukin- (il- ), il- , il- β, and interferon-β (ifn-β) were not significantly different between the infected chick groups and the control groups from day post hatch to days post hatch. however, il- , il- β, and ifn-β protein levels in the three clinical samples with hemangiomas were dramatically increased compared to the healthy samples. in addition, the anti-inflammatory cytokine il- increased sharply in two of three clinical samples. we also found a more than -fold up-regulation of isg - mrna at day post infection (d.p.i.) and a twofold up-regulation of zc hav mrna at d.p.i. however, there were no statistical differences in isg - and zc hav mrna expression levels in the tumorigenesis phase. alv-j infection induced a significant increase of toll-like receptor (tlr- ) at d.p.i. and dramatically increased the mrna levels of melanoma differentiation-associated gene (mda ) in the tumorigenesis phase. moreover, the protein levels of interferon regulatory factor (irf- ) and signal transducer and activator of transcription (stat ) were decreased in chickens with tumors. these results suggest that alv-j was primarily recognized by chicken tlr and mda at early and late in vivo infection stages, respectively. alv-j strain scau-hn did not induce any significant antiviral innate immune response in week old chicks. however, interferon-stimulated genes were not induced normally during the late phase of alv-j infection due to a reduction of irf and stat expression. avian leukosis virus (alv) is a member of the α-retrovirus genus of retroviridae, causing neoplastic disease, immunosuppression, and reproduction problems in the poultry industry worldwide. alv strains are divided into subgroups from a to j based on their viral envelope composition, host range, and cross-neutralization patterns (payne et al., ; barnard et al., ) avian leukosis virus subgroup (alv-j) isolates have been obtained from broiler breeders and laying chickens in most parts of china (cui et al., ) and these infections result in serious economic losses in commercial layer flocks and local chicken breeds (sun and cui, ; gao et al., ) . avian leukosis virus subgroup j can induce the formation of different types of tumors such as haemangiomas and myelocytomas and immunosuppression due to alv-j infection also increases susceptibility to other avian diseases (abolnik and wandrag, ). in the current study we analyzed the transcriptional level of selected immune-response genes we had previously identified from whole-transcriptome profiling of alv-j-induced tumors in chicken spleen samples (li et al., ) . these genes included those with known anti-viral properties such as the single copy mx gene, isg - as well as the toll-like receptor tlr- and other cytokine-related genes. we investigated the effects of alv-j infection on the in vivo mrna and protein expression of related immune-response genes and cytokines during the early and late phases of infection. our findings extend our current understanding of the host-response mechanism to exogenous alv infection. all animal research projects were sanctioned by the south china agriculture university institutional animal care and use committee. all animal procedures were performed according to the regulations and guidelines established by this committee and international standards for animal welfare. the alv-j strain scau-hn was kindly provided by dr. weisheng cao, south china agricultural university. infection with this virus leads to haemangioma development. thirty-six -day-old specific-pathogen-free (spf) white leghorn chickens were hatched from eggs (guangdong da hua nong animal health products co., ltd) and housed in isolator cages. three -day-old sick yellow chickens with neoplasms (designated sc , sc , and sc ) and three -day-old normal yellow chickens (designated nc , nc , nc ) were collected from the same flock from a farm in guangdong province, china. a total of -day-old spf chickens were randomly assigned to two groups: an scau-hn -infected group and control group, each with eighteen chickens per group. chickens were inoculated intraperitoneally at a dose of . ml ( . tcid / . ml) of strain scau-hn . the control group was injected with dmem media alone. spleens were aseptically collected from three infected chickens and three control chickens at , , , , , and d.p.i., respectively. plasma samples were aseptically collected and centrifuged at • c at rpm for min to isolate leukocytes. these were stored as viral stocks at − • c. spleens and livers from clinical samples were either stored at − • c or fixed in % buffered formalin until use. detection of spf chicken infected with alv-j rna was extracted from the spleens of spf chickens (fastagen, shanghai, china) and reverse transcribed into cdna (thermo-fisher scientific, usa) using commercial kits. rt-pcr analyses was employed to detect alv-j via specific primers (smith et al., ) . clinical sample detection, histopathology, and immunohistochemical staining dna was extracted from the spleens of the clinic samples using a commercial kit (omega, usa). specific primers were employed to differentiate between alv-j, alv-a/b, and other suspected viruses including marek's disease virus (mdv) and reticuloendotheliosis virus (rev) . according to a previously described method (li et al., ) , chicken plasma was used for virus isolation by inoculation into df cells. df- cells are only susceptible to exogenous alv virions (maas et al., ) . infected df cell culture supernatants from clinical samples were tested for alv group-specific antigen (p ) using the alv antigen test kit r (idexx, usa) according to the manufacturer's instructions. alv-j infection was further confirmed by immunofluorescence using standard techniques (venugopal et al., ) . infected cell images were collected using nis-elements br analysis software (nikon). liver tissue samples fixed in % buffered formalin were stained with hemotoxylin and eosin (he) and examined histopathologically (cheng et al., ) . immunohistochemical staining was used for further diagnoses with je monoclonal antibody (kindly provided by dr. kun qian, yangzhou university). binding of the je antibody was detected using anti-mouse-hrp (zhongshan goldenbridge, beijing, china). in our previous study, the transcriptome profiles of alv-j-induced tumors in spleen samples compared to healthy spleen samples from white recessive plymouth rock chickens were used to identify the genes related to alv-j invasion (li et al., ) . in this study, the related genes of innate immune transcriptional responses were analyzed according to the transcriptome profiles. expression of related genes of innate immunity were analyzed by quantitative real-time polymerase chain reaction (qrt-pcr). total rna was extracted from frozen spleens of spf chickens and clinical samples using the rnafast kit (fastagen), followed by cdna synthesis of mrna with the revertaid first strand cdna synthesis kit (thermo-fisher scientific) according to the manufacturer's instructions. qrt-pcr was performed on a biorad cfx real-time detection system using itaq tm universal sybr r green supermix kit reagents (biorad, ca, usa) according to the manufacturer's specifications. primers used for qrt-pcr were designed using the ncbi primer blast program and were based on published target sequences ( table ) . data analyses were performed using the − ct method (livak and schmittgen, ). spleen homogenates of spf and clinical chicken samples were used to detect cytokine protein levels. spleen tissues were rinsed in ice-cold pbs to remove excess blood thoroughly and weighed before homogenization. homogenized them in ml of pbs with a steel ball using tissuelyser ii (qiagen, germany). the resulting suspension was subjected to two freeze-thaw cycles to further break the cell membranes. the homogenates were centrifugated for min at × g and the supernatant was removed and was assayed immediately. enzyme-linked immunosorbent assay (elisa) kits for chicken interferon-β (ifn-β), interleukin- β (il- β), il- , and il- determination were obtained from wuhan uscn (cloud-clone corp. china). the elisa experiments were performed according to the manufacturer's specifications. tissue homogenates were prepared from spleens of chickens with tumors and normal chickens as above described. the lysates were collected and incubated on ice for min and then cleared by centrifugation at , g for min at • c. total protein content was determined with a bca protein assay kit (life technologies, usa). total protein ( µg) was resolved by % sds-page and transferred onto nitrocellulose membranes (whatman, maidstone, uk). membranes were blocked with % http://www.ncbi.nlm.nih.gov/tools/primer-blast/ w/v skim milk for h at • c, and then incubated overnight at • c with mouse anti-gapdh antibody (beyotime inst biotech, shanghai, china), rabbit anti-interferon regulatory factor (irf- ) and rabbit anti-signal transducer and activator of transcription (stat ) antibodies (lsbio, seattle, wa, usa). after three rinses with pbs tween (pbst) buffer, the membranes were incubated at • c for h with anti-rabbit-hrp or anti mouse-hrp (zhongshan goldenbridge, beijing, china) that had been diluted in pbst. membranes were washed three times with pbst and signals were detected using an ecl kit (zhongshan goldenbridge, beijing, china). statistical comparisons were made by graphpad prism (graphpad software inc., san diego, ca, usa) and statistical significance was represented by p values of > . , < . , . , or . . to determine whether the spf chickens were successfully infected, we measured gene expression levels of alv-j-specific genes from chickens infected with the scau-hn virus strain. rt-pcr tests of spleen samples from spf chickens at - d.p.i. using alv-j-specific primers were all positive ( figure a , panel ). alv-j could not be detected in the control animals (data not shown). to verify that the clinical tumors were induced by alv-j and no other oncogenic viruses, we used additional methods for pathogen detection. the presence of hemangiomas is a characteristic of alv-j infection. these tumors were evident on the skin of the joints and digits, in the ocular region and in the livers of birds from the same flock ( figure b , panels - ). histologically, the tumors were typical cavernous hemangiomas with malignant vessel hyperplasia visible as a closely packed meshwork of blood vessels. microscopically, the tumor cells were relatively uniform large myeloid cells and lymphoid cell hyperplasia was observed in liver ( figure c , panel ). bluish blisters were also observed and some of these lesions were associated with continuous bleeding. we also observed a significant up-regulation of viral gp expression in tumor cells from alv-j-infected livers using immunohistochemical staining with the je monoclonal antibody (figure c, panel ) . pcr tests on the genomic dna of the three sick chicken spleens using alv-j-specific primers were all positive (figure a, panel ) . we detected no related viral infections as judged by the absence of amplicons using primers specific to alv-a, alv-b, mdv, and rev ( figure a , panels , , , ). the pcr tests of three normal chickens from the same flock were negative using all the listed primers ( figure a , panels , , , ). we could also identify the presence of virion-encoded p from the sick, but not the healthy chickens (data not shown). furthermore, immunofluorescence of infected cells using an alv-j-specific monoclonal antibody were all positive ( figure d , panels - ) and the uninfected control completely lacked any according to the transcriptome profiles of alv-j-induced tumor spleen samples and healthy spleen samples from white (recessive) plymouth rock chickens in our previous experiments (li et al., ) , we analyzed the transcriptional level of related innate immune genes. as a guide, we compared healthy spleens to alv-j-infected spleen samples that possessed tumors and found a general decreasing trend except for tnfaip which increased slightly (figure a) . genes that were decreased two to eightfold included interferon-stimulated genes such as the single copy antiviral gene mx, ifnα-stimulated genes and (isg - , isg - ) and zc hav (zinc finger ccch-type antiviral protein ) (figure a) . considering this data, we examined whether alv-j induces or inhibits innate immune host responses during both early and late phases of infection. avian leukosis virus subgroup j infection in the early stages resulted in a more than -fold up-regulation of isg mrna at d.p.i. (p < . ) and greater than twofold up-regulation of zc hav mrna at d.p.i. (p < . ). however, alv-j late infection exhibited no statistical differences in isg - and zc hav mrna expression in the tumorigenesis phase (p > . ) ( figure b ). avian leukosis virus subgroup j also induced a significant increase of tlr- at d.p.i. (p < . ) as well as melanoma differentiation-associated gene (mda ) in the tumorigenesis phase (p < . ) (figure c) . these results suggest that alv-j was primarily recognized by chicken tlr and induced isg - expression at d.p.i. chicken mda was the main alv-jsensing pattern-recognition receptor during the late infection phase in vivo. to further explore the differences on innate immune responses between the early and late phases of alv-j infection, we measured cytokine levels in spleen homogenates comparing healthy chickens to chickens with alv-j infection. interestingly, il- , il- , il- β, and ifn-β showed no significant differences between the groups of spf chicks from to d.p.i. (p > . ) ( figure a) . however, clinical chickens with neoplasms showed il- , il- β and ifn-β levels that were significantly altered within the clinical group ( figure b ). in addition, the anti-inflammatory cytokine il- was also increased sharply in two of three clinical samples with neoplasms ( figure b) . taken together these results indicated that alv-j early infection induced no obvious antiviral innate immunity responses in chicks sampled from to d.p.i. however, this was not the case for late infections and there were significant increases in type i ifn, pro-inflammatory cytokines as well as il- . the jak-stat pathway as well as irf- are key regulators of viral immune responses. therefore we measured expression levels of irf and stat via western blotting. compared with the control uninfected chickens, irf and stat levels were decreased in the tumor samples. this was especially true with chicken sc that also displayed liver tumors (figures a,b and see figure a ). innate immunity plays a dominant role in antiviral responses of chickens at - days of age due to the incomplete structural organization of their secondary immune organs (mast and goddeeris, ) . alv transmission primarily occurs at hatching or in the st weeks of life (witter and fadly, ) . accordingly, we deliberately studied the innate immune response of chicks within a week of hatching in response to alv-j infection. our findings showed that cytokine levels were not significantly different between the infected chick group and the control group from to d.p.i. (p > . ). even so, il- , il- , and ifn-β levels were all increased immediately after infection at d.p.i. (figure a) . it was also at this time-point that isg - and tlr expression levels were dramatically increased. hence, day of infection plays a pivotal role in innate immune responses of chicks to some degree. retroviruses can selectively trigger an array of innate immune responses through various pattern recognition receptors (prrs) (van montfoort et al., ) . however, the nature of the exact innate sensors that detect alv-j had remained elusive until now. recognition of hiv- by tlr does not require retroviral replication, and only requires attachment and endocytosis (van montfoort et al., ) . our results showed that alv-j induced a significant increase of tlr- at d.p.i., so we speculated that alv-j was primarily recognized by chicken tlr at d.p.i. the cytoplasmic sensor rig-i can serve as a sensor for hiv genomic rna (van montfoort et al., ) and chickens lack rig-i, but the mda can partially compensate to generate an interferon response (magor et al., ) . as host mrnas, retroviral genomic rnas are capped and polyadenylated (solis et al., ) . during the tumor phase in vivo, alv-j had been integrated into the host genome (li et al., ) . alv-j induced a significant increase of mrna expression of mda in the tumorigenesis phase (p < . ), we speculated that mda was the main sensing receptor during the late phase in vivo and this is consistent with previous reports (hang et al., ) . since il- and ifn-β can induce the expression of ifn-stimulated genes by activating the jak-stat pathway (hoffmann et al., ; wang and zhang, ) , we speculated that alv-j was primarily recognized by chicken tlr . this would lead to regulation of isg - expression via jak-stat pathway activation at d.p.i. however, taken together, the early antiviral innate immune response was too weak to resist alv-j invasion as evidenced by the lack of obvious cytokine expression. in fact, the spf chicks were susceptible to alv-j within - days post hatch. during late infection stages, the secretion levels of il- , il- β, and ifn-β in the three clinical samples with neoplasms had significantly increased. of note was the anti-inflammatory cytokine (il- ) that possessed immunosuppressive effects (sabat et al., ) , and this cytokine was sharply increased in the two of three clinical samples. in other words, alv-j late infection caused significant immune responses including increasing type i ifn, pro-inflammatory as well as anti-inflammatory cytokines. however, there were no statistical differences in isg - and zc hav mrna expression in the tumorigenesis phase. we speculated that functional isgs cannot be induced in the late infection phase. as a tumor suppressor, irf expression is decreased in a variety of human cancers (connett et al., ; wang et al., ) . irf can also exert antiviral activity by inducing interferonstimulated gene expression directly (stirnweiss et al., ) . our previous study demonstrated that irf expression was decreased as a target of mir- b in the white (recessive) plymouth rock chickens with alv-j infection (li et al., ) . in that study we also showed that the antiviral activity of irf was inhibited during the late phase of alv-j infection. there are a number of reports concerning jak-stat pathway inhibition caused by reducing stat expression or by inhibiting its phosphorylation (precious et al., ; audsley and moseley, ) . our previous transcriptome results and the western blotting data presented here demonstrate that stat levels are decreased in chickens with tumors induced by alv-j infection. we hypothesize that alv-j escapes through inhibition of the host antiviral immune response by modulating the jak-stat signaling pathway. additional studies concerning this hypothesis are currently being conducted. in summary, the present study demonstrates that the alv-j strain scau-hn produces an almost undetectable antiviral innate immune response in week old chicks. cytokines were induced in the yellow chickens with tumors caused by alv-j infection. however, 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for spontaneous haemangiomas in layer chickens in china upregulation of chicken tlr , tlr and myd in heterophils and monocyte-derived macrophages stimulated with eimeria tenella in vitro key: cord- - vm authors: mikel, pavel; vasickova, petra; tesarik, radek; malenovska, hana; kulich, pavel; vesely, tomas; kralik, petr title: preparation of ms phage-like particles and their use as potential process control viruses for detection and quantification of enteric rna viruses in different matrices date: - - journal: front microbiol doi: . /fmicb. . sha: doc_id: cord_uid: vm the detection and quantification of enteric rna viruses is based on isolation of viral rna from the sample followed by quantitative reverse transcription polymerase chain reaction (rt-qpcr). to control the whole process of analysis and in order to guarantee the validity and reliability of results, process control viruses (pcv) are used. the present article describes the process of preparation and use of such pcv– ms phage-like particles (ms plp) – in rt-qpcr detection and quantification of enteric rna viruses. the ms plp were derived from bacteriophage ms carrying a unique and specific de novo-constructed rna target sequence originating from the dna of two extinct species. the amount of prepared ms particles was quantified using four independent methods – uv spectrophotometry, fluorimetry, transmission electron microscopy and a specifically developed duplex rt-qpcr. to evaluate the usefulness of ms plp in routine diagnostics different matrices known to harbor enteric rna viruses (swab samples, liver tissue, serum, feces, and vegetables) were artificially contaminated with specific amounts of ms plp. the extraction efficiencies were calculated for each individual matrix. the prepared particles fulfill all requirements for pcv – they are very stable, non-infectious, and are genetically distinct from the target rna viruses. due to these properties they represent a good morphological and physiochemical model. the use of ms plp as a pcv in detection and quantification of enteric rna viruses was evaluated in different types of matrices. the quantitative reverse transcription polymerase chain reaction (rt-qpcr) assay is nowadays considered as the gold standard method for detection and quantification of enteric rna viruses such as hepatitis a virus (hav), hepatitis e virus (hev) or human noroviruses (nov) (mattison et al., ; blaise-boisseau et al., ; di pasquale et al., ; vasickova et al., ; hennechart-collette et al., ) . these enteric viruses have a significant impact on human health throughout the world. worldwide, hav infections account for . million cases annually and about million asymptomatic and symptomatic cases occurred all together in (who, ; vos et al., ) , hev infections account for million cases annually (lozano et al., ; rein et al., ) and nov is responsible for approximately % of epidemic non-bacterial outbreaks of gastroenteritis around the world (lindesmith et al., ) . rt-qpcr is a fast and sensitive method capable of detecting as few as genome copies of viral nucleic acid in a sample (puig et al., ) . because of the drawbacks of rt-qpcr including the necessity of monitoring the efficiency of concentration and rna extraction steps, the removal of reverse transcription (rt) and pcr inhibitors there is a need of developing an entire rt-qpcr assay with a system of controls. effective control of all the analytical steps is now required for diagnostic assays and generally involves the utilization of a nonpathogenic virus -process control virus (pcv) -which is added in a defined amount to the sample prior to the processing. viral concentration and isolation procedures from complex matrices (e.g., food matrices or environmental samples) are often laborious and time-intensive, which increases the likelihood of mistakes that may lead to the failure of the analysis. therefore, in the european committee for standardization (cen) released iso technical specifications (iso/ts) iso/ts - and iso/ts - (the methods for determination of hav and nov in food using rt-qpcr), which require the use of pcv together with external control rna (eac) in rt-qpcr detection of these viruses in such complex matrices. according to these technical specifications, a cultivable non-enveloped positivesense single stranded rna (+ssrna) virus shall be used as such a control. furthermore, the pcv should be of a similar size to the target virus to provide a good morphological and physicochemical model, should be genetically distinct from the target virus to avoid cross-reactivity, and should not be naturally present in the analyzed sample. based on these recommendations, ms phage-like particles (ms plp) that could be used as pcv for rt-qpcr detection of enteric rna viruses were prepared. the technology for production of ms plp is theoretically well established and uses the knowledge gained from the study of the familiar bacteriophage ms (leviviridae, +ssrna) (pickett and peabody, ; dubois et al., ; pasloske et al., ; cheng et al., ; wei b.j. et al., ; yu et al., ) . the use of wildtype bacteriophage ms instead of ms plp has two major disadvantages. first, wild-type ms bacteriophage has the ability to proliferate -theoretically, in a specific samples such as those with fecal contamination that naturally contain escherichia coli (e. coli), ms bacteriophage can proliferate and exceed the number of detected pathogenic rna viruses present in the sample. second, the wild-type ms bacteriophage cannot be used as a highly specific pcv because its genome does not contain the specific target sequences. in contrast with wildtype bacteriophage ms , ms plp cannot replicate and allow packaging of the specific control rna sequence into their capsid. the advantages of ms plp include their ability to protect the control rna contained in their capsid from degradation by ubiquitous ribonucleases, their non-pathogenicity to humans, and their stability during long-term storage. the particles have similar physiochemical properties as the wild-type bacteriophage ms , which has been used many times as a pcv in the detection of enteric rna viruses (dreier et al., ; rolfe et al., ; blaise-boisseau et al., ; shulman et al., ) and as the surrogate virus in enteric rna virus environmental stability studies (shin and sobsey, ; bae and schwab, ; park and sobsey, ) . the present article is a followup study of a previously published theoretical concept (mikel et al., ) and describes a method of preparation of ms plp carrying a specific control sequence and their use as a pcv in rt-qpcr detection and quantification of enteric rna viruses in swab, liver tissue, serum, feces, and vegetable samples. the specific control sequence was derived from mitochondrial dna (mtdna) sequences of two extinct species -thylacine (thylacinus cynocephalus, genbank accession no. fj . ), and the moa bird (dinornis struthoides, genbank accession no. ay . ). due to the fact that the sequence was derived from mdna of two extinct species, its natural occurrence in the analyzed samples is highly unlikely. the control sequence with a total length of bp was synthesized de novo (elisabeth pharmacon, czech republic). the specificity of the control sequence was verified using blast . it was also analyzed using oligoanalyzer . software for the ability to form secondary structures, such as hairpins, which could result in unsuitability for rt or/and qpcr. the fragment encoding the control sequence derived from thylacine and the moa bird was obtained by pcr using a de novo template and the primer pairs tm-blpi f and tm-hindiii r, which included blpi and hindiii restriction enzyme sites ( table ) . the composition of the reaction mixture was . µl of fast start pcr master (roche molecular diagnostics, germany), . pmol of each primer and . pmol of template dna. the assay was run in a total volume of µl under the following conditions: • c for min, followed by cycles of • c for s, • c for s and • c for s and a final extension of • c for min. pcr products were examined using agarose gel electrophoresis ( %) staining with ethidium bromide, and were purified using the qiaquick pcr purification kit (qiagen, germany) and subsequently digested at • c for h. the composition of the restriction enzyme digest reaction was ng of pcr product, µl nebuffer (new england biolabs, uk; neb), u hindiii and u blpi endonucleases (both neb) in a final volume of µl. the cleaved pcr product was further purified using the qiaquick pcr purification kit (qiagen). recombinant plasmid dna (pau -tm) carrying the specific control sequence derived from thylacine and the moa bird was transformed into e. coli bl (de ) cells (neb) according to the manufacturer's instructions and the bacterial cells were grown in lb broth containing µg/ml of kanamycin at • c until the culture reached an optical density of nm (od ) = . . two milliliter of the culture were transferred to ml of lb broth containing µg/ml of kanamycin, cultivated at • c until od = . and centrifuged at × g for min at room temperature. subsequently, the pellet was resuspended in ml of lb broth containing µg/ml of kanamycin. protein expression was induced by addition of mm isopropyl-l-thio-d-galactopyranoside (iptg; sigma-aldrich) at • c for h. the cell suspension was centrifuged at × g for min at • c and cells were lyzed by ultrasonic disruption (bandelin vw sonicator, probe ms , bandelin, germany) at amplitude %, and with four pulses for a total length of min at • c. to verify the production of ms coat protein sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds-page) and immunoblot analysis was carried out using a phage ms coat protein polyclonal antibody (merck millipore, usa) as primary antibody and goat anti-rabbit igg, fc specific fragment (jackson immunoresearch, uk) as secondary antibody. intact ms plp were verified by transmission electron microscopy (tem; electron microscope philips em ; fei, czech republic) at , × magnification and an accelerating voltage of kv. to eliminate free nucleic acids the suspension containing lyzed cells and ms plp was briefly centrifuged at × g for min at • c and the supernatant ( ml) was incubated with u of dnase i (neb) and u of rnase a (qiagen) at • c for min. the particles were subsequently purified using sucrose density gradient ultracentrifugation. briefly, ml of supernatant was applied to the ultracentrifugation tubes, where two sucrose density layers of and % were layered. ultracentrifugation was conducted at × g (rotor sw ti, beckman, usa) for h at • c. a strong source of light -led -was used to distinguish three opalescent layers inside the ultracentrifugation tube. the three layers were checked for the presence of ms plp by agarose gel electrophoresis ( %) staining with ethidium bromide. the top layer was removed by a needle and ml syringe and the obtained ms plp were dialyzed using a float-a-lyzer g membrane (molecular weight cut off kda; spectrum laboratories, netherlands) against mm tris containing mm nacl and mm edta for h at • c with three buffer changes. the purity of obtained ms plp was controlled using agarose gel electrophoresis and tem as described above. the number of ms plp was determined by uv spectrophotometry using the avogadro constant, extinction coefficient of . mg/ml of ms bacteriophage per absorbance unit at nm and a molecular weight of . × as was previously described (cheng et al., ) . sample absorbance at nm was measured six times and mean and standard deviation values were calculated. on the basis of these data the quantity of ms plp was calculated as follows: where x = avogadro constant ( . × /mol), y = extinction coefficient of ms bacteriophage ( . × − g/ml) and z = molecular weight of ms bacteriophage ( . × g/mol). the result of this calculation revealed that the concentration of ms plp in a suspension of od was is . × particles/ml. as a second method for ms plp quantification tem was used according to (malenovska, ) with small modifications; the grid was stained with % ammonium molybdate (ph = . ) for min, the latex standard (agar scientific, stansted, england) had a concentration of . × particles/ml and the used magnification was , × to , × (malenovska, ) . ms plp was counted in randomly selected fields of view. a third method for ms plp was quantification by fluorimetric measurement of protein concentration using the qubit protein assay kit (life technologies) and qubit . fluorometer (life technologies). the suspension containing ms plp was diluted -fold in water and fluorimetric calculation of protein concentration in the samples was done according to manufacturer instructions. the samples were analyzed in hexaplicates and the mean and standard deviation values were calculated. as a fourth method for ms plp quantification rt-qpcr was used as described below. stability of ms plp was verified by their incubation with u of dnase i (neb) and u of rnase a (qiagen) at • c for h. as controls for the reaction, dna ( ng of plasmid pau-tm) and rna ( ng of iac in vitro transcript, see below) were incubated under the same conditions. the ability of ms plp to resist the action of nucleases was controlled using agarose gel electrophoresis ( %). prepared ms plp were diluted with mm tris containing mm nacl and mm edta supplemented with . mg/ml of bovine serum albumin (bsa; life technologies) to a final concentration of × /µl, verified by tem, aliquoted ( µl) and stored at − • c. a two-step, duplex, rt-qpcr was optimized for ms plp and internal amplification control (iac) detection and quantification. to distinguish false and truly negative results in vitro transcribed rna (iac) was included in the rt-qpcr assay. the iacspecific sequence was derived from the genomic sequences of two plants -nepenthes (nepenthes ampullaria, genbank accession no. gq . ), and potato (solanum tuberosum, genbank accession no. af ) and from mycobacterium avium subsp. paratuberculosis (genbank accession no. x ). the dna-containing iac sequence and dna-containing fragment encoding the specific control sequence were prepared according to vojkovska et al. ( ) . in vitro transcription of the iac and the fragment encoding the specific control sequence derived from thylacine and the moa bird was performed as was previously described vasickova et al. ( ) . in vitro transcripts were stored at − • c. reverse transcription was carried out using primescript reverse transcriptase (takara, shiga, japan) with slight modifications to the manufacturer's protocol. the rt mixture ( µl) contained . nmol of dntp mix (serva, heidelberg, germany), , molecules of iac rna, pmol of both reverse primers (tm r and np r; table ), µl of primescript reaction buffer, u of reverse transcriptase, u of rnase inhibitor (neb) and µl of isolated rna. the reaction was performed at • c for h followed by • c for min and a cooling step at • c. the optimized duplex qpcr assay was run in duplicate for each analyzed sample in a total volume of µl. the reaction mix contained µl of lightcycler probes master (roche), pmol of the np f and np r primers, pmol of the tm f and tm r primers, pmol of the iac probe and pmol of the np p probe, and µl template cdna. u of uracil dna glycosylase (roche) was used in each qpcr reaction to avoid possible carry-over contamination. qpcr was performed using the roche lightcycler under the following reaction conditions: initial denaturation at • c for min, followed by cycles at • c for s, • c for s and • c for s. the subsequent analysis of results (quantification) was carried out using the "fit point analysis" option of the lightcycler software release . . (version . . . ). quantification standards were prepared from a -fold dilution of in vitro-prepared rna transcripts of the fragment encoding the specific control sequence derived from thylacine and the moa bird in the range of × genome copies/ µl to × genome copies / µl. rna in vitro transcripts were quantified by fluorimetry using the qubit rna br assay kit (life technologies) and qubit . fluorometer (life technologies) according to the manufacturer's instructions. ms plp were lyzed in triplicate by heating at • c for min (cheng et al., ) and µl of undiluted, ten-fold and hundred-fold diluted (rnase-free h o, u/ml rnase inhibitor, neb; ng/µl carrier rna, life technologies) lyzed ms plp were used as template rna for the rt-qpcr reaction. rt was carried out in triplicate for each dilution and qpcr was done in triplicated for each sample. swab, liver tissue, serum, feces, and vegetable samples were artificially contaminated with the µl of ms plp ( × particles). rna from liver tissue ( mg aseptically taken from three different inner-liver locations) was isolated using the rneasy mini kit (qiagen), while rna from serum ( µl) and feces ( µl from mg of feces resuspended in . ml of phosphate-buffered saline buffer -pbs) was isolated using the qiaamp viral rna kit (qiagen) with slight modifications as described previously (vasickova et al., (vasickova et al., , . rna from leafy green vegetables ( g) and swabs ( cm , cotton swab rinsed in ml of pbs) was isolated using nuclisens magnetic extraction reagents (biomérieux, durham, nc, usa) according to iso/ts - . elution volumes of isolated rna were µl for swab and vegetable samples and µl for liver tissue, serum and feces samples. rt-qpcr for detection of ms plp was performed as described above. subsequently, µl of ms plp from aliquots which were used for artificial contamination of each sample were thermally lyzed ( • c for min) and µl of lyzed solution was directly added in duplicate to rt-qpcr. this approach allowed calculation of the exact z-value (see below). the extraction efficiency of each sample was calculated as follows: extraction efficiency (%) = where x = number of ms plp in the sample quantified in rt-qpcr, y = volume in which the isolated rna was eluted and z = number of ms plp added to the sample prior to nucleic acid isolation. to determine the quantity of isolated rna molecules, rt-qpcr quantity value (x) is divided by to obtain the quantity of rna molecules in µl (rt-qpcr uses µl of template rna), followed by multiplication by elution volume (y). the pau-tm expression vector carrying all the necessary information for the production of ms plp together with a unique control sequence was successfully prepared. the sequence of the expression vector was verified by sequencing (data not shown). the production of ms coat protein in cell culture after induction was controlled by sds-page and western blot analysis (figure ) . the results of these analyzes showed that the kda ms coat protein was produced in massive amounts in the iptg-induced cell culture carrying the pau -tm expression vector. in contrast, only basal production of ms coat protein was observed in the control iptg non-induced cell culture and no ms coat protein production was observed in the control e. coli bl (de ) without the pau-tm plasmid. the formation of intact ms plp in the supernatant was inspected by tem after ultrasonic disruption of collected e. coli cells (figure ) . these results clearly showed spontaneous assembly of ms plp in the induced cell culture. three opalescent layers were distinguishable inside the ultracentrifugation tube after ultracentrifugation. all layers were removed and the presence of ms plp in these layers was controlled by agarose gel electrophoresis ( %) and staining with ethidium bromide (figure ) . it was found that the lower layer contained no ms plp, the middle layer only a trace amount of ms plp and that the top layer contained the highest amount of ms plp. collected ms plp were further purified by dialysis. dialyzed ms plp was also controlled by agarose gel electrophoresis ( %) and staining with ethidium bromide. the band containing purified ms plp was clearly visible (data not shown). the integrity of purified ms plp was inspected by tem (figure ) . the number of ms plp was determined by uv spectrophotometry, tem (figure ) , fluorimetric measurement of protein concentration and rt-qpcr. the results of ms plp quantification experiments are summarized in table . values of ms plp concentrations obtained by uv spectrophotometry, tem and fluorimetry were comparable. in the case of rt-qpcr quantification of ms plp, undiluted, -fold and -fold diluted suspensions of thermally lyzed ms plp were tested in order to detect possible inhibitory effects of heat denatured proteins on rt-qpcr results. this dilution was done only in rt-qpcr quantification of ms plp experiment in which high quantity of ms plp was expected. because described rt-qpcr system prefers to amplify control sequence before iac sequence, in case that the quantity of ms plp was very high no iac was detected. when the sample was diluted, iac could be detected and the results were therefore valid. in calculation of extraction efficiency of ms plp from spiked matrices no dilution was done because the ms plp quantity was not so high and the iac was reliably detected even in undiluted samples. amplification of iac had to be successful otherwise the results were not valid. rt-qpcr quantification method was chosen as the most accurate method because it was the only method capable of identifying a specific control rna encapsulated inside the ms plp. the stability of ms plp was checked by agarose gel electrophoresis ( %) and staining with ethidium bromide (data not shown). these analyzes clearly showed that only the ms plp were stable in the solution with high concentrations of dnase and rnase whereas naked dna and rna molecules were quickly degraded in this suspension. according to results of rt-qpcr quantification prepared ms plp were diluted to a final concentration of × /µl. the prepared solution was verified by tem. tem inspection revealed that prepared ms plp did not form aggregates and that the suspension was homogeneous (figure ) . tem, transmission electron microscopy; rt-qpcr, reverse transcription polymerase chain reaction. sd, standard deviation. ms plp were added in the amount of × particles to the different types of matrices (swabs, liver tissue, serum, feces, and leafy green vegetables) to reveal their ability to serve as pcv in the rt-qpcr detection of enteric rna viruses. according to results of rt-qpcr the extraction efficiencies were calculated for each individual matrix. the obtained efficiencies from swabs, liver tissue, serum, feces and leafy green vegetables were . , . , . , . , and . %, respectively (table ) . rt-qpcr-based detection and quantification of rna viruses for diagnostic purposes requires strict control to avoid inaccurate results. bacteriophage ms and its derivatives (including ms plp) are not the only possible useful pcv for the rt-qpcr detection of enteric rna viruses. many different viruses were used as pcv for the rt-qpcr detection of hav and nov from food matrices, e.g. . moreover, evaluation of two commercially available mengovirus suspensions showed significantly different rna extraction efficiency results. it was clear that differences in virus source (e.g., method of propagation) can drastically impact the efficiency of rna extraction (gentry-shields and jaykus, ). also in this respect, ms plp have an additional advantage, because they are produced strictly in e. coli bl (de ) cells, which excludes the differences in virus source and provides for high quality, standardized pcv stocks. ms plp are appropriate candidates for pcv only in the case of detection of structurally similar rna viruses -small, non-enveloped ssrna viruses with icosahedral structure, among which the majority of common enteric viruses belong -to closely mimic their physiochemical properties during the nucleic acid extraction step. therefore, ms plp can be good pcv in the detection of hav, hev or nov from food matrices. although their production is not a trivial task, once they can be produced, a high amount is obtained. in comparison with commercially available pcv the price of self-producing ms plp is significantly lower. moreover, ms plp can be stored for a long time without degradation (walkerpeach et al., ; beld et al., ; hietala and crossley, ; wei y.x. et al., ; yu et al., ; zhan et al., ; song et al., ) . another positive feature of ms plp is that they are not infectious and thus working with them is safe for laboratory personnel. their advantage is the possibility to pack any ssrna sequence into a capsid which can then serve as a specific control sequence. on the other hand, a negative feature of ms plp is that they do not form plaques because they do not carry a gene for lysis. therefore, their number cannot be easily determined using a conventional plaque assay. for this reason, indirect methods for ms plp quantification are used. the number of ms plp may be determined using the avogadro constant, extinction coefficient of od = . mg/ml of ms bacteriophage and the molecular weight of × (dubois et al. , ; cheng et al., ; wei b.j. et al., ) . this indirect method of enumeration of ms plp was used together with flourimetry and with direct calculation of viral particles by tem. the tem quantification has not been used in any previous publications dealing with ms plp. this was probably due to their relatively small size (around nm) because quantitation of virus particles with tem was used mostly for viruses larger than nm (stinski et al., ; kwon et al., ; wei et al., ; weidmann et al., ) . the concentrations of ms plp obtained using uv spectrophotometry, fluorimetry and tem were consistent. using an rt-qpcr method, the measured number of ms plp was about log lower ( . × ms plp/ml) than by previously described methods. this indicates that the ratio of ms plp carrying specific control rna sequence in a population of prepared ms plp is only around %. this result can be explained on the basis of the latest findings regarding the capsid assembling mechanism of bacteriophage ms which use a sophisticated coassembly process where the rna genome interacts at multiple sites with the capsid proteins during assembly (dykeman et al., ) . therefore, it is probable that the majority of the prepared ms plp contain non-specific cellular rna rather than specific control rna, which is in concordance with previous findings (pickett and peabody, ) . based on these findings, the rt-qpcr method was selected for quantification of prepared ms plp because it was the only method capable of identifying a specific control rna encapsulated inside the particle. addition of a specific number of ms plp -pcv -to each analyzed sample should provide information about the process of the sample analysis and therefore, it is possible to determine the extraction efficiency of rna from each sample. because false negative results may be caused not only by the failure of viral concentration steps and the nucleic acid extraction step but also by the inhibition of enzymatic reactions steps or failures in the elution, another control rna molecule (iac) was added to the rt-qpcr to detect possible inhibition on the level of rt-qpcr. therefore, each isolated rna sample should be first subjected to a rt-qpcr assay for detection and quantification of ms plp with iac. this first assay allows exact determination of the rna extraction efficiency and also allows detection of potential inhibition in each analyzed sample. then, additional rt-qpcr assays can be performed to detect various types of enteric rna viruses. based on this approach, it is possible to obtain reliable results for rt-qpcr detection of enteric rna viruses. to demonstrate the usefulness of ms plp as pcv, ms plp were added to different types of matrices, which commonly harbor viruses. the exact number of ms plp added to samples ( × particles) was chosen according to iso/ts - and iso/ts - . nucleic acid was isolated from different types of matrices using commercially available isolation kits. all isolation procedures were able to isolate viral rna (pcv rna) with an efficiency of higher than %, which was recognized as the threshold of successful isolation (iso/ts - ). when the extraction efficiency is lower than %, sample results are not valid and the sample must be retested. although the purpose of this study was not to evaluate or to compare the performance of various rna isolation procedures, some interesting findings were obtained. first, the extraction efficiency does not depend on the used isolation kit, as is obvious from the extraction efficiency of swab samples ( . %) and vegetable samples ( . %) both isolated with nuclisens magnetic extraction reagents (biomérieux), or serum samples ( . %) and fecal samples ( . %), both isolated with qiaamp viral rna kit (qiagen). the rna extraction protocols are therefore well suited and the resulting extraction efficiency is much more dependent on concentration steps, which precede the isolation of viral rna. for example, in the case of vegetable matrix, samples undergo a relatively complicated, time-consuming rinsing and concentration procedure prior to the nucleic acid isolation step, resulting in relatively low extraction efficiency. identical isolation applied to swab samples without any pre-processing steps results in higher extraction efficiency of rna (table ) . second, the composition of the analyzed sample has a crucial impact on extraction efficiency. the extraction efficiency from the serum matrix ( . %), which is a relatively homogenous matrix, is almost -fold higher than extraction efficiency from feces ( . %), which are not as homogeneous as serum samples and are expected to contain a higher concentration of inhibitory substances. a similar impact of sample composition is also obvious in the case of swab and vegetable samples (abu al-soud and radstrom, ; radstrom et al., ; schrader et al., ) . the use of pcv in rt-qpcr detection of rna viruses in complex matrices is highly beneficial, since further increases the validity of the results. iso/ts emphasizes the use of pcv (mengovirus) in rt-qpcr detection of rna viruses from food matrices (iso/ts - , ; iso/ts - , ) . the use of appropriate pcv in rt-qpcr detection of rna viruses from clinical samples is also necessary, but so far there is no iso/ts. therefore, the potential of ms plps as pcvs was tested in rt-qpcr detection of rna viruses from food matrices together with clinical samples. since there is still no universally accepted standard for clinical samples, some requirements (exact number of ms plps added to the samples, threshold of successful isolation) were adopted from standard applied in the analysis of food matrices (iso/ts - ). moreover, ms plps also fulfill some additional requirements of iso/ts -are non-enveloped, carry +ssrna, have a similar size to the target viruses, are genetically distinct from the target viruses and their natural occurrence in tested sample is highly unlikely. on the other hand, ms plps do not meet the requirement of iso/ts - that pcv shall be a cultivable virus. this requirement is based on need to be able to maintain a sufficient supply of pcv in the laboratory by cultivating the pcv in cell culture and get more in-house prepared supplies of it. this can be a problem in laboratories where cultivation of viruses is restricted or in laboratories which do not have experience with the cultivation of viruses in cell cultures. in these cases, ms plps represents an alternative approach because they can be easily produces through bacterial cultures in high quantity. moreover, this production approach of ms plps excludes the difference in virus source and provides for high quality, standardized pcv stocks. this work took some generally accepted recommendations used in rt-qpcr detection of rna viruses from food matrices and applied them also for clinical samples. this work does not represent a competitive approach for iso/ts . in case of food samples, using of ms plps in rt-qpcr detection exactly according to iso/ts would require comparison of the pcv used in iso/ts (mengovirus) with the ms plps and also discussion regarding additional specific requirements for pcv with technical committee that developed the standard. the described rt-qpcr system including ms plp represents a potent tool for the complex monitoring of sample analysis. the design of the system with the inclusion of control rna and iac sequences allows their usage in any diagnostic procedure for the molecular detection of non-enveloped rna viruses from different types of matrices. the testing of the developed rt-qpcr system on different matrices of clinical and food origin artificially contaminated with ms plp showed that it is robust enough to provide reliable and precise data. conception and design of the work: pm, pv, and pek; acquisition of data: pm, rt, hm, pk, and tv; interpretation of data: pm, pv, and pek; drafting the work: pm; revision of the manuscript: pv and pek; all authors approved the version to be published in frontiers in microbiology and agreed to be accountable for all aspects of the work. the results of the project lo were obtained with financial support from the meys of the cr under the npu i program and project ro from the ma of the cr. this work was supported by grant no. nt - / of the ministry of health of the czech republic. the funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication. effects of amplification facilitators on diagnostic pcr in the presence of blood, feces, and meat evaluation of murine norovirus, feline calicivirus, poliovirus, and ms as surrogates for human norovirus in a model of viral 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characterization of influenza virus subpopulations using field flow fractionation and multiangle light scattering: correlation of particle counts, size distribution and infectivity quantitative analysis of particles, genomes and infectious particles in supernatants of haemorrhagic fever virus cell cultures prevention and control of viral hepatitis infection: framework for global action preparation of armored rna as a control for multiplex real-time reverse transcription-pcr detection of influenza virus and severe acute respiratory syndrome coronavirus armored long rna controls or standards for branched dna assay for detection of human immunodeficiency virus type the authors would like to thank neysan donnelly (max-planck-institute of biochemistry, germany) for grammatical corrections of the manuscript. key: cord- -r jl authors: bhuvaneshwar, krithika; song, lei; madhavan, subha; gusev, yuriy title: vigen: an open source pipeline for the detection and quantification of viral rna in human tumors date: - - journal: front microbiol doi: . /fmicb. . sha: doc_id: cord_uid: r jl an estimated % of cancers worldwide are associated with infectious causes. the extent and biological significance of viral presence/infection in actual tumor samples is generally unknown but could be measured using human transcriptome (rna-seq) data from tumor samples. we present an open source bioinformatics pipeline vigen, which allows for not only the detection and quantification of viral rna, but also variants in the viral transcripts. the pipeline includes major modules: the first module aligns and filter out human rna sequences; the second module maps and count (remaining un-aligned) reads against reference genomes of all known and sequenced human viruses; the third module quantifies read counts at the individual viral-gene level thus allowing for downstream differential expression analysis of viral genes between case and controls groups. the fourth module calls variants in these viruses. to the best of our knowledge, there are no publicly available pipelines or packages that would provide this type of complete analysis in one open source package. in this paper, we applied the vigen pipeline to two case studies. we first demonstrate the working of our pipeline on a large public dataset, the tcga cervical cancer cohort. in the second case study, we performed an in-depth analysis on a small focused study of tcga liver cancer patients. in the latter cohort, we performed viral-gene quantification, viral-variant extraction and survival analysis. this allowed us to find differentially expressed viral-transcripts and viral-variants between the groups of patients, and connect them to clinical outcome. from our analyses, we show that we were able to successfully detect the human papilloma virus among the tcga cervical cancer patients. we compared the vigen pipeline with two metagenomics tools and demonstrate similar sensitivity/specificity. we were also able to quantify viral-transcripts and extract viral-variants using the liver cancer dataset. the results presented corresponded with published literature in terms of rate of detection, and impact of several known variants of hbv genome. this pipeline is generalizable, and can be used to provide novel biological insights into microbial infections in complex diseases and tumorigeneses. our viral pipeline could be used in conjunction with additional type of immuno-oncology analysis based on rna-seq data of host rna for cancer immunology applications. the source code, with example data and tutorial is available at: https://github.com/icbi/vigen/. an estimated % of cancers worldwide are associated with infectious causes. these infectious agents include viruses, bacteria, parasites and other microbes. examples of viruses include human papilloma viruses (hpvs) in cervical cancer, epstein-barr virus (ebv) in nasopharyngeal cancer, hepatitis b and c in liver cancer (hbv and hcv), human herpes virus in kaposi sarcoma (ks); human t-lymphotrophic virus- (htlv- ) in adult t cell lymphocytic leukemia (atl) and non-hodgkin lymphoma; merkel cell polyomavirus (mcv) in merkel cell carcinoma (acs, ) . bacteria such as helicobacter pylori have been implicated in stomach cancer. parasites have also been associated with cancer, examples are opisthorchis viverrini and clonorchis sinensis in bile duct cancer and schistosoma haematobium in bladder cancer (acs, ) . detection and characterization of these infectious agents in tumor samples can give us better insights into disease mechanisms and their treatment (hausen, ) . vaccines have been developed to help protect against infection from the many cancers. but these vaccines can only be used to help prevent infection and cannot treat existing infections (acs, ) . there are several screening methods widely used to detect viral infections, especially for blood borne viruses including hbv, hcv, hiv and htlv. these include the enzyme linked immunosorbent assay (elisa or eia) (yoshihara, ) , chemluminescent immunoassay (chlia), indirect fluorescent antibody (ifa), western blot (wb), polymerase chain reaction (pcr), and rapid immunoassays . elisa and wb test detects and measures antibodies in serum taken from the patient's blood, and are typically prescribed after certain symptoms are observed in the patient. there are several challenges in detection of viruses in tumors including loss of viral information in progressed tumors and limited or latent replication resulting in low transcription of tumors (schelhorn et al., ) . the extent and biological significance of viral presence/infection in actual tumor samples is generally unknown but could be measured using human transcriptome data from tumor samples. the popularity of next-generation sequencing (ngs) technology has exploded in the last decade. ngs technologies are able to perform rapid sequencing, and in a massively parallel fashion (datta et al., ) . in recent years, applications of ngs technologies in clinical diagnostics have been on the rise fda complete list of donor screening assays for infectious agents and hiv diagnostic assays (accessed march , ) . available online at: https:// www.fda.gov/biologicsbloodvaccines/bloodbloodproducts/approvedproducts/ licensedproductsblas/blooddonorscreening/infectiousdisease/ucm .htm abbreviations: hbv, hepatitis b virus; hcv, hepatitis c virus; herv k , human endogenous retrovirus k ; tcga, the cancer genome atlas; hcc, hepatocellular carcinoma; nafld, nonalcoholic fatty liver disease; hep b, hepatitis b; hep c, hepatitis c; hepb + hepc, coinfected with both hepatitis b and c virus; hbsag, hepatitis b surface antigen; hbeag, hepatitis b type e antigen; ngs, next-generation sequencing; rna-seq, whole transcriptome sequencing; bam, binary version of sequence alignment/map format; cds, coding sequence; cox ph, cox proportional hazard; hbx, viral gene x; sts, sequence-tagged sites; ncbi, national center for biotechnology information; gff, general-feature-format. (barzon et al., ; byron et al., ) . amongst the various ngs technologies, whole-transcriptome sequencing, also called rna-seq, has been very popular with methods and tools being actively developed. exploring the genome using rna-seq gives a different insight than looking at the dna since the rna-seq would have captured actively transcribed regions. every aspect of data output from this technology is now being used for research, including detection of viruses and bacteria (khoury et al., ; salyakina and tsinoremas, ; wang et al., ) . they are also independent of prior sequence information, and require less starting material compared to conventional cloning based methods, making them powerful and exciting new technologies in virology (datta et al., ) . these high throughput technologies give us direct evidence of infection in the tissue as compared to elisa-based assays, which only proves presence of infection somewhere in the human body. rna-seq technology has hence enabled the exploration and detection of viral infections in human tumor samples. this technology also enables detection of variants in viral genome, which have been connected to clinical outcome (moyes et al., ; downey et al., ) . in recent years, us regulators approved a viral based cancer therapy (ledford, ) , proving that the study of viruses in the human transcriptome has biomedical interest, and is paving the way for promising research and new opportunities. in this paper, we present our pipeline vigen to not only detect and quantify read counts at the individual viral-gene level, but also detect viral variants from human rna-seq data. the characterization of viral variants helps enable better epidemiological analysis. the input file to our pipeline is a fastq (wikipedia, ) file, so our vigen pipeline can be extended to work with genomic data from any ngs technology. our pipeline can also be used to detect and explore not only viruses, but other microbes as well, as long as the sequence information is available in ncbi . we applied our vigen pipeline to two case studies as a proof of concept -a dataset of cervical cancer patients, and a set of liver cancer patients, both from the tcga collection. we first applied the pipeline to the transcriptome of cervical cancer patients to see if we are able to detect the human papilloma viruses. we also performed additional in-depth analyses on a small focused study of liver cancer patients. in this cohort, we performed viral-gene quantification, viral-variant extraction and survival analysis. from our analyses, we show that we were able to successfully detect the human papilloma virus among the tcga cervical cancer patients. we compared the vigen pipeline with two metagenomics tools and demonstrate similar sensitivity/specificity. we were also able to quantify viraltranscripts and extract viral-variants using the liver cancer dataset. this enabled us to perform downstream analysis to give us new insights into disease mechanisms. in addition to the two case studies, we have made available an end-to-end tutorial demonstrated on a publicly available we also provided step-by-step instructions on how to run our vigen pipeline on this sample data, along with the code at https://github.com/icbi/vigen/ and demonstrated the detection of hbv transcripts in this sample. this allows other users to apply this pipeline to explore viruses in their data and disease of interest. we are currently implementing the vigen pipeline in the seven bridges cancer genomics cloud . there are a number of existing pipelines that detect viruses from human transcriptome data. of these, very few pipelines offer quantification at the gene expression level. a comprehensive comparison of these pipelines is provided in table . our goal was not to compete with these other tools, but to offer a convenient and complete end-to-end publicly available pipeline to the bioinformatics community. to the best of our knowledge there are no publicly available pipelines or packages that would provide this type of complete analysis in one package. customized solutions have been reported in the literature however were not made public. in the future, our plan is to package this pipeline and make it available to users through bioconductor (lawrence et al., ) , allowing users to perform analysis on either their local computer or the cloud. in this paper, we applied our vigen pipeline to two case studies as a proof of concept -a dataset of cervical cancer patients, and a set of liver cancer patients, both from the tcga collection (nci, ) . we first applied the pipeline to the transcriptome of cervical cancer patients to see if we are able to detect the human papilloma viruses. we also performed additional in-depth analyses on a small focused study of liver cancer patients afflicted with hepatitis b virus. in this cohort, we perform viral-gene quantification, viral-variant extraction and survival analysis. the results from these analyses allowed us to compare experimental and control groups using viral-gene expression data and viral-variant data, and give us insights into their impacts on the tumor, and disease mechanisms. in the following sections, we describe the vigen pipeline, and the two case studies. the vigen pipeline includes major modules. figure shows an image of our vigen pipeline. in module (labeled as "filtered human sample input"), the human rna sequences were aligned to the human-reference genome using the rsem (li and dewey, ) tool. one of the outputs of rsem includes sequences that did not align to the human genome (hence the name "filtered human sample input"). these un-aligned sequences were taken and aligned to the viral reference file using popular alignment tools bwa (li and durbin, ) and bowtie (langmead and salzberg, ) . in module (labeled as "unfiltered human sample input"), the rna seq sequences were directly aligned to the viral reference using bowtie without any filtering. the reason for using two methods to obtain the viral genomes in human rna-seq data (module and module ) was to allow us to be as comprehensive as possible in viral detection. the aligned reads from module and were in the form of bam files (center-for-statistical-genetics, ), from which read counts were obtained for each viral genome species (referred to as "genome level counts") using samtools idxstats or picard bamindexstats tools. using the genome level counts, we estimated the number of reads that covered the genome, a form of viral copy number. viral copy number was defined as in equation below: viral copy number = number of mapped reads × read length genome length only those viral species with copy number more than a threshold are selected for the next module. the bam files from module and (from bowtie and bwa) were input into module (referred to as "viral gene expression level analysis"), which calculated quantitate read counts at the individual viral-gene level. we found existing rnaseq quantification tools to be not sensitive enough for viruses, and hence developed our own algorithm for this module. our in-house algorithm used region-based information from the general-feature-format (gff) files of each viral genome, and the reads from the bam file. it created a summary file, which had a total count of reads within or on the boundary of each region in the gff file. this is repeated for each sample and for each viral gff file. at the end, a matrix is obtained where the features (rows) are regions from the gff file, and the columns are samples. the read count output from module (viral gene expression module) allowed for downstream differential expression analysis of viral genes between case and controls groups. the source code for our in-house algorithm, written using the r programming language (r core team, ), has been made public at available at github.com/icbi/vigen. the bam files from module and (from bowtie ) were also input to module to detect mutations in the transcripts from these viruses (referred to as "viral rna variant calling module"). the bam files were first sorted coordinate-wise using samtools ; pcr duplicates were removed using tool picard , then the chromosomes in the bam file were ordered in the same way as the reference file using picard. the viral reference file was created from combining all known and sequenced human viruses obtained from ncbi . because viral variants are known to be low frequency, we have selected a variant calling tool varscan (koboldt et al., ) , which allows detection of low-frequency variants (spencer et al., ) . low quality and low depth variants were flagged, but not filtered out, in case these low values were due to low viral load. once the variants were obtained, they were merged to form a multi-sample vcf file. only variants that had a variant in two or more samples were retained. plink was used to perform case-control association test (fishers exact test) to compare groups. the vigen pipeline is easy to implement because our pipeline incorporates existing best practices and tools available. for module , we developed our own algorithm for viral-gene quantification. the major motivation for this paper was to build on existing viral detection tools, and to build a quantification tool in order to quantify, explore and analyse the genes detected in viruses. the source code for the in-house algorithm, along with a tutorial on how to execute the code on sample data has been made public at https://github.com/icbi/vigen/. since access to tcga raw data is controlled access, we could not use this dataset to create a publicly available tutorial. so we used a publicly available rna-seq dataset to demonstrate our pipeline with an end-to-end workflow. we chose one sample (srr ) from publicly available hbv liver cancer rna-seq dataset from ncbi sra (http://www. ncbi.nlm.nih.gov/bioproject/prjna ). this dataset is also available through ebi sra (http://www.ebi.ac.uk/ena/data/view/ srr ). the dataset consisted of hbv liver cancer patients, and adjacent normal liver tissues. we downloaded the raw reads for one sample, and applied our vigen pipeline to it and were able to successfully detect hbv transcripts in this sample. a step-by-step workflow that includes -description of tools, code, intermediate and final analysis results are provided in github: https://github.com/icbi/vigen/. this tutorial has also been provided as additional file . we were interested in exploring all viruses existing in humans. so we first obtained reference genomes of all known and sequenced human viruses obtained from ncbi ( viruses) and merged them into one file (referred to as the "viral reference file") in fasta file format (wikipedia, ) . this file has been shared in our github page. cervical cancer is caused by the human papilloma virus (hpv). this dataset consisted of cervical cancer patients in the tcga data collection. these samples were primary tumors from either cervical squamous cell carcinoma or endocervical adenocarcinoma where rna-seq data was available. we applied our vigen pipeline on these samples using the seven bridges platform (https://cgc.sbgenomics.com). among the cervical cancer patients, patients had virus detection confirmed by pcr or other lab methods and made available through the clinical data. so we used this information from the patients to estimate the sensitivity and specificity of our vigen pipeline. this dataset consisted of liver cancer patients in the tcga data collection. of these patients were afflicted with hepatitis b virus (labeled "hepb"), while the rest of the patients had a co-infection of both hepatitis b and c viruses (labeled "hepb+c"). information about viral presence was obtained from "viral hepatitis serology" attribute from the clinical information. we first applied the vigen pipeline on the samples, using the globus genomics platform (bhuvaneshwar et al., ) . once the viral genomes were detected, we then chose only the high abundance viral species for the gene quantification step and viral variant detection steps (module and respectively). we then performed a focused analysis on this dataset. we used the viral-gene expression read counts, to examine the differences between "dead" and "alive" samples. the dead/alive status of the samples was obtained from the clinical data and refers to patients in the cohort that died or not from cancer. we performed this analysis on the patients in the hepb only group to prevent any confounding with the hepb+hepc group. out of hepb patients, were alive (baseline group), and dead (comparison group) as per the clinical data. the analysis was performed using a bioconductor software package called edger (robinson et al., ) in the r programming language (http://www.r-project. org). cox proportional hazards (cox ph) regression model (cox and oakes, ) was then applied to look at the association of viral-gene expression data with overall survival. thie cox model was applied on all samples in the cohort (i.e., hep b and hepb+hepc) samples to maximize power. we also compared the dead and alive samples at the viral rna variant level in the hepb group using a tool called plink to see if it can add valuable information to the tumor landscape in humans. we used our vigen pipeline to detect viruses in the rna of human cervical tissue and obtained viral copy number for each species. we used a threshold copy number of as a "positive" viral detection for both hpv- , hpv- and hpv- viruses. based on this criterion, hpv- was detected in % of the samples, hpv- in % of the samples and hpv- in . % of the samples (figure ). the threshold copy number limit that defines a "positive" detection is one of the parameters of the software which could be set by the user depending on the specifics of the experiment. we obtained the clinical data for this tcga cervical cancer cohort from the cbio portal (cerami et al., ) . among the patients, patients had virus detection confirmed by pcr figure | the hpv viruses detected in cervical cancer patients using the vigen pipeline. frontiers in microbiology | www.frontiersin.org or other lab methods and made available through the clinical data. out of the patients, patients had the hpv- virus, patients had hpv- , and the rest had other hpv viruses. so we used this information from the clinical data to estimate the sensitivity and specificity of our vigen pipeline. we got a sensitivity of % and specificity of % for hpv- detection ( table a) ; and a sensitivity of % and specificity of % for hpv- detection (table b ). we applied our vigen pipeline (modules and ) on the rnaseq data from the tcga liver cancer tumors, and obtained genomelevel read counts for each viral species. we used a threshold copy number of to define a positive detection of the hepatitis b virus. once the viral genomes were detected, we short-listed the high abundance viral species for the viral-gene quantification step and viral-variant detection steps (module and respectively). high abundance was defined as those virus species that were detected in at-least samples. in addition to hepatitis b and c viruses, several other viruses came up in this short list including human endogenous retrovirus k (herv k ) and others. a complete list is provided in table . to get a more detailed overview of the viral landscape, we applied module of the vigen pipeline to the liver cancer dataset. this allowed us to quantify viral-gene expression regions in the rna of liver tumor tissues. we then used those results to examine the differences between dead and alive samples. it is known that these patients were afflicted with the hepatitis b virus and hence many of the differentially expressed regions were from this viral genome. but as we know, other viruses also coexist in humans. this was confirmed by the presence of differentially expressed viral-regions from other viruses. the differentially expressed regions that were significant among the results are shown in tables a,b. table a lists only the differentially expressed regions from hepatitis b virus and table b shows the differentially expressed regions from other viruses. from the differential expression analyses, the two most informative results were ( ) a region of the hepatitis b genome that produced the hbeag and hbcag proteins were overexpressed in the dead patients and ( ) another region of the hepatitis b genome that produced hbsag protein was overexpressed in the alive patients. in detail, we saw several important findings as described below: (a) region nc_ . _cds_ _ of the hepatitis b genome was . times overexpressed (log fold change = + . ) in dead patients. this region contains gene c that produces pre-code protein external core module ) , were the same. we collated the significant common results (p-value ≤ . ) in tables a,b . among these results, we saw several missense and frameshift variants in gene x of the hepatitis b genome (nucleotide ), gene p ( , , ) , and a region that overlaps gene p and pres (nucleotides , , , ) . all these variants were found mutated more in the cases than controls. other significant common results included variants in gene c (nucleotide , ) and variants in pres region (nucleotide positions , and ) ( table a ). in addition, there were two missense variants that were common among the top results, but not significant (p-value = . ). they were variants in the x gene of the hepatitis b genome (nucleotides and ) ( table a) . among the significant common results to both, were a few variants of the human endogenous retrovirus k complete genome (herv k ). these include nucleotide positions , , and . these map to frameshift and missense mutations in the putative envelope protein of this virus (q _gp , also called "env") ( table b) . (c) the overall model is significant with p-value < . from the log rank test (also called score test). the table is sorted based on annotation. annotation includes gene name, protein name, etc., separated by commas, multiple annotations separated by semi-colon. table a shows variants in the hepatitis b virus only while table b shows variants in other species. (shows only common results between two possible analysis steps). frontiers in microbiology | www.frontiersin.org the seven bridges team used two metagenomic tools,centrifuge (kim et al., ) and kraken (wood and salzberg, ) , to detect hpv viruses on the same cohort of tcga patients (bridges, ; malhotra et al., ) , and shared the results with us. they used an abundance of . as a positive viral detection (bridges, ; malhotra et al., ) . we compared vigen with kraken and centrifuge in terms of the percentage of samples where the species was detected ( table ) . we can see that the results are in the same range for all three tools. we also estimated the sensitivity and specificity of these tools using the same patients and compared with that of the vigen pipeline. the centrifuge tool had a sensitivity of % and specificity of % for hpv- detection; and a sensitivity of % and specificity of % for hpv- detection. the kraken tool had a sensitivity of % and specificity of % for hpv- detection; and a sensitivity of % and specificity of % for hpv- detection (detailed in additional file ). it shows that our vigen pipeline was able to match the sensitivity and specificity of centrifuge tool and surpassed that of kraken (detailed in additional files , ). we used our vigen pipeline to get genome-level read counts obtained from viruses detected in the rna of human liver tissue. in our results, hbv was detected in % of the samples. this is similar to earlier analyses of tcga liver cancer cohort study (khoury et al., ; tang et al., ; the cancer genome atlas research network, ) , which detected the hbv virus in and % (with typically low counts range) of cases respectively. it has also been reported that the viral gene x (hbx) was the most predominately expressed viral gene in liver cancer samples (tang et al., ) which is in concordance with our findings where the peak number of reads were observed for gene x region of the hbv genome. to get a more detailed overview of the viral landscape, we examined the human rna-seq data to detect and quantify viral gene expression regions. we then examined the differences between dead and alive samples at the viral-transcript level on the hepatitis b sub-group (tables a,b) . from the differential expression analyses, the two most informative results were ( ) a region of the hepatitis b genome that produced the hbeag protein was overexpressed in the dead patients and ( ) another region of the hepatitis b genome that produced hbsag protein was overexpressed in the alive patients. presence of hbeag or hbcag is an indicator of active viral replication; this means the person infected with hepatitis b -jensen et al., ; liang, ). so our results, showing that antigens hbeag and hbcag were overexpressed in dead patients compared to alive patients makes sense, indicating that these patients never recovered from acute infection. the results also indicate a higher level of hbsag in the alive patients compared to the dead patients. the highest levels of hbsag in the virus are known to occur in the "immunotolerant phase." this pattern is seen in patients who are inactive carriers of the virus i.e., they have the wild type dna, and the virus has been in the host for so long, that the host does not see the virus as a foreign protein in the body, and hence there's no immune reaction against the virus. in this phase, there is known to be minimal liver inflammation and low risk of disease progression (park, ; tran, ; locarnini and bowden, ) . this could explain why we saw higher level of hbsag in the alive patients compared to the dead patients. also among the significant results were three regions from the human endogenous retrovirus k (herv k ) genome (with negative log fold change) that were overexpressed in the alive patients. two of these regions were sequence-tagged sites (sts) and the third region was in the gag-pro-pol region that has frameshifts. herv could protect the host from invasion from related viral agents through either retroviral receptor blockade or immune response to the undesirable agent (nelson et al., ) . overall, we found that our results from viral-gene expression level make biological sense, with much of the results validated through published literature. we performed variant calling on the viral data to see if it can add valuable information to the tumor landscape in humans. we then compared the dead and alive samples at the viral-variant level on the patients in the hepatitis b sub-group. among the significant results (tables a,b) included variants in gene c (nucleotide , ) and variants in pres region (nucleotide positions , and ). the gene c region creates the pre-capsid protein, which plays a role in regulating genome replication (tan et al., ) . the mutation in the position lies in a known cpg island (ranging from to ), whose methylation level is significantly correlated with hepatocarcinogenesis (jain et al., ) . mutations in pres are associated with persistent hbv infection, and emerge in chronic infections. the pres and pres regions are known to play an essential role in the interaction with immune responses because they contain several epitopes for t or b cells (cao, ) . mutations in the / positions of the x gene are known to be associated with greater risk of hcc (cao, ; wang et al., ) , and is independent of serum hbv dna level (wang et al., ) . this mutation combination is also known to be associated with hepatitis b related acute-on-chronic liver failure (xiao et al., ) . it is predicted that mutations associated with hcc variants are likely generated during hbv-induced pathogenesis. the a t/g a combined mutations was shown to be a valuable biomarker in the predicting the risk of hcc (cao, ; wang et al., ) ; and are often detected about years before the diagnosis of hcc (cao, ). among the significant common results to both, were a few variants of the human endogenous retrovirus k complete genome (herv k ). these variants map to frameshift and missense mutations in the putative envelope protein of this virus (q _gp , also called "env"). studies have shown that this envelope protein mediates infections of cells (robinson and whelan, ) . herv k is a provirus and is capable of producing intact viral particles (boller et al., ) . studies have shown a strong association between herv-k antibodies and clinical manifestation of disease and therapeutic response (moyes et al., ; downey et al., ) . it is hypothesized that retroviral gene products can be "reawakened" when genetic damage occurs through mutations, frameshifts and chromosome breaks. even though the direct oncogenic effects of hervs in cancer are yet to be completely understood, it has shown potential as diagnostic or prognostic biomarkers and for immunotherapeutic purposes including vaccines (downey et al., ) . we compared various viral detection pipeline using the several criteria (table ) . our pipeline provides similar functionality as the tools listed in table for the detection of viruses from human rnaseq data; but also has an advantage of enabling gene-level expression analysis and quantification, as well as variant analysis of viral genomes in a single open source publicly available package. one limitation of our vigen pipeline is that it is dependent on sequence information from reference genome. this makes it challenging to detect viral strains where reference sequence information is not known. in the future, we plan to explore de novo assembly incorporating more sophisticated methods like hidden markov models (hmm) (alves et al., ) . this would enable us to provide in-depth analysis of strain pathogenicity in the context of clinical outcome. in recent years, us regulators approved a viral based cancer therapy (ledford, ) , proving that the study of viruses in the human transcriptome has biomedical interest, and is paving the way for promising research and new opportunities. we show that our vigen pipeline can thus be used on cancer and non-cancer human ngs data to provide additional insights into the biological significance of viral and other types of infection in complex diseases, and tumorigeneses. our viral pipeline could be used in conjunction with additional type of immuno-oncology analysis based on rna-seq data of host rna for cancer immunology applications. detection and characterization of these infectious agents in tumor samples can give us better insights into disease mechanisms and their treatment (hausen, ) . with the decreasing costs of ngs analysis, our results show that it is possible to detect viral sequences from whole-transcriptome (rna-seq) data in humans. our analysis shows that it is not easy to detect dna and rna viruses from tumor tissue, but certainly possible. we were able to not only quantify them at a viral-gene expression level, but also extract variants. our goal is to facilitate better understanding and gain new insights in the biology of viral presence/infection in actual tumor samples. the results presented in this paper on two case studies are in correspondence with published literature and are a proof of concept of our pipeline. this pipeline is generalizable, and can be used to examine viruses present in genomic data from other next generation sequencing (ngs) technologies. it can also be used to detect and explore other types of microbes in humans, as long as the sequence information is available from the national center for biotechnology information (ncbi) resources. this pipeline can thus be used on cancer and non-cancer human ngs data to provide additional insights into the biological significance of viral and other types of infection in complex diseases, and tumorigeneses. we are planning to package this pipeline and make it open source to the bioinformatics community through bioconductor. the tcga liver cancer dataset was used in the analysis and writing of this manuscript. the data can be obtained from https:// cancergenome.nih.gov/. since access to tcga raw data is controlled access, we could not use this dataset to create a publicly available tutorial. so we looked for publicly available rna-seq dataset to demonstrate our pipeline with an end-to-end workflow. we chose one sample (srr ) from publicly available liver cancer rna-seq dataset from ncbi sra (http://www. ncbi.nlm.nih.gov/bioproject/prjna ). this dataset is also available through ebi sra (http://www.ebi.ac.uk/ena/data/view/ srr ). the dataset consists of liver cancer patients, and adjacent normal liver tissues. we downloaded the raw reads for one sample, and applied our vigen pipeline to it. a step-by-step workflow that includes -description of tools, code, intermediate and final analysis results are provided in github: https://github.com/icbi/vigen/. project name: vigen project home page: https://github.com/icbi/vigen/ operating system(s): the r code is platform independent. the shell scripts can run on unix, linux, or ios environment programming language: r, bash/shell other requirements: n/a license: n/a any restrictions to use by non-academics: n/a infections that can lead to cancer genseed-hmm: a tool for progressive assembly using profile hmms as seeds and its application in alpavirinae viral discovery from metagenomic data applications of next-generation sequencing technologies to diagnostic virology rapid identification of non-human sequences in high-throughput sequencing datasets a case study for cloud based high throughput analysis of ngs data using the globus genomics system human endogenous retrovirus herv-k is capable of producing intact viral particles identifying viral sequences in tcga data using kraken and centrifuge translating rna sequencing into clinical diagnostics: opportunities and challenges clinical relevance and public health significance of hepatitis b virus genomic variations available online at the cbio cancer genomics portal: an open platform for exploring multidimensional cancer genomics data second-generation plink: rising to the challenge of larger and richer datasets virusseq: software to identify viruses and their integration sites using next-generation sequencing of human cancer tissue analysis of survival data next-generation sequencing in clinical virology: discovery of new viruses human endogenous retrovirus k and cancer: innocent bystander or tumorigenic accomplice? infections causing human cancer comprehensive dna methylation analysis of hepatitis b virus genome in infected liver tissues landscape of dna virus associations across human malignant cancers: analysis of , cases using rna-seq centrifuge: rapid and sensitive classification of metagenomic sequences varscan : somatic mutation and copy number alteration discovery in cancer by exome sequencing pathseq: software to identify or discover microbes by deep sequencing of human tissue fast gapped-read alignment with bowtie software for computing and annotating genomic ranges cancer-fighting viruses win approval rsem: accurate transcript quantification from rna-seq data with or without a reference genome fast and accurate short read alignment with burrows-wheeler transform the sequence alignment/map format and samtools viralfusionseq: accurately discover viral integration events and reconstruct fusion transcripts at single-base resolution hepatitis b: the virus and disease hepatitis b surface antigen quantification: not what it seems on the surface enabling scalable and rapid metagenomic profiling of the transcriptome with the seven bridges cancer genomics cloud the distribution of the endogenous retroviruses herv-k and herv-k in health and disease the cancer genome atlas demystified. human endogenous retroviruses r: a language and environment for statistical computing infectious entry pathway mediated by the human endogenous retrovirus k envelope protein edger: a bioconductor package for differential expression analysis of digital gene expression data viral expression associated with gastrointestinal adenocarcinomas in tcga high-throughput sequencing data sensitive detection of viral transcripts in human tumor transcriptomes performance of common analysis methods for detecting lowfrequency single nucleotide variants in targeted next-generation sequence data immunosuppressive treatment of hbsag-positive chronic liver disease: significance of hbeag the interface between hepatitis b virus capsid proteins affects self-assembly, pregenomic rna packaging, and reverse transcription the landscape of viral expression and host gene fusion and adaptation in human cancer comprehensive and integrative genomic characterization of hepatocellular carcinoma immune tolerant hepatitis b: a clinical dilemma using small rna deep sequencing data to detect human viruses virusfinder: software for efficient and accurate detection of viruses and their integration sites in host genomes through next generation sequencing data detection of hepatitis b virus a t/g a mutant by amplification refractory mutation system fasta format kraken: ultrafast metagenomic sequence classification using exact alignments hepatitis b virus genotype b with g a and a t/g a mutations is associated with hepatitis b related acute-on-chronic liver failure kb and yg designed the pipeline. kb and ls implemented the pipeline. kb and yg wrote the manuscript with editorial comments from sm. this work was funded by the lombardi cancer center support grant (p ca ). the supplementary material for this article can be found online at: https://www.frontiersin.org/articles/ . /fmicb. additional file | vigen github tutorial.additional file | detailed results from analysis of tcga cervical cancer patients.additional file | output from kraken and centrifuge shared by the seven bridges team. the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © bhuvaneshwar, song, madhavan and gusev. this is an openaccess article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- -mu jt ul authors: tong, mingwei; yi, li; sun, na; cheng, yuening; cao, zhigang; wang, jianke; li, shuang; lin, peng; sun, yaru; cheng, shipeng title: quantitative analysis of cellular proteome alterations in cdv-infected mink lung epithelial cells date: - - journal: front microbiol doi: . /fmicb. . sha: doc_id: cord_uid: mu jt ul canine distemper virus (cdv), a paramyxovirus, causes a severe highly contagious lethal disease in carnivores, such as mink. mink lung epithelial cells (mv. .lu cells) are sensitive to cdv infection and are homologous to the natural host system of mink. the current study analyzed the response of mv. .lu cells to cdv infection by itraq combined with lc–ms/ms. in total, and differentially expressed proteins (deps) were markedly up-regulated or down-regulated, respectively. thirteen deps were validated via real-time rt-pcr or western blot analysis. network and kegg pathway analyses revealed several regulated proteins associated with the nf-κb signaling pathway. further validation was performed by western blot analysis and immunofluorescence assay, which demonstrated that different cdv strains induced nf-κb p phosphorylation and nuclear translocation. moreover, the results provided interesting information that some identified deps possibly associated with the pathogenesis and the immune response upon cdv infection. this study is the first overview of the responses to cdv infection in mv. .lu cells, and the findings will help to analyze further aspects of the molecular mechanisms involved in viral pathogenesis and the immune responses upon cdv infection. canine distemper virus (cdv), a negative-sense, single-stranded rna virus, belonging to the genus morbillivirus, family paramyxoviridae, causes a severe highly contagious lethal disease in carnivores, such as dogs, lions, ferrets, raccoon dogs, foxes, and minks (williams et al., ; deem et al., ; martella et al., ; zhao et al., ; viana et al., ) . the disease is distributed worldwide and is characterized by respiratory and gastrointestinal tract symptoms with generalized immunosuppression (blancou, ; decaro et al., ). the immune system dysfunction of cdv infection favors opportunistic secondary pathogens, resulting in high morbidity and mortality in a wide range of carnivore species (appel et al., ; kauffman et al., ; blixenkrone-moller, ) . generally, in domestic dogs, cdv establishes a systemic infection, initiating transmission from immune cells, such as alveolar macrophages and/or dendritic cells, of the upper respiratory tract to the local lymphatic tissues by immune-mediated progression, and ultimately propagates to most organs and tissues, including epithelial tissues via cell-associated viremia (appel et al., ) . epithelial cells are susceptible to cdv infection and play a role in transmission during the late stages of cdv pathogenesis (pratakpiriya et al., ; noyce et al., ) . the virus is amplified and secreted from the epithelial cells of the respiratory, gastrointestinal, and urinary systems of the infected host (ludlow et al., ) . the infection of various viruses has been demonstrated to interact widely with numerous host cell proteins. some interactions elicit changes in the host proteome, as illustrated by the capacity of the virus to both induce and evade the host immune response (kash et al., ) , effecting autophagy and apoptosis (ludwig et al., ; gunnage and munz, ). for measles virus (mv), another morbillivirus closely similar to cdv, cell cycle arrest in lymphocytes (naniche et al., ) and apoptosis in t lymphocytes (fugiervivier et al., ) have also been reported. many studies have reported the effects of cdv infections on the host cell proteins, such as inhibiting stat and stat nuclear import (rothlisberger et al., ) , inducing cytokine responses in pbmcs (nielsen et al., ) , and inducing lymphocytes apoptosis (kumagai et al., ) . however, most of these reports have primarily investigated a single host cell protein or partially selected proteins and the mechanisms of cdv pathogenesis and immunomodulation have not been fully elucidated. thus, a new approach for further understanding the pathogenic mechanism and immunomodulation of cdv infection is needed, and the identification of global host cell proteins that interact with cdv infection represents one option. more details associated with host responses to cdv infection should also shed some light on potential targets for antiviral agents. for decades, proteomic assays have been applied as significant tools to analyze the interaction of host responses to viral infection. investigation of the changes in the proteome upon virus infection is becoming an effective instrument for providing potential targets for antiviral research. this approach has revealed the specific insights into the cellular mechanisms involved in viral pathogenesis for several viral pathogens, including transmissible gastroenteritis virus (tgev) , human influenza a (vester et al., ) , canine parvovirus (cpv) (zhao et al., ) , marek's disease virus (mdv) (chien et al., ) and infectious bronchitis virus (ibv) (emmott et al., ) . isobaric tags for relative and absolute quantification (itraq) combined with lc-ms/ms analysis have emerged as a powerful quantitative proteomic technique, which has been used for various virus-host interaction studies (zhang et al., ; liu et al., ; luo et al., ) . the present study is the first global view of the changes in the mink proteome upon cdv infection. based on itraq combined with lc-ms/ms, a quantitative proteomic analysis was performed to identify differentially expressed proteins (deps) in mink lung epithelial cells (mv. .lu cells) infected with cdv at hours post infection (hpi). these findings will help to analyze further aspects of the molecular mechanisms involved in viral pathogenesis and systematically understand the host immune responses challenged by cdv infection. mink lung epithelial cells (mv. .lu cells) were purchased from the type culture collection of the chinese academy of sciences (shanghai, china) and grown in minimum essential medium (gibco r invitrogen, u.s.a.), supplemented with % fetal bovine serum (invitrogen) at • c and % co . the canine distemper virus strain cdv-ps (genbank accession no. jn ), a low passage isolate (< passages) from a morbid dog in (yi et al., ) , was preserved in our laboratory. the virus was propagated in vero cells. in the study, three additional passages of the virus were performed in mv. .lu cells, resulting in the virus suspension with a titer of . tcid /ml determined by a % tissue culture infectious dose (tcid ) assay (yamaguchi et al., ) . briefly, monolayers of mv. .lu cells in -well plates were infected with a -fold serial dilution of the supernatant fluids and further incubated for up to h. the wells were assessed for cytopathic effects (cpe) after - days, and the tcid was calculated using the reed-muench formula. because of the low virus titer and the impurity of the virus suspension, virus concentration and purification were performed to improve the virus titer and avoid the effect of non-viral components. the clarified suspension was concentrated by polyethylene glycol , precipitation and purified by ultracentrifugation in a gradient of sucrose according to standard procedures. sucrose-purified viruses were then titrated using the tcid assay as described above, and the titer of the virus stocks increased to . tcid /ml. the attenuated cdv vaccine cdv strain was treated the same as ps. the virus stocks were aliquoted and stored at − • c until further use in the following experiments. for the establishment of viral kinetics, mv. .lu cells were grown in -well plates and subsequently challenged by the virus (ps) at a multiplicity of infection (moi) of , calculated based on the infectious virus particle concentration determined as tcid . at , , , , , , and hpi, viral propagation was confirmed by observation of the cpe and viral replication and production of ps nucleoprotein for the different time points analyzed was tested by anti-cdv np antibody. the one-step growth curve, indicating the viral load with the time, was generated according to chuzo ushimi with slight modifications (ushimi et al., ) . briefly, µl of culture medium was collected at indicated time, followed by the extraction of total rna from all samples. qrt-pcr was then applied to detect the viral rna at each indicated time. for itraq labeling, mv. .lu cells were grown in t flasks to - % confluence and subsequently infected with the virus (ps) at an moi of . as an uninfected control, a mock-infection was performed. the cells were collected at hpi for the protein extraction. three biological replicates were prepared for all samples. all experiments were performed under biosafety level conditions. the collected cells were lysed in lysis buffer containing a protease inhibitor cocktail. the lysate was sonicated and centrifuged at , g for min, and the supernatant was quantified with the bca protein assay kit (bio-rad, u.s.a.). subsequently, µg of protein for each sample was digested with µg of trypsin (promega, wi) overnight at • c. according to the protocol of the itraq reagents ( plex, applied biosystems), µg of peptide mixture from each sample was labeled follows: the three mock-infected samples were each labeled with itraq , , or , and the three ps-infected samples were labeled with itraq , , or . the labeled samples were then mixed and dried with a rotary vacuum concentrator. to reduce the complexity of the peptide mixtures, itraqlabeled peptides were fractionated by scx chromatography using the akta purifier system (ge healthcare). briefly, the dried peptide mixture was reconstituted and acidified with buffer a ( mm kh po in % of acn, ph . ) and loaded onto a polysulfoethyl . × mm column ( µm, Å, polylc inc., u.s.a.). the peptides were eluted at a flow rate of ml/min with a gradient of buffer b ( mm kcl, mm kh po in % of acn, ph . ). the elution was monitored by absorbance at nm, and fractions were collected every min. a total of fractions were collected with screening, and then desalted on c cartridges (empore tm spe cartridges c (standard density), bed i.d. mm, volume ml) and concentrated by vacuum centrifugation. each fraction was injected for nanolc-ms/ms analysis. the peptide mixture was loaded onto a reverse phase trap column (thermo scientific acclaim pepmap , µm * cm, nanoviper c ) connected to the c -reversed phase analytical column (thermo scientific easy column, cm long, µm inner diameter, µm resin) in buffer a ( . % formic acid) and separated with a linear gradient of buffer b ( % acetonitrile and . % formic acid) at a flow rate of nl/min controlled by intelliflow technology. the lc-ms/ms analysis was performed on a q exactive mass spectrometer (thermofisher, u.s.a.) coupled to the easy nlc chromatography system (thermofisher, u.s.a.). the mass spectrometer was operated in positive ion mode. ms data was acquired using a data-dependent top method, dynamically selecting the most abundant precursor ions from the survey scan ( - , m/z) for hcd fragmentation. the automatic gain control (agc) target was set to e , and the maximum inject time was set to ms. dynamic exclusion duration was . s. survey scans were acquired at a resolution of , at m/z and resolution for hcd spectra was set to , at m/z , and the isolation width was m/z. normalized collision energy was ev and the underfill ratio, which specifies the minimum percentage of the target value likely to be reached at maximum fill time, was defined as . %. the instrument was run with the peptide recognition mode enabled. all ms raw data files were analyzed by proteome discoverer software . (thermofisher, u.s.a.) using the mascot . search engine against a database of mustela putorius furo protein sequences (ncbinr, released march , , containing , sequences). for protein identification, a mass tolerance of . da was allowed for fragmented ions, with permission of two missed cleavages in the trypsin digests: itraq -plex (y), oxidation (m) as the potential variable modifications, and carbamidomethyl (c), itraq -plex (n-term), and itraq plex (k) as fixed modifications. the strict maximum parsimony principle was performed, and only peptide spectra with high or medium confidence were considered for protein grouping. a decoy database search strategy was also used to estimate the false discovery rate (fdr) to ensure the reliability of the proteins identified. for relative quantitation, proteins that involved at least one unique peptide were considered a highly confident identification and used for quantification. additionally, to guarantee the accuracy of quantification, the proteins with coefficient of variation values < % for three biological repeats were considered deps. the quantitative protein ratios were calculated and normalized by the median ratio in mascot. for comparison, three identical mock samples, labeled with itraq , , and , were used as references. between samples, the proteins with fold-change ratios ≥ . or ≤ . and a p < . were considered deps according to the t-test. to further explore the impact of the dep on cell physiological processes and discover internal relations between deps, an enrichment analysis was performed. go enrichment on three ontologies [biological process (bp), molecular function (mf), and cellular component (cc)] was applied based on the fisher's exact test, considering the whole quantified protein annotations as the background dataset. benjamini-hochberg correction for multiple testing was further applied to adjust derived p-values. only functional categories with p-values under a threshold of . were considered significant. kegg pathway annotation was extracted from the online kegg pathway database (http:// www.kegg.jp/kegg/pathway.html). the protein-protein interaction information involved in the immune response process of the studied proteins was subsequently retrieved from string software (http://string-db. org/). then, the results were imported into cytoscape software (http://www.cytoscape.org/, version . . ) to visualize and further analyze functional protein-protein interaction networks. total rna was isolated using trizol reagent (invitrogen, u.s.a.) from mv. .lu cells infected with moi ps or mockinfected cells at and hpi. after treatment with gdna removal (transgen biotech, china), µg of each total rna was used for cdna synthesis. real-time rt-pcr (qrt-pcr) assays were performed on an applied biosystems r quantstudio r system (thermo fisher scientific, u.s.a.) employing the transstart top green qpcr supermix kit (transgen biotech, china) according to the manufacturer's protocol. the primers for amplifying traf , traf , irak , irak , nfκb , ccl , tnfα, il- , and gapdh are presented in table . each experiment was performed in triplicate. the relative gene expression was calculated using the − ct model, which is representative of n-fold changes compared with mock-infected samples. the data was analyzed by two-way anova followed by duncan's test. for testing the production of ps nucleoprotein for the different time points analyzed, cell lysates were harvested at for confirmation of the itraq-ms data by western blotting, cell lysates were harvested at and hpi from ps-, cdv -, and mock-infected cultures. after measuring the protein concentrations, equivalent amounts of cellular proteins from the triplicates were separated by sds-page and electrophoretically transferred onto nitrocellulose pvdf membranes (millipore, u.s.a.). the membranes were blocked with % bsa dissolved in tbs, containing . % tween- , for h at room temperature, followed by incubation with the corresponding primary antibodies (see below) at • c overnight and incubation with hrp-conjugated goat anti-rabbit or antimouse igg secondary antibodies (sangong biotech, china) at room temperature for h. the protein bands were detected using the ecl detection kit (beyotime, china). the gapdh protein was used as an internal control. the following primary polyclonal antibodies were used: anti-cdv np mouse monoclonal antibody (prepared in our laboratory), nf-κb p (rela) rabbit polyclonal antibody (an , beyotime, china), nfκb p rabbit polyclonal antibody ( , cst, u.s.a), nfκbib (iκb-β) rabbit polyclonal antibody (pa - , thermofisher, u.s.a), mhc-i mouse monoclonal antibody (ab , abcam, uk), rps rabbit polyclonal antibody (pa - , thermofisher, u.s.a), iκb-α rabbit polyclonal antibody ( , cst, u.s.a), phospho-nf-κb p rabbit polyclonal antibody (ma - , thermofisher, u.s.a), and gapdh rabbit polyclonal antibody (cw m, cwbio, china). mv. .lu cells were cultivated on cover glasses in -well plates, followed by infection with ps or cdv at an moi of when the cells reached ∼ % confluence. the mock-infected cells were treated with pbs as a negative control. next, at hpi, the cells were fixed with % paraformaldehyde and subsequently permeabilized with . % triton x- . further, the cells were incubated with an nf-κb p rabbit polyclonal antibody (beyotime, china) and a mouse monoclonal antibody specific to cdv n protein and incubated with cy -labeled goat anti mouse igg (beyotime, china) and fitc-conjugated goat anti-rabbit igg secondary antibody (thermofisher, u.s.a) prior to staining with dapi. the fluorescent images were analyzed under confocal microscopy (leica, germany). a previous study demonstrated the capacity of cdv growth in mv. .lu cells (lednicky et al., ) , thus, we initially confirmed the ability of ps replication in mv. .lu cells and established the growth kinetics of ps replication. an optimal time point under ps infection for proteomic analysis was then identified. as shown in figure a , cpes in the infection groups became visible at hpi and progressed thereafter. up to hpi, an obvious cpe was observed and nearly percent of the cells were detached at hpi. the one-step growth curve revealed that the virus load reached a plateau of ∼ . log copy numbers/µl between and hpi, followed by a gradual decline ( figure b) . collectively, hpi was considered the optimal time-point for further proteomic analysis, at which a high viral load was maintained and most cells showed little cpe. virus replication at and hpi was additionally ensured through rt-pcr. the abundance of the cdv-n gene increased as the infection progressed ( figure c ). further validation was performed by sequencing analysis of the pcr products (data not shown). moreover, the production of nucleoprotein for the different time points analyzed was tested by anti-cdv np antibody, the result showed quite similar tendency of the viral one-step growth curve ( figure d ). the host response to ps infection at hpi was analyzed by examining differences in protein expression. based on a combination of three biological replicates from mock-infected and ps-infected samples, the itraq-coupled lc-ms/ms analysis identified and measured a total of , peptides and proteins. the proteins were designated deps based on the following criteria: a p < . and fold-change ratios ≥ . or ≤ . . among all the deps, and proteins were markedly up-regulated or down-regulated, respectively. partial deps are shown in table and more detailed information for all deps is collated in table s . to characterize the biological functions of the deps, canonical gene ontology (go) enrichment were performed using david (dennis et al., ) and uniprot databases to obtain relevant annotations about the cellular components (cc), molecular functions (mf), and biological processes (bp). first, the putative subcellular localizations of the deps were analyzed. as depicted in figure , a majority of the deps were mainly distributed in the nucleus ( . %) and cytoplasm ( . %), followed by extracellular space ( . %), mitochondria ( . %), and plasma membrane ( . %), and a smaller portion were localized in the chloroplast ( . %), lysosome ( . %), golgi ( . %), cytoskeleton ( . %), peroxidase ( . %), and er ( . %) (more detailed information is collated in table s ). interestingly, the go analysis showed that most proteins were assigned to functions involved in similar molecular functions and biological processes. as shown in figure a , most deps were closely related to binding and catalytic activity when infected by ps infection (more detailed information is provided in table s ). the bp annotation showed that deps associated with various biological processes, including cellular process, metabolic process, biological regulation, immune system process and process of response to stimulus ( figure b ) (more detailed information is provided in table s ). collectively, these categories consisted of the following proteins: ccl , irak , ube l , nfκb , nfκb , tnf-a, irak , il- , traf , apoa , tnfaip , traf , rela, and vcam (up-regulated proteins) and ccr , cxcr , smurf , nfκbib, mapk , rbm , igf , tsc , and cd (down-regulated proteins). to further investigate the pathways involving the identified deps, kegg pathway analysis was performed. according to the results, deps were mainly involved in the nf-κb and nod-like receptor (nlr) signaling pathways. in addition, several proteins could be mapped to apoptosis and specific disease associations, consisting of infectious and respiratory diseases ( figure c ) (more detailed information is shown in table s ). in the present study, we detected a total of deps involved in the immune response process. to further investigate the interaction network associated with the immune response, these proteins were imported into string software and further analyzed by cytoscape . as shown in figure , strongly interacting proteins were interestingly grouped into a functional set chiefly associated with the nf-κb signaling pathway. the interaction network provides clues for further illumination of the pathogenic mechanism and immunomodulation between cdv and the mink host. to confirm the itraq-ms data, we selected significantly changed proteins, including nfκb , rela, mhc-i, rps , and nfκbib, which reliably cross-reacted with polyclonal antibodies to the corresponding human proteins for western blotting analysis. as shown in figure a , the five representative proteins showed up-regulated or down-regulated expression in ps-infected mv. .lu cells at and hpi (the original blots are shown in figure s ), in accordance with the results of the itraq analysis ( figure b ). however, due to the limitation of the availability of antibodies to neovison vison proteins, the confirmation of deps by immunoblotting was restricted. thus, eight other proteins involved in the immune response process were selected and tested using real-time rt-pcr. as illustrated in figure c , compared to the mock group, mrna expression of traf , traf , irak , irak , nfκb , ccl , tnf-a, and il- in ps-infected cells was significantly up-regulated in a timedependent manner, which further confirmed the itraq-ms data. the activation of the nf-κb signaling pathway requires a series of cascade reactions, followed by the recruitment and phosphorylation of nf-κb protein and subsequent translocation from the cytoplasm to the nucleus, as well as the proteasome degradation of iκb proteins, which ultimately induces the production of inflammatory cytokines and type i ifn. therefore, the degradation of iκb proteins (typically represented by iκbα) and phosphorylation and nuclear accumulation of the nf-κb proteins (typically represented by nf-κb p ) are distinct features of nf-κb signaling pathway activation. the network analysis of the deps involved in the immune response has preliminarily indicated the induction of the nf-κb pathway by ps infection. to further validate this speculation, mv. .lu cells were infected with ps at moi, after incubation for or h, total proteins were collected to measure the expression of iκb-a and phosphorylated nf-κb p proteins. as shown in figure a , compared to that in mock-infected cells, phosphorylated nf-κb p (p-p ) and iκb-a proteins were obviously increased and decreased in ps-infected cells, respectively (the original blots are shown in figure s ). to assess whether ps infection facilitates nf-κb p nuclear translocation, mv. .lu cells were infected with ps at an moi of or mock infected for h. as shown in figure b , nf-κb p showed evident nuclear translocation in ps-infected cells but remained in the cytoplasm of mockinfected cells. further, to determine whether other cdv strains could activate nf-κb p , the expression of phosphorylated p and iκb-α was also detected in cdv -infected cells, which was increased and decreased, respectively ( figure a ). additionally, the nuclear translocation of nf-κb p was also observed in cdv -infected cells ( figure b ). cdv infection commonly causes a severe lethal disease in carnivores, including minks. however, the molecular mechanisms involved in viral pathogenesis and host immune responses have not been fully elucidated. to date, no research has focused on differential proteome analysis of host cells in response to cdv infection. therefore, we utilized an itraq approach to identify the deps to further explore the pathogenic mechanism and immunomodulation of cdv infection through an analysis of the effects on host cell proteins in the mink. the present study is the first to use mv. .lu cells for itraq analysis due to their ability to efficiently support cdv replication in vitro, and this cell line is homologous to the natural host system of minks. as a starting point, we determined an optimal time to perform proteomic analysis by monitoring the cpes and analyzing the one-step viral growth curve in ps-infected mv. .lu cells. the results revealed that ps infection induced serials cpe changes from to hpi, with the virus load exhibiting a plateau between and hpi. considering the high virus load was maintained at hpi and most cells showed little cpe, we conducted the following proteomic analysis based on hpi. in total, we identified up-regulated and downregulated proteins. notably, an interesting observation in the present study was that cdv infection induces nf-κb activation in mv. .lu cells. the nf-κb pathway regulates the expression of numerous immune system components to efficiently modulate the innate immune, inflammatory, and antiviral responses (bose et al., ; bours, ) and comprises a hub of cellular signal transduction pathways involved in host immune responses to viral challenge (moynagh, ) . so far, nf-κb has been reported as activated following various viral infections of porcine parvovirus , type porcine circovirus (wei et al., ) , and herpes simplex type (patel et al., ) . additionally, nf-kb activation has previously been shown in mv infection (helin et al., ) and was postulated as one of the mechanisms by which cdv might induce osteoclastogenesis (mee and sharpe, ) . moreover, nf-kb was subsequently demonstrated as induced by cdv (onderstepoort strain) infection in human osteoclast precursors (selby et al., ) ; however, these observations are all cases in humans or found in case of one single cdv strain. no reports of different cdv strains affecting nf-κb signaling in mink cells have been previously demonstrated. in the present study, nine nf-κb signaling regulators and downstream cytokines, including tnfa, irak , traf , traf , nfκb , nfκb , rela, tnfaip , and vcam , were significantly up-regulated, and the nf-κb complex inhibitory protein iκb-β was obviously down-regulated. further, kegg pathway and network analyses of the deps involved in the immune response process also indicated the induction of the nf-κb signaling pathway. these results preliminarily indicated the activation of the nf-κb pathway by ps infection in mv. .lu cells. more profound confirmation was observed by the detection of the phosphorylation and nuclear translocation of the nf-κb p subunit and the proteasome degradation of iκb-α protein in ps-infected mv. .lu cells. moreover, the activation of nf-κb p in cdv -infected mv. .lu cells also confirmed these findings. together with the previous finding that nf-κb activation was found in human cells after cdv (onderstepoort strain) challenge, these findings enriched the current knowledge of nf-κb activation by cdv infection, suggesting that nf-κb activation was not specific for a certain cdv strain or a certain species cells, but was suitable at least in part for several cdv strains and different species cells. further validation is needed to compare the ability of various cdv strains to activate nf-κb signaling in other cell lines. in addition, some deps involved in the nf-κb pathway, containing irak , rela, traf , nfκb , and tnf-a together with irak and il- , were also identified as associated with measles and respiratory diseases, such as tuberculosis and pertussis, which are similar to the respiratory symptoms of cdv infection. the causative agent of measles is mv. in dogs and ferrets, cdv causes a disease that is highly similar to measles in humans (hutchins et al., ; perry and halsey, ) . several theories have proposed that il- is a critical inducer in the development of pagetic osteoclasts and bone lesions in paget's disease induced by mv (roodman et al., ; ehrlich and roodman, ) . mice expressing il- and tnf-a in astrocytes suffer ataxia, inflammation and neurodegeneration after mv infection (akassoglou et al., ; raber et al., ) . therefore, the expression of these cytokines could contribute, in part, to mink pathological symptoms during cdv infection. furthermore, in the present study, nlr signaling pathway was closely associated with ps infection. this innate immunity signaling pathway may play essential roles in the production of type i interferon and in promoting inflammasome assembly upon virus activation (kobayashi et al., ; sabbah et al., ) . recent studies have suggested that the inflammasome nlrp , known as the nod-like-receptor-family, pyrin domaincontaining , recognizes several rna viruses, such as influenza virus (allen et al., ; ichinohe et al., ) , vsv (rajan et al., ) , and emcv (poeck et al., ) . mv also activates the nlrp inflammasome, resulting in the caspase- -mediated maturation of il- β (zilliox et al., ; komune et al., ) . the nf-κb-induced activation of nlrp and pro-il- β gene expression is requisite for activating caspase- by the nlrp inflammasome to further regulate the secretion of the inflammatory cytokines il- β and il- (motta et al., ) . however, whether there is signaling crosstalk between nf-κb activation and the nlr signaling pathway during cdv infection is an open question. collectively, the findings suggested that activation of the innate immune nf-κb signaling pathway and the nlr signaling pathway was involved in mink immune responses against cdv infection, and the nf-κb signaling was associated with the pathological respiratory or other symptoms figure | confirmation of the itraq-ms data by western blotting or real-time rt-pcr. (a) western blot analysis of nf-κb , rela, mhc-i, rps , and nfκbib in ps-infected and control samples at and hpi. gapdh was served as internal reference. (b) the intensity ratio of the corresponding bands (infection/mock) was quantified using imagej software and normalized against gapdh. (c) eight selected differently expression proteins related to nf-κb pathway were testified using real-time rt-pcr method. each gene was performed in three independent experiments. the relative gene expression was calculated using -ct model, representative of n-fold changes in comparison with mock-infected samples. error bars represent the standard error for triplicate samples. *p < . ; **p < . ; ***p < . . the data was analyzed by two-way anova followed by duncan's test. in mink after cdv infection. further research may answer these questions. cdv infection could cause gastrointestinal symptoms or severe diarrhea after secondary infection. the nherf, na + /h + exchanger regulatory factor, commonly locates or becomes enclosed in the intestinal brush border, thereby binding to the renal proximal tubule brush border na+/h+ exchanger nhe protein, which is mainly responsible for the absorption of electroneutral salt in the intestine and is the most essential sodium absorptive transporter (donowitz et al., ) . therefore, nherf plays a crucial part in establishing and maintaining the functional integrity of the intestinal barrier. previous reports have demonstrated that nherf down-regulation leads to reduced na + absorption though affecting nhe activity, ultimately increasing intestinal epithelial permeability and the risk of inflammatory bowel disease (ibd) (sartor, ; strober et al., ) . butler et al. discovered that the dysregulation of sodium transit contributed to piglet diarrhea and the pathogenicity of tgev after infection (butler et al., ) . in the present study, nherf is significantly down-regulated, consistent with a previous observation of the significant downregulation of nherf (a member of nherf family) protein in lu cells were infected with moi ps, cdv or mock-infected. at hpi, the cells were fixed and incubated with rabbit polyclonal antibody specific to mink nf-κb p and mouse monoclonal antibody specific to cdv n protein, then incubated with fitc-labeled goat anti rabbit igg and cy -labeled goat anti mouse igg, respectively. cell nuclei were stained by dapi. the fluorescent images were analyzed under a confocal microscopy (leica, germany). tgev-infected pk- cells using quantitative proteomic analysis . accordingly, the observation suggested that the down-regulation of nherf by ps infection induced disordered salt and water transit through nhe dysfunction and further leaded to in the malfunction of the sodium pump in the intestinal barrier, ultimately resulting in gastrointestinal symptoms or severe diarrhea in infected minks. the present study provides a new view of the pathogenesis of diarrhea in cdv-infected minks. ubiquitination, the covalent conjunction of ubiquitin to the target protein substrate, is the first of two successive steps associated with ubiquitin-proteasome pathway, which is responsible for a wide variety of cellular functions, including the activation of nf-κb signaling and type i ifn pathways (ciechanover, ; glickman and ciechanover, ) . accumulated evidence has suggested that various viruses have evolved complicated mechanisms to exploit or manipulate the ubiquitin-proteasome pathway (gao and luo, ) . for example, the activation of the ubiquitin-proteasome pathway is required for influenza virus replication (widjaja et al., ) and is also required other viruses, such as rotavirus (lopez et al., ) , human cytomegalovirus (tran et al., ) , and porcine reproductive and respiratory syndrome virus (zhou et al., ) . the present study identified traf , traf , ube l (e ubiquitin isg -conjugating enzyme), usp (an isg specific isopeptidase enzyme) and trim (e ubiquitin-ligase) as up-regulated proteins involved in protein ubiquitination. traf and traf are well-recognized as signal transducers in the nf-κb signaling pathway that function together with a dimeric ubiquitin-conjugating enzyme complex to catalyze the synthesis of k -linked polyubiquitin chains and ultimately activate iκb kinase (ikk) and the downstream nf-κb pathway (deng et al., ; yang et al., ) . as an ifn-induced ubiquitin-like protein, isg plays a role in immunomodulation and imparting a direct antiviral activity against a wide spectrum of virus (pincetic et al., ; dai et al., ; sooryanarain et al., ) . although the present study failed to detect the isg protein, we identified the significantly up-regulated proteins ube l and usp , which are strongly related to the isgylation of isg . similar to the mechanism of ubiquitination, isgylation involves the sequential co-operation of e , e , e and an isg -specific isopeptidase enzyme (here identified as usp ) to facilitate isg combination with target proteins for the execution of antiviral responses (kroeker et al., ; falvey et al., ) . the tripartite-motif family (trim) of proteins plays essential roles in the innate immune responses to antimicrobial infections. trim , a member of the trim family and previously known as transcriptional intermediary factor gamma (tif -γ), functions in monocyte/macrophage mediated inflammation (gallouet et al., ) and inflammasome activation (weng et al., ) . our results provided the first evidence of multiple differentially up-regulated immune-related proteins associated with protein ubiquitination in response to ps infection in mv. .lu cells, indicating that ubiquitination appeared to be a pivotal regulatory mechanism in the immune responses to cdv infection in mink. apoptosis plays a role in regulating the pathogenesis of various infectious diseases, which oppositely affect viral pathogenesis by either restraining viral transmission or accelerating viral propagation by the release of the virus particles (pastorino et al., ). in the present study, seven up-regulated proteins, including tnf-a, rela, nfκb , traf , a-tubulin, ctsk (cathepsin k), and ctsv (cathepsin v), were identified as apoptosis-related, suggesting the induction of apoptosis in ps infection in mv. .lu cells. ctsk and ctsv are associated with a mitochondria-dependent intrinsic pathway to trigger the apoptosis of host cells, while tnf-a participates in an extrinsic receptor-mediated pathway (benedict et al., ) . this finding was consistent with previous reports showing that cdv induces apoptosis in the cerebellum and lymphoid tissues of the natural infection of dogs and in vero cells in vitro (moro et al., ; del puerto et al., . the mechanisms of apoptosis in the pathogenesis of cdv have not yet been clearly illuminated, and the extensive study of these proteins should enhance the current understanding of the mechanisms underlying apoptosis regulation during cdv infection. in summary, the present study provides the first overview of the protein alterations in cdv-infected mv. .lu cells using itraq analysis. the identification of differently expressed proteins reflects a comprehensive interaction network of mv. .lu cells and cdv during infection. although some significantly regulated proteins were suggested to be related to the pathological symptoms and the immune responses to cdv infection, further functional elucidations are needed to clarify the pathogenic mechanisms and the immune responses to additionally identify new therapeutic targets for preventing cdv infection. mt and sc designed the study; mt, ly, ns, and yc performed the experiments; zc and jw analyzed the data; sl, pl, and ys prepared the figures and tables; mt wrote the manuscript. this study was supported by agricultural science and technology innovation project (no. ny) and jilin provincial science and technology development project (no. jh). astrocytespecific but not neuron-specific transmembrane tnf triggers inflammation and degeneration in the central nervous system of transgenic mice the nlrp inflammasome mediates in vivo innate immunity to influenza a virus through recognition of viral rna quantitative proteomic analysis reveals that transmissible gastroenteritis virus activates the jak-stat signaling pathway lymphocyte-mediated immune 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https://www.frontiersin.org/articles/ . /fmicb. . /full#supplementary-material key: cord- -ow aez v authors: ismail, ashrafali m.; lee, ji sun; lee, jeong yoon; singh, gurdeep; dyer, david w.; seto, donald; chodosh, james; rajaiya, jaya title: adenoviromics: mining the human adenovirus species d genome date: - - journal: front microbiol doi: . /fmicb. . sha: doc_id: cord_uid: ow aez v human adenovirus (hadv) infections cause disease world-wide. whole genome sequencing has now distinguished distinct genotypes in species (a-g). over half of these hadvs fall within species d, with essentially all of the hadv-d whole genome sequences generated in the last decade. herein, we describe recent new findings made possible by mining of this expanded genome database, and propose future directions to elucidate new functional elements and new functions for previously known viral components. human adenovirus (hadv) infections represent a significant source of morbidity and mortality, world-wide and at all ages, through highly transmittable infections at mucosal sites, including the eye, and urinary, respiratory, and gastrointestinal tracts (horwitz, ) . hadv causes fatal acute respiratory distress syndrome in healthy adults and is especially lethal in infants and the immune compromised (bhanthumkosol, ; ryu et al., ; wallot et al., ; engelmann et al., ; tan et al., ; zhang et al., ) . no fda-approved therapy for acute hadv infection is available. at resolution of acute infection, persistence may develop within nasopharyngeal lymphoid tissue (neumann et al., ; garnett et al., garnett et al., , zhang et al., ; assadian et al., ) , as yet uncharacterized cells in the gastrointestinal tract (roy et al., ) , and possibly the ocular surface (kaye et al., ) , permitting evolution of new hadvs through homologous recombination between two or more hadvs infecting the same cell(s) (lee et al., (lee et al., , echavarria et al., ; mccarthy et al., ; seto et al., ) . hadvs are divided phylogenetically into seven species (a-g), with a total of recognized genotypes with whole genome sequences in genbank, including the original "serotypes"-determined by serum neutralization-which now all have been fully sequenced ( table ) (robinson et al., a) human adenovirus species d (hadv-d) is the largest and most rapidly growing among all hadv species, and contains viruses associated with epidemic keratoconjunctivitis (ekc), a severe, hyperacute ocular surface infection (butt and chodosh, ) . a collaboration funded by the american recovery and reinvestment act of came to fruition with the complete whole genome sequencing and analysis of all previously unsequenced hadv-d serotypes (robinson et al., a) , leading to a new understanding of adenovirus ontogeny (jones et al., ; robinson et al., robinson et al., , a robinson et al., b,c; robinson et al., a,b; walsh et al., walsh et al., , a arnold et al., ; torres et al., ; dehghan et al., dehghan et al., , a walsh et al., ; liu et al., ; seto et al., seto et al., , singh et al., singh et al., , zhou et al., )-including those hadv-ds associated with ekc (robinson et al., (robinson et al., , b (robinson et al., , b walsh et al., ; zhou et al., ) -and ultimately to a new typing system for hadv based on genomics . recent published work demonstrates how genome "mining, " in-depth analyses of the growing hadv genome database, can bring about new realizations and add critical new information to prior ones. the trimeric fiber protein on adenoviruses mediates viral entry through interaction of the distal most "knob" structure on the fiber with host cell receptors. in a phylogenetic analysis of hadv-d fiber genes, hadv-d types associated with ekc were recently shown to form a unique clade (ismail et al., ) . by proteotyping, a new in silico methodology described in detail below, ekc virus-associated fiber knobs were uniquely shared, and signature amino acid positions distinguished ekc from non-ekc types. remarkably, human corneal epithelial cell tropism could be predicted by the presence of a lysine or alanine at residue , and this amino acid residue in ekc viruses showed evidence for positive selection. these data added to the prior observation by huang and coworkers that artificial mutation to a lysine at residue in a non-ekc virus could confer infection of chang cells, a conjunctiva derived continuous cell line (huang et al., ) . however, because chang cells came later known to be contaminated by hela cells, the importance of residue to ocular tropism was until this new observation, in some doubt. another recently published effort provided further evidence of the importance and potential for hadv genome mining. late adenoviral gene expression is initiated by the adenovirus major late promoter (ramke et al., ) , followed by splicing of mrnas to the viral tripartite leader for translation (chow et al., ; akusjärvi and pettersson, ; chow and broker, ; logan and shenk, ) . the hadv tripartite leader is a nucleotide ' noncoding region that circumvents the requirement for eukaryotic initiation factor f or cap binding protein complex (ziff and evans, ; akusjärvi and pettersson, ; dolph et al., ; zhang et al., ) , and permits translation of hadv mrnas at late times in infection when cap-dependent translation is blocked due to shut down of host cellular capdependent mrna translation. hadv ′ untranslated regions ( ′ utrs) are critical for cap-independent initiation, and impact mrna localization and stability. the hadv tripartite leader (tpl), composed of three introns (tpl - ), drives translation of hadv late mrna. the annotation of hadv genotypes for the hadv tpl and another previously described leader, the i-leader, let to identification of newly identified polycistronic mrnas for rid-α and rid-β within the e transcription unit, and a potential new open reading frame (orf) within the i-leader sequence, with termination of this potential protein in tpl (ramke et al., ) . in addition, the authors also identified a potential new leader sequence embedded within the e region, tentatively named the j-leader (figure ). the hadv is non-enveloped, icosahedral in shape, and contains a double stranded dna genome of ∼ , base pairs (bp) frontiers in microbiology | www.frontiersin.org figure | putative "j"-leader located within the cr -α e gene. (a) schematic for the location of a newly detected leader ("j"-leader) embedded within the e cri-α gene, experimentally determined to be spliced to some, but not all mrnas of the e genes. (b) gel photomicrograph of mrna transcripts amplified with forward primer from tpl and reverse primers from cr -γ, cr -β, and rid-α. primers were chosen to elicit similarly sized bands to facilitate subsequent sequencing. (c) nucleotide sequence of the pcr product for cr -β. the putative j-leader sequence and splice sites are shown in yellow and green, respectively. note an additional nucleotide ′ utr (aacc) prior to the cr -β start site (red). the ′ utr in (c) prior to the splice site for the j-leader is from tpl . adapted from ramke et al. ( ) with permission. with ∼ open reading frame (orf) for every nucleotides. viral dna is associated with four (interior) core proteins including mu, vii, v, and terminal protein. the histonelike protein (p) vii protects viral dna from cellular dna damage responses (lischwe and sung, ; karen and hearing, ; avgousti et al., ) . the outer protein coat (capsid) of the virus consists of hexon capsomers and penton capsomers, along with several minor capsid proteins. the latter include pvi, piiia, pviii, and pix and are important to capsid stability. each penton capsomer contains a ring of five penton base proteins which bind and support the trimeric fiber protein with its distal fiber knob. during viral infection, the fiber knob binds to one of several host cell receptors (nemerow, ; goosney and nemerow, ; nemerow et al., ). the penton base protein contains two hypervariable loops. the interaction between fiber knob and a host cell receptor brings about secondary contact between the hypervariable loop (hvl ) arginine-glycineaspartic acid (rgd) motif in each penton base protein (five per penton base capsomer) with host cell integrins α v β , α v β , and α v β , that in turn induce endocytosis of the virus (li et al., a,b; li et al., ) . hadv structural proteins can serve multiple functions. for example, the minor capsid structural protein vi (pvi) plays a critical role in at least three distinct aspects of the viral "life" cycle: endosomal escape during cell entry, nuclear assembly during viral replication, and stability of the intact, infectious virus outside the host (wodrich et al., ; wiethoff et al., ; moyer et al., moyer et al., , . these findings suggest that, as with pvi, other hadv structural proteins may have multiple functions yet to be elucidated. the relatively large genome database for hadv-d (over unique viruses with available whole genome sequences) (tables , ) has permitted detailed analyses of genome relationships within this clinically important adenovirus species. hadv-d genomes are highly conserved (> %). however, whole genome analyses of hadv-d have revealed specific loci of genetic hypervariability in the hexon, penton base, fiber, and e cr α, β, and γ genes (figure ), dictating nonsynonymous amino acid changes in corresponding proteins (figure ). gc content confers genome stability and resistance to recombination (gruss et al., ) , and the genomes of hadv-d have among the highest gc content among hadv species (∼ %). the hypervariable regions in hadv-d were found to be sharply reduced in gc nucleotide content relative to the rest of the genome (robinson et al., a) . mutations in hadv are relatively infrequent, with genome stability now documented in some types across decades (hofmayer et al., ; mahadevan et al., ; seto et al., ; dehghan et al., b; robinson et al., a; alkhalaf et al., ) . however, those regions of the genome shown to be hypervariable and relatively low in gc content are the very same also shown to undergo homologous recombination (robinson et al., a (robinson et al., , b walsh et al., ; zhou et al., ; singh et al., ) , driving the evolution of new genotypes. adenoviruses recombine specifically during viral replication (williams et al., ; meinschad and winnacker, ; munz et al., ) , and do so by both homologous and heterologous mechanisms (young et al., ; epstein and young, ; crawford-miksza and schnurr, ) . however, the evidence for homologous recombination as the major mechanism driving hadv-d evolution is unassailable (robinson et al., a; singh et al., ) . specifically, recombination occurs in the two penton base hypervariable regions (these code for two hypervariable loops (hvls) on the penton base protein, separated from one another by ∼ conserved amino acids), seven hexon hypervariable regions (these are closely adjacent in the hexon gene and determine two adjacent hvls on the hexon protein), fiber (fiber gene and protein are entirely hypervariable), and e cr α, β, and γ (each also entirely hypervariable). for homologous recombination between two hadvs to occur, at least two virus types with high nucleotide sequence homology at corresponding locations in both genomes must co-infect the same cell, and viral dna replication should be ongoing. coinfection by two or more hadvs has been well documented (lee et al., ; echavarria et al., ; vora et al., ; mccarthy et al., ; halstead et al., ; seto et al., ) , as has the presence of two hadv types in archived clinical samples (singh et al., ) . "proteotyping" is a novel approach to the study of genome evolution (obenauer et al., ) , and has been applied to characterize recombination among hadv-d (robinson et al., a; singh et al., ) .in this method, maximum likelihood trees are used to align amino acid sequences of hypervariable, frequently recombined proteins. each amino acid is assigned a unique, arbitrary color. consensus residues are colored white, and gaps in the alignment are colored black. a threshold of < % sequence divergence is used to distinguish unique proteotypes. an example of proteotyping is shown (figure ) , comparing an amino acid alignment from e . k, a highly conserved gene with one distinct proteotype, with the hypervariable e cr α (singh et al., ) , with six distinct proteotypes observed among hadv-ds. e . k is therefore not hypervariable and not recombinant. e cr α is hypervariable and recombinant. another way to interpret the analyses for those proteins like e cr α, with more than one proteotype is that those proteotypes containing more than one hadv type have previously recombined in nature, while those proteotypes with only one hadv type are those that have not (yet) been shown to recombine in nature. hadv-d and fall within different hexon proteotypes (figure ) . hadv-d shares a hexon proteotype with hadv-d and (robinson et al., a) , while hadv-d shares a hexon proteotype with hadv-d , , and (singh et al., ) . these two hexon proteotypes therefore have undergone prior homologous recombination. in contrast, the hexon proteins of hadv-d and are each in a proteotype with only one member; hexon recombination for these two viruses has therefore not yet been documented in nature. in sum, these data show by independent means that homologous recombination within hadv-d is common, and confirm previously recognized patterns of homologous recombination among hadv-d (robinson et al., a (robinson et al., , b (robinson et al., , a walsh et al., walsh et al., , a singh et al., singh et al., , zhou et al., ; gonzalez et al., ) . the local sequence and/or structure of dna in regions flanking recombinogenic sites is significant for directing cellular recombination machinery to those regions. in bacteria, a signal for recombination between homologous dna is the crossover hotspot instigator, or chi nucleotide sequence. this was first discovered in bacteriophage lambda, then in bacterial dna, and later shown to mediate recombination between them (stahl, ) . the chi sequence in e. coli (chi ec ) is ′ -gctggtgg- ′ (smith et al., ; bianco and kowalczykowski, ) , and its presence induces the conversion of the recbcd enzyme from a helicase to an exonuclease, producing ssdna that can invade homologous dsdna during recombination (taylor et al., ) . the reca enzyme of e. coli is loaded onto unwound ssdna by recbcd and promotes ssdna exchange/recombination with homologous dsdna (cox, ; smith, ) . reca has significant homology to eukaryotic rad and its paralogs (suwaki et al., ) , enzymes that repair dsdna breaks in human cells, facilitate homologous recombination, and during adenovirus infection, bind to the e dna binding protein (tookman et al., ) . in our study of the region just ′ to hvl on the penton base gene, a recombination hot-spot for hadv-d (robinson et al., a) , we found chi-like sequences (chi ad ), e.g., ′ -acttctga- ′ in the proteotype containing hadv-d , and ′ -tctcctga- ′ in the proteotype including hadv-d (lee et al., ) . the putative chi ad sequences we identified in hadv-d were found within the gc-rich component of gc/at transition zones that precede and include hvl , and were conserved within each proteotype. in vitro, e. coli lysates containing reca protein increased recombination of two hadv-d genotypes with the same penton base hvl proteotype. reca was shown by chip to bind specifically to chi ad nucleotide sequence in the same regions, and also colocalize with adenovirus dna within infected cell nuclei. these data suggest that chi-like nucleotide sequences adjacent to the junction of conserved and hypervariable gene segments in hadv-d may be an important signal for homologous recombination, and provide evidence in support of the idea that local bacterial flora might enhance natural recombination through chi-like nucleotide sequences at hadv-d recombination hotspots. another explanation for homologous recombination between hadv, not exclusive of a role for chi ad , is the potential for gclow (at-rich) single stranded dna (ssdna) to form hairpin loops (nagy and bujarski, ; ohshima et al., ) , a physical nonlinearity that would facilitate binding of ssdna of one hadv-d type to a homologous segment of ss or dsdna from a physically adjacent but different hadv-d type during coinfection of the same cell. hairpin loops and other alterations in the physical configuration of ssdna during dna replication might also contribute to polymerase jumping (jennings et al., ; spaan et al., ; pääbo et al., ; viswanathan et al., ) , in which physical constraints to polymerization lead to translocation of the dna polymerase to an adjacent dna from a different virus, resulting in a recombined dna. polymerase jumping has been shown to occur during hadv dna replication (king et al., ; de jong et al., ) , although it has not been suggested previously as a mechanism for hadv-d evolution. analysis of hadv-d whole genome sequences identified instances of nucleotide-long gc-rich sequence adjacent to nucleotide-long at-rich sequence (sometimes with a or nucleotide-long gc-moderate sequence intervening), located just ′ and ′ to frequently recombined gene segments, and which were shown by in silico analysis of their corresponding ssdna to form hairpin loops (robinson et al., a) . taken together, these data suggest covariant effects of nucleotide sequence and ssdna secondary structures on homologous recombination between two hadv-ds. regions of the hadv-d genome currently thought to be "noncoding, " may contain functional elements. because viruses exist on the nano-scale, viral genomes are by necessity constrained by size, and "junk" nucleotide sequences represent an extravagance. the national human genome research institute project to identify functional elements in the human genome (encyclopedia of dna elements, or encode) identified functionality in much of the human genome previously without known utility (consortium et al., ; qu and fang, ; kellis et al., ) the double-stranded dna genomes of hadv also contain regions with no known function. transcriptional profiling of host gene expression has been studied after hadv infection (dorer et al., ) however, although viral transcriptomes have been reported for several viruses, most notably dengue, varicella zoster, and epstein-barr viruses (ortmann et al., ; ertl et al., ; nagel et al., nagel et al., , arvey et al., ; sujayanont et al., ) , a de novo hadv transcriptome has not been reported. wu and coworkers used deep rna sequencing to confirm known bat adv transcripts (wu et al., ), but did not investigate "noncoding" regions. in silico orf prediction in hadv can be difficult due to splice variants and inconsistencies in banked gene annotations (davison et al., ) , but in a prior annotation of hadv-d , ∼ new additional orfs were predicted using in combination, the ncbi orf finder, tigr annotation engine, and genemark heuristic model (robinson et al., ) putative genes were found within the large regions of noncoding dna on the complementary strand opposite to established hadv genes figure | proteotyping analysis comparing the hadv-d e . k (a) and cr α (b) proteins. the . k protein was conserved, while cr α demonstrated unique proteotypes. maximum likelihood phylogenetic trees are shown to the left for each putative protein, and amino acid signatures to the right. the scale bar at the bottom left of each sub-figure denotes the phylogenetic distance reflected in horizontal dimension of the respective tree. to construct the amino acid signatures shown, each amino acid was assigned a unique color (upper right corner), consensus amino acids at each position across all viruses were assigned white, and gaps in the alignment were colored black. horizontal red lines delineate distinct proteotypes. adapted from. singh et al. ( ) with permission. (figure ) , in smaller regions on the coding strand within established transcription units but between confirmed genes, and overlapping or completely within established genes. work is in progress in our laboratories to identify putative new genomic elements in hadv by high-throughput sequencing of the viral transcriptome of hadv-d . hadv uses host tfs nuclear factor i and iii (nf-i and nf-iii) as part of the viral dna replication complex (pruijn et al., ; mul et al., ; hearing, , ) simian advs typically lack the nf-i binding site, while human viruses express it. it was previously reported that hadv-e , originally isolated in , is a product of recombination between hadv-b and the simian adv, sadv-e . clinical strains of hadv-e isolated recently contain a nf-i binding site in the inverted terminal repeat (houng et al., ; dehghan et al., a,b) that is absent in the original isolate (purkayastha et al., ) , suggesting that nf-i binding may be important to viral fitness in humans. to further elucidate mechanisms of viral gene expression, we are exploring novel tf binding sites on hadv-d dna, using encode validated methodologies (gerstein et al., ; landt et al., ) . the major hadv capsid structural proteins-hexon, penton base, and fiber-interact directly with extracellular mediators of host immunity (gahéry-ségard et al., ; molinier-frenkel figure | proteotyping for hadv-ds, sorted for the hexon proteotype column. numbers and colors are arbitrary, and distinguish distinct proteotypes. recombinants can be identified by rows. for example, hadv-d , -d , and -d fall within the same proteotype and are predicted to share highly similar nucleotide sequences for their respective hexon hypervariable regions (as confirmed by singh et al., ) . for hadv-d and -d , the recombination event extended through the e cr β orf gene and then ended. figure | transcription map for hadv-d . genes are divided by early (shaded) or late expression. red brackets denote large areas of "noncoding" dna, but many additional, smaller, potential coding regions exist between and within known genes. adapted from robinson et al. ( ) with permission. frontiers in microbiology | www.frontiersin.org september | volume | article figure | comparison of e transcription unit from hadv-c and -d. note in particular the difference in orf size between cr β of the two hadv species. adapted from robinson et al. ( c robinson et al. ( ) with permission. et al., schoggins and falck-pedersen, ; tamanini et al., ; kalyuzhniy et al., ; chintakuntlawar et al., ; bradshaw et al., ; flatt et al., ) . the hexon, penton base, and fiber proteins also exhibit distinct amino acid signatures, characterizing discrete proteotypes (robinson et al., a) . gene products from the e transcription unit of hadv function in viral immune evasion (horwitz, ; lichtenstein et al., b; windheim et al., ) . in hadv-d, the open reading frames for three of eight e genes-cr α, cr β, and cr γ-are uniquely hypervariable compared to the other orfs within the e transcription unit, and also segregate into discrete proteotypes (singh et al., ) . highly conserved genes, such as dna binding protein, dna polymerase, and e . k, show no such variability (robinson et al., a; singh et al., ) . while it may be assumed that hypervariablity in major capsid and e proteins is driven through evolutionary selection by the extracellular interactome, amino acid differences in a hypervariable protein can also lead to differences in that protein's intracellular interactome, the set of intrinsic host cell proteins which network with the viral protein, as was recently confirmed for e cr genes across hadv species (martinez-martin et al., ) . viral capsid structural proteins are critical to virion stability. for the nonenveloped hadv, fiber and penton base proteins on the external surface of the capsid serve as ligands for attachment to the host cell (huang et al., ) and initiate viral entry (wickham et al., ) , respectively. hadvs are typically internalized via endosomes. endosomal acidification leads to structural instability of the capsid and endosomal release into the cytosol. hadv capsid is then transported by microtubules to the nuclear membrane. viral dna then enters the nucleus through nuclear pores, leaving almost all the viral structural proteins in the cytosol (henaff et al., ) . viral capsid proteins within the cell are eventually targeted for ubiquitination (ko et al., ; marvin and wiethoff, ; horan et al., ; li et al., ) and degraded (greber et al., ) , but there are many opportunities for interaction with intracellular host cellular proteins during entry, trafficking, translation, assembly, and egress. penton base hvl , with its rgd motif, is critical to viral internalization through the interaction with host cell integrins,; (wickham et al., ) but function of penton base hvl is unknown, and might be revealed though knowledge of its protein interactome. the closely adjacent hexon hvl and form the epsilon epitope that determines serum neutralization, and interactions between the hexon protein and serum coagulation factor x confers liver tropism to hadv-c (sumarheni et al., ) . however, nothing is known about potential hexon interactions with intracellular proteins during infection. the e transcription unit of hadv codes for proteins that mediate immune evasion by the virus (horwitz, ) . although e is labeled as an early transcription region, its transcripts are expressed both early and late during viral infection (chow et al., ; chow and broker, ; bhat and wold, ) , and there is evidence for at least one e protein that late transcripts are translated (robinson et al., a) . e gene products are not required for viral replication in cultured cells (morin et al., ) , but inhibit cellular and cytokine mediated host immune responses to infection (horwitz, ; lichtenstein et al., b; windheim et al., ) . almost all of what is known about the function of specific e proteins derives from studies on hadv-c. for example, hadv-c e cr α directs another e protein ( k) to the endoplasmic reticulum of cytotoxic t cells (wilson-rawls et al., ) , where k binds to and retains mhc class i proteins (jefferies and burgert, ) , preventing presentation of viral peptides within mhc class i at the cell surface kvist, , ; andersson et al., ; cox et al., ) . cr α, ridα, and ridβ proteins cooperate to evade tnfα-related apoptosis through trail (elsing and burgert, ; tollefson et al., ; benedict et al., ; lichtenstein et al., a) . cr β (wold et al., ) , also called the adenovirus death protein (tollefson et al., ) , is required for cell lysis (tollefson et al., ) and viral spread (doronin et al., ) . the orf size of each e gene varies across hadv species (figure ) (robinson et al., c) . similarly, immune evasion functions of e gene products may not be the same across hadv species, or function similarly in all cell types (routes and cook, ). windheim and coworkers recently showed that the cr β protein of the eye pathogen hadv-d suppresses natural killer cell function (windheim et al., ) . the e cr genes are uniquely hypervariable within hadv-d, and as predicted, overlapping but distinguishable intracellular interactomes across proteotypes were recently reported by martinez-martin and colleagues, who used protein microarrays to identify novel cr β binding partners (martinez-martin et al., ) . hadv was critical to the dual discoveries of viral oncogenesis and rna splicing (berget et al., ; chow et al., ; whyte et al., ) . hadv is also a significant agent of disease for which there is no approved treatment. recent mining of hadv genomes has been highly productive, and there is ample evidence to suggest that further whole genome analyses will elucidate new and fundamental mechanisms in hadv biology. in the last decade, of newly identified hadvs, were hadv-ds, suggesting the continuing evolution of new pathogens from species d. analyses of fully sequenced hadv-d whole genomes identified homologous recombination of specific regions within the hexon, penton base, fiber, and e cr genes as the major mechanism behind hadv-d evolution, a new finding (robinson et al., a; singh et al., ) . stereotypical reductions in gc content at the junction of conserved and hypervariable regions, along with chi-like sequence motifs (also a new finding), appear likely to augment the intrinsic tendency of hadv to undergo homologous recombination in vivo (lee et al., ) . recently, the whole genome sequences of hadvs from archives and current collections were determined, including both historical and circulating strains, respectively . of these, novel recombinants within hadv-b and within hadv-d were identified. only two of the hadv-ds were found to contain novel genes (penton base and fiber); these were subsequently typed as hadv-d and . isolates of hadv-d and hadv-d , two novel genotypes recently recognized, were also identified, adding confidence in their clinical importance. fully genotyped hadvs now number , with more awaiting type numbers, and the scientific community has a -fold larger database of unique hadv genomes than available only years ago. published and validated encode methodologies can now be applied, and comparisons made across disparate hadv genomes. we suggest that the hadv genome contains previously uncharacterized functional elements, and that every hadv protein has 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assembly by cleavage of nuclear transport signals mapping a new gene that encodes an , -molecular-weight protein in the e transcription unit of adenovirus deep rna sequencing reveals complex transcriptional landscape of a bat adenovirus replication and recombination in adenovirus-infected cells are temporally and functionally related fatal pneumonia cases caused by human adenovirus in immunocompetent adults secondary structure analysis of adenovirus tripartite leader modeling adenovirus latency in human lymphocyte cell lines analysis of human adenovirus type associated with epidemic keratoconjunctivitis and its reclassification as adenovirus type coincidence of the promoter and capped ' terminus of rna from the adenovirus major late transcription unit key: cord- -e xwb g authors: yamashita, akifumi; sakamoto, tetsuya; sekizuka, tsuyoshi; kato, kengo; takasaki, tomohiko; kuroda, makoto title: dgv: dengue genographic viewer date: - - journal: front microbiol doi: . /fmicb. . sha: doc_id: cord_uid: e xwb g dengue viruses (denvs) and their vectors are widely distributed throughout the tropical and subtropical regions of the world. an autochthonous case of denv was reported in tokyo, japan, in , for the first time in years. a comprehensive database of denv sequences containing both serotype and genotype data and epidemiological data is crucial to trace denv outbreak isolates and promptly respond to outbreaks. we constructed a denv database containing the serotype, genotype, year and country/region of collection by collecting all publically available denv sequence information from the national center for biotechnology information (ncbi) and assigning genotype information. we also implemented the web service dengue genographic viewer (dgv), which shows the geographical distribution of each denv genotype in a user-specified time span. dgv also assigns the serotype and genotype to a user-specified sequence by performing a homology search against the curated denv database, and shows its homologous sequences with the geographical position and year of collection. dgv also shows the distribution of denv-infected entrants to japan by plotting epidemiological data from the infectious agents surveillance report (iasr), japan. this overview of the denv genotype distribution may aid in planning for the control of denv infections. dgv is freely available online at: (https://gph.niid.go.jp/geograph/dengue/content/genomemap). dengue viruses (denvs) are members of the genus flavivirus in the family flaviviridae and consist of four serotypes (denv- to - ) (lanciotti et al., ; kuhn et al., ) . each serotype can be divided into five to six genotypes. however, there is no standard genotyping classification [denv- , (goncalvez et al., ) ; denv- , (anez et al., ; khan et al., ) ; denv- , (lanciotti et al., ; wittke et al., ; klungthong et al., ) ; denv- , (abubakar et al., ) ]. denv has a positive-sense, single-stranded rna genome that is ∼ kb in length and encodes a capsid protein (c), premembrane protein (prm), and envelope glycoprotein (e) in addition to seven non-structural proteins (nss, ns , ns a, ns b, ns , ns a, ns b, and ns ) . denv infection causes dengue illness, which may range from dengue fever (a mild illness) to dengue hemorrhagic fever and dengue shock syndrome (the severe forms of the illness) in addition to asymptomatic cases abbreviations: denv, dengue virus; c, capsid protein; prm, premembrane protein; e, envelope glycoprotein; ns, nonstructural protein; ncbi, national center for biotechnology information; iasr, infectious agents surveillance report. (centers for disease control and prevention; http://www. cdc.gov/dengue/clinicallab/clinical.html). infection results in lifetime immunity against the same serotype, but successive exposure to different denvs increases the likelihood of contracting a severe form of dengue illness, such as dengue hemorrhagic fever or dengue shock syndrome (chiappelli et al., ) . denv and its vectors have become widely distributed throughout the tropical and subtropical regions of the world (murray et al., ). an autochthonous case of denv infection was reported in tokyo, japan, in for the first time in years (kutsuna et al., ) . to ensure prompt action in response to a denv outbreak, a comprehensive denv database based on the genotypes would be essential for tracing the outbreak source. to date, only two denv databases provide genotype information. the web service vipr (pickett et al., ) supports genetic analysis based on the viral genome for a tested input sequence, including denv sequences. the second database is the dengue virus genotyping database (yamashita et al., ) , which provides a summary table containing the denv serotype/genotype, year and country of collection and accession number. there are many other denv databases; however, no other sites provide summarized genotype information. the dengue virus resource facilitates the retrieval of denv sequences deposited in genbank according to serotype, disease symptom, host, region/country, genome region, and collection and/or release data (resch et al., ) . denvirdb provides sequence information and computationally curated information of dengue viral proteins (asnet et al., ) . denvdb focuses on the dengue virus sequence database for keyword searches (no publication: http://proline.bic.nus.edu.sg/denvdb/). finally, the dengue virus portal is a sequence collection with metadata (no publication: https://www.broadinstitute.org/ annotation/viral/dengue/home.html). here, we constructed the website dengue genographic viewer (dgv), which presents denv information based on the genotype and epidemiological data by using the geographic tool google maps © to update the recent dissemination of denv genotypes from a global perspective. the denv genotype database was constructed as follows: ( ) all accessible denv nucleotide sequences were collected; ( ) the complete sequences of each protein region (c, prm, e, ns , ns a, ns b, ns , ns a, ns b, and ns ) were extracted from the sequences; ( ) a blastn homology search was performed against the genotype database and the genotype of the most homologous sequence was assigned; ( ) and the genotype data were stored in a database using sqlite (https://www.sqlite.org/). . the denv nucleotide sequences were downloaded from the ncbi database using key words ("dengue virus"[porgn:__txid ]). . a blastx homology search was performed to detect nucleotide regions that corresponded to each mature denv protein; nucleotide regions that exhibited more than % sequence coverage to the protein were used for the subsequent analysis. . to reduce the time required to obtain the most homologous sequences in the genotype database, we reduced the number of sequences used in the blast search by clustering highly homologous sequences. we performed a uclust search (edgar, ) against the nucleotide sequences of each protein region and selected one representative sequence for each homologous sequence group with a clustering threshold of % identity; then, the representative sequences were subjected to a homology search against the genotype database. the original genotype database was constructed according to the method proposed by previous report (yamashita et al., ) . briefly, the representative sequences were aligned by using the mafft (katoh and standley, ) program and neighbor joining (nj) phylogenetic trees were constructed using the mega program (tamura et al., ) . the genotype of each gene was assigned manually according to the previous genotype database (yamashita et al., ) . . sequence id, country/region and year of collection were extracted from the deposited genbank data and integrated into the sql database by using an in house perl script. the above processes except for the original database construction are performed automatically every night to update the recent denv database. we implemented a set of viewer applications on dgv by using google maps © , which shows the data in a temporal and spatial manner. one application presents the geographical distribution of each denv genotype on the map in a userspecified time span. another option is a homology search program that searches for the most homologous denv sequence in the dgv database and show the geographical positions of closely related sequences on the map. the other interface shows the sources of imported dengue cases on the map, according to the infectious agents surveillance report (iasr), japan (http://www.nih.go.jp/niid/en/iasr-e.html). this set of applications is available at the dgv web site (https://gph.niid.go.jp/geograph/dengue/content/genomemap). on march , , dgv included a total of , denv sequences, which consisted of , , , and denv serotype- , - , - , and - sequences, respectively ( table ) . some genotypes have been abundantly sequenced and deposited in the public database, whereas other genotypes have rarely been sequenced (i.e., denv- genotype ii was reported in only seven records from to , denv- genotype iii was also reported in only seven records from to , and denv- genotype iv has not been reported since ). these rare genotypes may have become minor populations or may be undergoing a silent transmission cycle (lanciotti et al., ; chen and vasilakis, ; santiago et al., ) . fifteen years' worth of data from to for all serotypes showed that denv sequences were primarily reported from south to southeast asia, central to south america, and the countries of oceania (figure a) . some biases in denv serotype compositions were observed in several countries. for instance, the dominant serotypes were denv- and - in mexico, denv- and - in polynesian countries with the exception of fiji, and denv- and - in pakistan. in contrast, all serotypes were sampled in brazil and thailand. intriguingly, when focusing on the genotype instead of the serotype, the data from to showed at least three potential geographical genotype distribution border lines in asia (figures b, ) . the first border is between the american continents and other regions (figure b) , the second is located between bangladesh and myanmar for the genotype distributions of denv- and - and india and myanmar for denv- , and the third is located between indochina and the malay peninsula (figure ) . there seem to be differences in the denv- and - distributions between malaysia, singapore and indonesia; however, the border line is not clear because malaysia and indonesia consist of many islands and share kalimantan island and the deposited sequence data do not specify the original island isolation site. although, some boundaries are not clear, these boundaries are roughly conserved among all serotypes except for the bangladesh-myanmar border line for denv- , suggesting potential barriers against the vector mosquitos' movements or human activities between the countries. we also found a timeline change in the predominant genotypes. from to , the dominant genotype in asia was cosmopolitan, although india-pakistan-sri lanka and southeast-oceania belonged to different lineages (khan et al., ) . the major genotypes in the indochina countries were different from those of the other asian countries; genotype figure | a screenshot of the denv sequence similarity search. an env sequence derived from an autochthonous case in japan (lc or gi: ) was used as a sample query. the query was assigned as the env region of denv- genotype i. asian i was predominant in thailand, whereas genotype asian american was predominant in cambodia and vietnam (figure and movie s ). from , asian i increased in cambodia and vietnam until finally in asian i became the predominant genotype in indochina. the genotype asian i viruses in thailand seemed to be widely disseminated into vietnam via cambodia but did not reach malaysia and bangladesh (figure ) . thus, the asian american genotype was replaced by asian i in cambodia and vietnam between and . this example also suggests the idea of genotype transition, which probably reflects the mosquito vector habitat and human activities in the indochinese peninsula. dgv currently does not support the prediction of dengue epidemics, because number of deposited sequence data does not always reflect the actual number of events, in addition, it takes long time to be a public sequence through isolation, sequencing, and publication. dgv provides a search engine for the assignment of the denv serotype, genotype, and origin country according to the most homologous sequence on the basis of a blastn search against the denv database. the search results are shown as text and are also plotted through google maps © . subsequently, the query sequence is divided into mature protein regions and displayed with a serotype/genotype assignment. the homology search results and the divided nucleotide sequences in fasta format can be downloaded. here, we present an example similarity search for an env sequence derived from an autochthonous case in japan (lc or gi: ). dgv assigned the sequence as the env region of the denv- genotype i and identified homologous sequences from japan, china, singapore and indonesia. these results are consistent with those from a previous study (figure ; kutsuna et al., ) . to aid in visualizing the source countries of dengue infection cases imported to japan, the number of annual imported cases was also mapped on google maps © . the serotype (but not genotype), year, and visiting country/area are also indicated based on the infectious agents surveillance report (iasr), which releases monthly data and information obtained from prefectural and municipal public health institutes and quarantine stations to the public (figure ). ay performed the experimental design, participated in the analysis and drafted the manuscript. ts implemented the application, performed the data collection, constructed the original genotype database, and participated in the analysis. ts , kk, and tt reviewed the application and participated in the discussion. mk contributed to the experimental design, performed the analysis and drafted the manuscript. all authors read and approved the final manuscript. this work was supported by a grant for research on emerging and re-emerging infectious diseases (h shinko-ippan- /h shinko-gyosei-shitei- ) from the ministry of health, labor and welfare, japan, and was also supported by the research program on emerging and re-emerging infectious diseases ( fk h and fm h ) from the japan agency for medical research and development, amed. this work was also partially supported by jsps kakenhi grant number k .the funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. we are grateful to ms. inamine from niid for drawing the dgv icon. we thank prof. ikuta and prof. yasunaga from biken for allowing us to use the original dataset "dengue virus genotyping database." the supplementary material for this article can be found online at: http://journal.frontiersin.org/article/ . /fmicb. movie s | genotype transition of denv- in asia from - to - . frontiers in microbiology | www.frontiersin.org emergence of dengue virus type genotype iia in malaysia circulation of different lineages of dengue virus type in central america, their evolutionary time-scale and selection pressure analysis denvirdb: a web portal of dengue virus sequence information on asian isolates dengue-quo tu et quo vadis? viruses viral immune evasion in dengue: toward evidence-based revisions of clinical practice guidelines search and clustering orders of magnitude faster than blast diversity and evolution of the envelope gene of dengue virus type mafft: iterative refinement and additional methods emergence and diversification of dengue cosmopolitan genotype in pakistan molecular genotyping of dengue viruses by phylogenetic analysis of the sequences of individual genes structure of dengue virus: implications for flavivirus organization, maturation, and fusion autochthonous dengue fever rapid detection and typing of dengue viruses from clinical samples by using reverse transcriptase-polymerase chain reaction molecular evolution and epidemiology of dengue- viruses epidemiology of dengue: past, present and future prospects virus pathogen database and analysis resource (vipr): a comprehensive bioinformatics database and analysis resource for the coronavirus research community virus variation resources at the national center for biotechnology information: dengue virus reemergence and decline of dengue virus serotype in puerto rico mega : molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods extinction and rapid emergence of strains of dengue virus during an interepidemic period origin and distribution of divergent dengue virus: novel database construction and phylogenetic analyses key: cord- -pad nww authors: banerjee, arinjay; pérez-lópez, edel; mossman, karen title: commentary: phyllostomid bat microbiome composition is associated to host phylogeny and feeding strategies date: - - journal: front microbiol doi: . /fmicb. . sha: doc_id: cord_uid: pad nww nan phyllostomid bat microbiome composition is associated to host phylogeny and feeding strategies by carrillo-araujo, m., tas, n., alcantara-hernandez, r. j., gaona, o., schondube, j. e., medellin, r. a., et al. ( ) . front. microbiol. : . doi: . /fmicb. . in their article, carrillo-araujo et al. show that phyllostomidae (new world leaf-nosed bat family) gut microbiome composition is closely associated with host phylogeny. they provide evidence that feeding-strategy plays a role in the microbiome composition of these bats (carrillo-araujo et al., ) . we were particularly intrigued by their detection of deoxyribonucleic acid (dna) from firmicutes in intestinal sections from these bats. the authors use a previously published approach of sequencing s rrna for taxonomic assignment of the microbial community (caporaso et al., ) . s rrna is a good predictor of taxonomic classification (mizrahi-man et al., ; chaudhary et al., ) , but it is not foolproof (reviewed here by janda and abbott, ) . data on the probability of correct taxonomic assignment will bolster the findings in this article and allow researchers to confidently design follow-up studies. although not necessary for this study, testing a subset of the microbial population using an alternate sequencing-taxonomic grouping pipeline would further substantiate the taxonomic classification of microbial communities. mollicutes (phylum firmicutes) are a class of microorganisms that include phytoplasmas ('candidatus phytoplasma') that are being increasingly recognized for their role in plant diseases such as sapodilla little leaf, yellow leaf roll disease of peach, strawberry green petal and sugarcane white leaf syndrome. these diseases affect a diverse array of economically and ecologically important plant hosts around the world (vesterinen et al., ; pérez-lópez et al., . mollicutes are highly diverse and are made up of five orders, acholeplasmatales, anaeroplasmatales, entoplasmatales, haloplasmatales, and mycoplasmatales. all members of these orders are obligate parasites. genera 'candidatus phytoplasma' and spiroplasma, from the order acholeplasmatales and entoplasmatales, respectively, consist of plant pathogens. 'candidatus phytoplasma' and spiroplasma represent % of the class mollicutes (zhao et al., ) . 'candidatus phytoplasma' consists of over species that are known to be pathogenic in plants (miyazaki et al., ) . in the genus spiroplasma, at least two species have been identified as plant pathogens, spiroplasma citri, the causative agent of citrus stubborn disease (saglio et al., ) and spiroplasma kunkelii, which is associated with corn stunt disease (whitcomb et al., ) . (miller et al., ) , c. perspicillata (barquez et al., a) , l. yerbabuenae (medellin, ) , and g. soricina (barquez et al., b; iucn, ) sampled in this study by carrillo-araujo et al. (f) schematic representation of the possible role of bats as vectors and sentinels of phytoplasmas. fruit and nectivorous bats could potentially move phytoplasmas around through guano or seeds that are part of the guano. alternatively, microbiome analysis of insectivorous bats that feed on fruit eating insects could allow us to monitor phytoplasma prevalence and spread. in their article, the authors indicate that artibeus jamaicensis have the highest relative abundance of mollicutes in their intestinal contents (carrillo-araujo et al., ) . in their article, supplementary table s also indicates the presence of nucleic acids from firmicutes in all bat species that were sampled. looking at their data from a plant disease perspective, we wondered if frugivorous and nectivorous bats could play a role in the transmission of plant pathogens. could this data set and the sampling methods established allow us to monitor bats as sentinels of plant pathogens? here, we speculate upon the role of frugivorous and nectivorous bats as possible vectors of plant pathogens. we outline limitations of this study that do not allow us to fully establish the dynamics of plant pathogen-bat interactions. we also discuss future directions to firmly establish the role of bats as potential vectors of plant pathogens. recently, bats have been implicated as the reservoirs of several emerging viruses that cause serious disease in humans and agricultural animals (calisher et al., ; moratelli and calisher, ; zhou et al., ) . these viruses fail to cause disease symptoms in experimentally or naturally infected bats (munster et al., ; hu et al., ; schuh et al., ) . we and others have since identified several adaptations in innate immune signaling molecules that might allow bats to control virus propagation more effectively than other mammals (zhou et al., ; banerjee et al., ; xie et al., ) . bats are also recognized as potential reservoirs of pathogenic bacteria (loftis et al., ; becker et al., ) . plant pathogens do not generally infect mammalian hosts, but after careful analysis of the data in carrillo-araujo et al.'s article (carrillo-araujo et al., ) , the role of bats as potential vectors/carriers of plant pathogens cannot be ruled out. we compared the extent of spread of phytoplasmas in the americas ( figure a ) and observed that it overlapped with the spread of frugivorous bats a. jamaicensis ( figure b) and carollia perspicillata ( figure c ) and nectivorous bats leptonycteris yerbabuenae ( figure d) and glossophaga soricina (figure e ) that were sampled by the authors. the authors mention that frugivorous and nectivorous bats diverged - million years ago (mya). evolutionary reconstructions show that the divergence of mollicutes into two major branches occurred about mya, placing phytoplasmas and their closest ancestor, ancholeplasma in the same branch (maniloff, ) . there is further evidence that phytoplasmas diverged from an acholeplasma-like ancestor around mya (zhao et al., ) . thus, phytoplasmas and frugivorous bats have co-existed for at least million years. could bats have acquired phytoplasmas as part of their microbiome after their divergence into frugivorous and nectivorous bats? could bats have played a role in the spread of phytoplasmas? we do not know. phytoplasmas have been detected in fruits in the united states and canada (bagadia et al., ; rosete et al., ) . there is a need to sample additional bat species to fully elucidate the overlap in the spread of phytoplasmas and frugivorous bats. multiple studies have established the role of bats as reservoirs of certain mammalian viruses (corman et al., ; plowright et al., ; ng and tan, ; noh et al., ; widagdo et al., ) . similar studies are needed for plant pathogens. in this study, the authors analyzed the microbiome of bats using dna sequencing. the ability to culture mollicutes from bat intestinal samples would identify if these bacteria remain viable while they pass through the harsh environment of the digestive tract. unfortunately, many of the plant pathogenic mollicutes, including phytoplasmas, are unculturable in axenic media. although mollicutes are unlikely to replicate in bat gut cells, they could potentially proliferate within the intestinal micro-community. however, this remains to be tested. other questions about the possible excretion of viable bacteria through bat guano and the ability to infect plants remain unknown ( figure f ). seeds could be part of bat guano, but phytoplasma transmission through seeds has not been confirmed or disproved yet. alongside birds, bats are capable of true flight. the possibility to deposit phytoplasma-contaminated guano from one area to another and within the same area is high. aryan et al. showed that phytoplasmas are transmissible through graft (aryan et al., ) . thus, mechanical transmission is another possibility. this form of transmission occurs when feeding animals cause tissue damage in plants, aiding the spread of microorganisms. while bats are speculated as reservoirs of multiple microbes, this study brings up the possibility of using bat microbiome data as predictors of pathogen spread and prevalence ( figure f) . although our knowledge about the bat microbiome is limited, it does provide us with an opportunity to study bats as sentinels of plant pathogens. this opportunity extends to insect-eating bats, since insects such as leafhoppers and planthoppers are known vectors for phytoplasmas (weintraub and beanland, ; pérez-lópez et al., ) . future studies focused on identifying neglected vectors of plant pathogens will elucidate the likely role played by herbivorous wildlife in the dispersal of these microorganisms. results from such studies will have agricultural policy implications for plant diseases. phytoplasmas continue to cause losses to local farmers and has an impact on the economy. in our opinion, this study by carrillo-araujo et al. that identified mollicutes in the intestinal content of phyllostomid bats opens up an alternate and intriguing line of investigation in to wildlife vectors and sentinels of plant pathogens. ab and ep-l wrote the commentary. km edited the commentary. phytoplasma transmission by heterologous grafting influences viability of the scion and results in early symptom development in periwinkle rootstock characterization and molecular differentiation of sri-e and srix-e phytoplasmas associated with blueberry stunt disease in new jersey lack of inflammatory gene expression in bats: a unique role for a transcription repressor carollia perspicillata. the iucn red list of threatened species glossophaga soricina. the iucn red list of threatened species genetic diversity, infection prevalence, and possible transmission routes of bartonella spp. in vampire bats bats: important reservoir hosts of emerging viruses ultra-high-throughput microbial community analysis on the illumina hiseq and miseq platforms phyllostomid bat microbiome composition is associated to host phylogeny and feeding strategies s classifier: a tool for fast and accurate taxonomic classification of s rrna hypervariable regions in metagenomic datasets rooting the phylogenetic tree of middle east respiratory syndrome coronavirus by characterization of a conspecific virus from an african bat discovery of a rich gene pool of bat sars-related coronaviruses provides new insights into the origin of sars coronavirus the iucn red list of threatened species. version - s rrna gene sequencing for bacterial identification in the diagnostic laboratory: pluses, perils, and pitfalls phytoplasma: phytopathogenic mollicutes detection of rickettsia, borrelia, and bartonella in carios kelleyi (acari: argasidae) phylogeny and evolution leptonycteris yerbabuenae. the iucn red list of threatened species artibeus jamaicensis. the iucn red list of threatened species candidatus phytoplasma noviguineense' , a novel taxon associated with bogia coconut syndrome and banana wilt disease on the island of new guinea taxonomic classification of bacterial s rrna genes using short sequencing reads: evaluation of effective study designs bats and zoonotic viruses: can we confidently link bats with emerging deadly viruses? replication and shedding of mers-cov in jamaican fruit bats (artibeus jamaicensis) understanding bat sars-like coronaviruses for the preparation of future coronavirus outbreaks -implications for coronavirus vaccine development simultaneous detection of severe acute respiratory syndrome, middle east respiratory syndrome, and related bat coronaviruses by real-time reverse transcription pcr the underestimated diversity of phytoplasmas in latin america molecular diagnostic assays based on cpn ut sequences reveal the geographic distribution of subgroup srxiii-(a/i)i phytoplasma in mexico detection of maize bushy stunt phytoplasma in leafhoppers collected in native corn crops grown at high elevations in southeast mexico transmission or within-host dynamics driving pulses of zoonotic viruses in reservoir-host populations identification and molecular characterization of the blueberry stunt phytoplasma in canada spiroplasma citri gen. and sp. n.: a mycoplasma-like organism associated with "stubborn" disease of citrus modelling filovirus maintenance in nature by experimental transmission of marburg virus between egyptian rousette bats next generation sequencing of fecal dna reveals the dietary diversity of the widespread insectivorous predator daubenton's bat (myotis daubentonii) in southwestern finland insect vectors of phytoplasmas spiroplasma kunkelii sp. nov.: characterization of the etiological agent of corn stunt disease tissue distribution of the mers-coronavirus receptor in bats dampened stingdependent interferon activation in bats should 'candidatus phytoplasma' be retained within the order acholeplasmatales? fatal swine acute diarrhoea syndrome caused by an hku -related coronavirus of bat origin contraction of the type i ifn locus and unusual constitutive expression of ifn-alpha in bats key: cord- -nph ezii authors: zhu, zixiang; du, xiaoli; li, pengfei; zhang, xiangle; yang, fan; cao, weijun; tian, hong; zhang, keshan; liu, xiangtao; zheng, haixue title: early growth response gene- suppresses foot-and-mouth disease virus replication by enhancing type i interferon pathway signal transduction date: - - journal: front microbiol doi: . /fmicb. . sha: doc_id: cord_uid: nph ezii early growth response gene- (egr ) is a multifunctional transcription factor that is implicated in viral infection. in this study, we observed that foot-and-mouth disease virus (fmdv) infection significantly triggered egr expression. overexpression of egr suppressed fmdv replication in porcine cells, and knockdown of egr considerably promoted fmdv replication. a previously reported fmdv mutant virus (with two amino acids mutations in sap domain) that displays a strong type i interferon (ifn) induction activity was used in this study. we found that sap mutant fmdv infection induced a higher expression of egr than wildtype fmdv infection, and also triggered higher ifn-β and ifn-stimulated genes (isgs) expression than wildtype fmdv infection. this implied a link between egr and type i ifn signaling. further study showed that overexpression of egr resulted in sendai virus (sev)-induced ifn-stimulated response element (isre) and nf-κb promoter activation. in addition, the sev-induced isgs expression was impaired in egr knockdown cells. egr upregulation promoted type i ifn signaling activation and suppressed fmdv and seneca valley virus replication. suppression of the transcriptional activity of egr did not affect its antiviral effect against fmdv. this study reveals a new mechanism evolved by egr to enhance type i ifn signaling and suppress fmdv replication. foot-and-mouth disease virus (fmdv) is a non-enveloped virus with positive-sense and singlestranded rna genome. the viral genome is approximately . kb nucleotides in length, including a single large open reading frame that encodes a polyprotein. the polyprotein is subsequently processed by viral proteases during protein synthesis, generating several intermediates and mature proteins (sobrino and domingo, ; grubman and baxt, ) . during co-evolution with the hosts, these viral proteins have acquired many functions to counteract host antiviral responses, cause immunosuppression, and promote viral replication and infection (mason et al., ; rodriguez pulido and saiz, ) . therefore, fmdv causes an acute vesicular disease of infected animals, which is called foot-and-mouth disease (fmd). fmd is a highly contagious disease that can lead to significant economic losses to the local livestock industry (rweyemamu et al., a; paton and taylor, ; zai-xin, ; bouguedour and ripani, ) . the understanding of host-fmdv interaction as well as the involved mechanism contributes to the planning of new strategies for fmd prevention (domingo et al., ; rweyemamu et al., b; rodriguez pulido and saiz, ) . accordingly, many researches on host responses in fmdv-infected cells have to be investigated. early growth response gene- (egr ), also designated zif , is a host transcriptional regulator that expresses rapidly after a number of stimuli like oxygen deprivation, growth factors, cytokines, shear stress and injury (brand et al., ; khachigian et al., ; nishi et al., ; jo et al., ; li et al., ) . egr is involved in diverse biologic functions and a broad variety of host signal transduction cascades that mediates cell growth, survival, differentiation, apoptosis and proliferation (pagel and deindl, ; papanikolaou et al., ) . different pathways have been identified that participate in egr induction and then regulates several biological behaviors. such as, the ras homologue gene family (rho) genes are involved in cell cycle progression, and the rho/rho-kinase pathway has been shown to regulate egr expression (barrientos et al., ; pagel and deindl, ) . as a zinc-finger dna-binding protein, egr also regulates expression of diverse gene families by binding to promoter sequences of target genes (papanikolaou et al., ) . therefore, egr is involved in activation of signal transduction of many pathways. several studies indicate that egr is linked to viral infection and immune response. egr modulates pro-apoptotic pathway and promotes venezuelan equine encephalitis virus (veev) replication (baer et al., ) . knockdown of egr in rhabdomyosarcoma cells decreases enterovirus (ev ) replication (song et al., ) . it seems that egr might play a positive role in these viruses replication. however, egr also appears critical for the initiation of immune response in b cells and t cells. egr plays roles in regulation of the expression of sever cytokines including interleukin- , cd , icam- and tumor necrosis factor genes (skerka et al., ; mcmahon and monroe, ; shin et al., ; cubero and nieto, ) . an sap domain [scaffold-attachment factor (saf)-a/b, apoptotic chromatin-condensation inducer in the nucleus (acinus) and pias (protein inhibitor of activated signal transducer and activator of transcription) domain] previously was identified within the fmdv l pro by de los santos et al. ( ) . mutation of l pro sap domain promotes type i ifn signaling activation and decreases virus growth. in addition, animals inoculated with the fmdv sap mutant display strong neutralizing antibody response and t cell response comparing with infection with wildtype fmdv (de los santos et al., ; diaz-san segundo et al., ) . in this study, a robust egr upregulation was observed in both wildtype and sap mutant fmdv-infected cells comparing with the mock-infected cells by viewing the protein abundance of egr . therefore, we investigated the correlation between fmdv infection and egr , and determined the antiviral role of egr against fmdv. sap mutant fmdv infection induced a higher expression of egr than wildtype fmdv infection. sap mutant fmdv infection also triggered higher ifn-β and ifn-stimulated genes (isgs) expression than wildtype fmdv infection. we also found that overexpression of egr enhanced sendai virus (sev)-induced interferon (ifn)-stimulated response element (isre) activation. sev-induced isgs expression was impaired in egr knockdown cells, which may serve as a link between upregulation of egr and type i ifn signaling. further study showed that egr enhanced tbk phosphorylation during fmdv infection. it indicated that egr upregulation promoted type i ifn signaling activation by enhancing tbk phosphorylation and resulted in decreased fmdv replication. this study reveals a link between egr and innate immune response during fmdv infection. porcine kidney pk- cells, human embryonic kidney t cells (hek t) cells described previously were maintained in dulbecco's modified eagle's medium supplemented with % heat-inactivated fetal bovine serum, u/ml penicillin, and µg/ml streptomycin sulfate. all the cells were cultured at • c under % co . sendai virus (sev), a model rna virus widely used to activate type i ifn signaling in cells, was kindly provide by hongbing shu's laboratory (wuhan university, china) (zhou et al., ; . fmdv strain o/by/cha/ (genbank number: jn ) described previously was used for virus infection (zheng et al., ) . commercial antibodies used in this study include an anti-egr mouse monoclonal antibody (abcam, cambridge, ma, united states), anti-tbk rabbit antibody from cell signaling technology (cst) inc. (beverly, ma, united states), anti-phospho-tbk rabbit antibody (cst), anti-c-myc mouse antibody (santa cruz biotechnology, santa cruz, ca, united states) and anti-β-actin mouse antibody (santa cruz biotechnology). anti-ifn-β and anti-ifn-α antibodies ( nu/ml, pbl biomedical laboratories) and igg isotype antibodies were used in the type i ifn-blocking experiments as previously described (trottier et al., ) . anti-fmdv vp protein polyclonal antibody was previously produced in our laboratory . transfection reagents include opti-mem medium and the lipofectamine that were purchased from invitrogen. poly (i:c) was purchased from invivogen. ifn-β was purchased from pbl biomedical laboratories. the full-length porcine egr cdna fragment was cloned into a pcdna tm . /myc-his(-)a vector (invitrogen) to construct a myc-tagged egr eukaryotic expressing plasmid (myc-egr , including a c-terminal myc tag). the constructed plasmid was analyzed and verified by dna sequencing. a series of plasmids expressing ha-tagged type i ifn pathway-related proteins [including mda , rig-i(card), visa, tbk , irf and irf ], and the ifn-β promoter luciferase reporter plasmids and control plasmid renilla luciferase prl-tk were kindly provided by hongbing shu's laboratory (zhou et al., ; . the plasmids were transfected into cells using opti-mem medium and the lipofectamine (invitrogen) reagent according to the manufacture's protocol. trizol reagent (invitrogen) was used to extract cellular or viral rna following the instruction of the protocol. the firststrand cdna was synthesized by reverse transcription reaction with the extracted rnas as templates. reverse transcription was performed with m-mlv reverse transcriptase (invitrogen) and random hexamer primers (takara) according to the manufacturer's recommendations. the quantification of the cdna was performed by qpcr. the relative amounts of the synthesized cdna was determined as an indicator of the target transcripts. qpcr was carried out using sybr premix ex taq (takara) on a quantstudio real-time pcr instrument (applied biosystems) according to the manufacturer's instructions. the glyceraldehyde- -phosphate dehydrogenase (gapdh) gene was used for normalization in qpcr analysis. relative transcript levels were calculated using − ct method as described previously . all the primers used in this study were listed in table . for western blotting, the cells were collected at the indicated time points and. the lysed cell extracts were resolved by % sds-page and transferred onto a nitrocellulose membrane (pall). the nitrocellulose membrane was then blocked with % skim milk powder in tbst ( mm tris, mm nacl, . % tween ) overnight at • c. the membrane was incubated with primary and secondary antibodies as described previously (zhu et al., ) . the membrane was washed × min, before being protein abundance analysis. the antibody-antigen complexes were visualized using enhanced chemiluminescence detection reagents (thermo). small interfering rna (sirna) was used to knockdown egr protein expression. sirna fragments were chemically synthesized by genepharma company (china). the sequences of the sirnas used in this study include: -ccaugga caacuacccuaatt- (egr sirna- ), -gccuaguga gcaugaccaatt- (egr sirna- ), and -gcuguca ccaacuccuucatt- (egr sirna- ). a non-targeting sirna (nc sirna) was used as a negative control. sirna fragments were transfected into cells using lipofectamine as described previously . forty-eight hours after sirna transfection, cells were used for further experiments. to examine the effect of sirna on egr expression, egr mrna and protein abundance were measured by qpcr and western blotting respectively. hek t cells seeded on -well plates were co-transfected with ng luciferase reporter plasmid with ng internal control renilla luciferase reporter plasmid (to normalize for transfection efficiency) prl-tk (promega), together with the indicated plasmids and/or empty vector controls using lipofectamine according to the manufacture's instruction. to make the cells receive the same amounts of total plasmids, the empty vector plasmids were used in all transfection experiments. as for sevmediated type i ifn signaling pathway activation, the cells were mock-infected or infected with sev ( hau/ml) for h; and the dual luciferase assays were then performed according to the promega dual-luciferase reporter assay system protocol. the relative luciferase activity was expressed as arbitrary units by normalizing firefly to renilla luciferase activity. as for type i ifn pathway adaptor molecules-induced ifn-element activation assay, the hek t cells were co-transfected with the reporter plasmids with the indicated plasmid or vector plasmid for h; and the luciferase activities were measured. all experiments were performed at least in triplicate. the measured values are represented as mean ± sd from three independent experiments. the statistical significance analyses were performed using the student's t-test. data considered significant when * p < . , and highly significant when * * p < . . pk- cells were infected by equal amounts of wildtype or sap mutant fmdv for h as previously described (zhu et al., ) . the expression levels of egr and viral vp protein were detected by western blotting. we observed that egr protein level is significantly upregulated both in wildtype and sap mutant fmdv-infected cells at h postinfection (hpi) ( figure a) . therefore, the correlation between fmdv infection and egr was further investigated. the dynamics of egr in fmdv-infected cells were determined. transcripts of egr were considerably upregulated after fmdv infection and reached to the highest level at hpi. no significant changes were observed in mock-infected cells ( figure b) . egr protein expression was also gradually upregulated as the infection progressed ( figure c ). this indicates that fmdv infection triggers upregulation of egr . to investigate whether egr is an ifn inducible gene, hek t and pk- cells were incubated with ifn-β to induce the expression of ifn inducible genes. the expression of two ifn inducible genes isg and isg was highly induced by incubation of ifn-β. however, the expression of egr was not changed by treatment of ifn-β ( figure d ). this indicated that egr expression was not induced by ifnβ treatment; however, fmdv infection could induce egr expression. to investigate the potential role of egr during fmdv infection, we evaluated the viral replication level in egr overexpressed cells. pk- cells were transfected with different doses of egr expressing plasmids, the cells were incubated with equal amounts of fmdv ( . moi) at h post-transfection (hpt). the viral protein and viral rna expression level was measured at hpi. overexpression of egr significantly suppressed both the viral protein expression and viral rna replication. the viral titers in both vector and egr plasmids ( µg) transfected cells were measured and compared, which showed that fmdv yields were also decreased by overexpression of egr (figure a) . to further confirm the antiviral role of egr during fmdv infection, the sirnas that target egr were designed and evaluated. pk- cells were transfected with the nc sirna or egr sirna for h, the interference efficacy of the sirnas was determined by qpcr analysis. the egr sirna- showed the highest efficacy and was used for egr knockdown assay ( figure b) . pk- cells were transfected with egr sirna- , the cells were infected with fmdv at hpt and incubated for another or h. the expression of egr and fmdv vp protein was detected using western blotting. knockdown of egr considerably increased vp protein expression during fmdv infection (figure c) . the relative fold-change in abundance of fmdv vp protein in fmdv-infected nc sirna or egr sirna cells was determined by densitometric analysis and normalized to β-actin, which confirmed that knockdown of egr enhanced fmdv vp protein expression (figure c , right panel). viral rna detection also suggested that knockdown of egr promoted viral replication ( figure d) . the viral titers were subsequently measured at hpi, which showed that knockdown of egr significantly promoted fmdv propagation ( figure d , right panel). these results suggest the antiviral role of egr against fmdv. both wildtype and sap mutant fmdv infection resulted in egr upregulation, however, sap mutant fmdv resulted in a higher upregulation of egr ( figure a) . previous study indicates sap mutant fmdv infection induces higher expression of ifn-β and isgs than wildtype fmdv infection (de los santos et al., ) . we also investigated the expression state of ifn-β and isgs (isg and mx ) in the cells infected by wildtype or sap mutant fmdv. at hpi, there was ∼ -fold difference in ifn-β transcripts for sap mutant fmdv-infected cells relative to wildtype fmdv-infected cells ( figure a) . a similar pattern was observed for isg and mx , varying from -to -fold higher for sap mutant fmdv compared to wildtype fmdv ( figure a) . these results were similar to the previous results reported by de los santos et al. ( ) . this showed that both egr expression and type i ifn signaling were enhanced in sap mutant fmdvinfected cells. this implied a link between egr and type i ifn pathway. to identify the role of egr on type i ifn signaling, egr is overexpressed in hek t cells, and the sev that is routinely used to induce type i ifns in cell culture was used to activate type i ifn signaling. overexpression of egr significantly promoted sev-induced type i ifn signaling, showing a dosedependent manner (figure b) . the expression of ifn-β, isg and mx in egr overexpressed cells were subsequently evaluated. the results showed that overexpression of egr considerably promoted sev-induced ifn-β, isg and mx expression ( figure c) . the effect of egr on sev-induced nf-κb activation was also evaluated by dual luciferase reporter assay, which also showed that egr positively enhanced nf-κb-mediated transcriptional activity ( figure d) . the role of egr on poly (i:c)-induced type i ifn signaling was further evaluated. overexpression of egr significantly promoted poly (i:c)-induced type i ifn signaling ( figure e) . the expression of poly (i:c)-induced isgs was also measured. the results figure | state of egr in fmdv-infected cells. (a) pk- cells were incubated with equal amounts of sap mutant fmdv or wildtype fmdv for h, the abundance of egr and viral vp protein was detected. (b) pk- cells were infected with wildtype fmdv or mock-infected for , , , , , or h. the transcripts of egr and viral rna were detected by qpcr. (c) pk- cells were infected with wildtype fmdv for , , , , , or h. the expression levels of egr and vp protein were detected by western blotting. (d) hek t or pk- cells were mock-treated or incubated with ifn-β at a concentration of ng/ml for h. the expression levels of isg , isg and egr was measured by qpcr. showed that overexpression of egr considerably promoted poly (i:c)-induced isg and isg expression ( figure f ). sevinduced isgs expression levels in egr knockdown cells were also analyzed. the sirna interference efficacy was also verified in hek t cells ( figure g) . egr was knocked down by transfection of sirna, and the cells were infected by sev at hpt and incubated for h. the expression of ifn-β, isg and mx were measured. the transcript levels of ifn-β, isg and mx remarkably decreased in egr knockdown cells comparing with that in nc sirna cell ( figure h) . these results suggested a positive regulatory role of egr on type i ifn signaling. the type i ifn blocking antibody experiments were also performed. anti-ifn-β and anti-ifn-α antibodies ( nu/ml) was used to block type i ifn signaling in pk- cells, the egr overexpressed cells were treated with ifn antibodies for h and then infected with fmdv. the viral yields were measured at hpi. type i ifn blocking antibodies obviously abrogated the inhibitory effects of egr on fmdv propagation ( figure i) . these results suggested that egr suppressed fmdv replication by enhancing type i ifn signaling. we also evaluated the antiviral role of egr against another picornavirus seneca valley virus (svv) which showed a close relationship with fmdv, and we found that upregulation of egr also enhanced isgs expression (isg and isg ) and suppressed svv replication ( figure j) (b) pk- cells were transfected with nc (negative control) or sirna (egr sirna- , egr sirna- or egr sirna- ) for h. the egr mrna levels were measured by qpcr. (c) schematic diagram of the strategy in egr knockdown experiment and investigation of the viral replication state in egr knockdown cells. pk- cells were transfected with nc sirna or egr sirna- for h. the cells were infected with fmdv for , , or h. viral protein abundance was measured by western blotting. relative fold-change in abundance of vp protein was determined by densitometric analysis using quantity one software (bio-rad) and normalized to β-actin. (d) viral rna levels in fmdv-infected nc sirna or egr sirna- cells at , , and hpi were measured by qpcr. viral yields in fmdv-infected nc sirna or egr sirna- cells at hpi were measured by tcid assay. * p < . was considered as statistically significant and * * p < . was considered as highly significant. i ifn signaling pathway. to screen the potential proteins that were targeted by egr , hek t cells were co-transfected with the myc-vector or myc-tagged egr plasmids and the indicated plasmids expressing rig-i, rig-i(card) (the card domain of rig-i), mda (helicase) (the helicase domain of mda ), visa, tbk , irf and irf , together with isre luciferase reporter plasmid and the internal control plasmid prl-tk. luciferase activity was measured at h after transfection. overexpression of adaptor proteins rig-i, rig-i(card), mda ), visa, tbk , irf or irf all activated the isre luciferase reporter system, and overexpression of mda (helicase) did not activate the isre luciferase reporter system (figure ) . tbk or its upstream proteins (rig-i, mda and visa) mediated type i ifn signaling was significantly enhanced by overexpression of egr . however, overexpression of egr did not promote irf and irf mediated type i ifn signaling (figure ) . irf and irf are the downstream proteins of tbk . therefore, we speculated that tbk or its upstream molecules (rig-i, mda and visa) were the target/targets of egr to enhance type i ifn signal transduction. to investigate whether egr interacted with the adaptors of type i ifn pathway, the coimmunoprecipitation assay was performed by co-transfection of the myc-egr plasmids and the plasmids expressing various ha-tagged adaptors of type i ifn pathway. the transfectants were immunoprecipitated with anti-ha antibodies and subjected to western blotting analysis. no interaction was observed between egr and the adaptors (figure a) . egr enhanced the activation of isre luciferase reporter stimulated by tbk and its upstream molecules. tbk might be a key adaptor to enhance type i ifn signaling. the influence of egr on tbk expression and tbk phosphorylation levels were evaluated in fmdv-infected cells. pk- cells were transfected with µg of myc-egr or its empty vector plasmids. the cells were infected with equal amounts of fmdv at hpt and collected at , , , or hpi. overexpression of egr had no influence on tbk expression during fmdv infection. however, it significantly promoted tbk phosphorylation levels after fmdv infection comparing with that in the empty vector transfected cells (figure b) . the vp was used as an indicator of viral replication. overexpression of egr also resulted in decreased vp abundance ( figure b ). this confirmed that upregulation of egr enhanced type i ifn signaling and suppressed fmdv replication. as a transcription factor, the transcriptional activity is significantly involved in the regulatory function of egr . the transcripts of ifn-β, isg and mx were detected by qpcr. (d) hek t cells were transfected with vector or myc-egr plasmids together with nf-κb luciferase reporter plasmid and prl-tk. the transfected cells were infected with sev, and the luciferase activity was measured by dual luciferase assay. (e) hek t cells were transfected with vector plasmids or myc-egr plasmids and solvent control or poly (i:c) together with isre luciferase reporter plasmid and prl-tk. the luciferase activity was measured by dual luciferase assay. (f) hek t cells were co-transfected with vector plasmids or myc-egr plasmids and solvent control or poly (i:c), the expression of isg and isg expression was measured by qpcr. (g) hek t cells were transfected with nc or egr sirna- for h. the egr transcripts were measured by qpcr. (h) hek t cells were transfected with nc sirna or egr sirna- for h. the cells were then mock-infected or infected with sev for h. the transcripts of ifn-β, isg and mx were detected by qpcr. (i) pk- cells were transfected with equal amounts of vector or egr expressing plasmids for h, the egr -transfected cell were mock-treated or treated with ifn antibodies for h and then infected with fmdv. the viral yields were measured by tcid assay at hpi. (j) pk- cells were transfected with vector plasmids or myc-egr plasmids for h. the transfected cells were infected with svv for h. the viral rna, isg and isg expression levels were detected by qpcr. * p < . was considered as statistically significant and * * p < . was considered as highly significant. to investigate whether the transcriptional activity is related to the antiviral function of egr , znegr , a previous reported dominant-negative mutant of egr that lacks a transcriptional function, was used as an inhibitor of the transcriptional activity of egr (levkovitz and baraban, ) . the myc-tagged znegr expressing plasmid was constructed ( figure a) . pk- cells were transfected with vector, myc-egr plasmids or cotransfected with myc-egr and myc-znegr figure | the target of egr in type i ifn pathway activation. hek t cells were co-transfected with myc-egr or empty vector plasmids and the constructs expressing rig-i, rig-i(card), mda , visa, tbk , irf or irf , together with isre luciferase reporter plasmid and the internal control plasmid prl-tk. dual luciferase activity was determined at hpt. * * p < . was considered as highly significant. plasmids and subjected to fmdv infection. overexpression of egr suppressed fmdv replication, and egr -mediated antiviral effect was not blocked by cotransfection with znegr ( figure b) . we further evaluated the localization of egr in mock-or fmdv-infected cells, and we found fmdv infection did not change the localization of egr compared with that in the mock-infected cells ( figure c) . these data suggest that the transcriptional activity is not involved in the antiviral function of egr . egr , as a multifunctional transcription factor, plays regulatory roles in a variety of cellular responses. in addition, egr shows an anti-tumor function. overexpression of egr decreases tumorigenesis in nude mice and various of human tumor cell lines (huang et al., (huang et al., , . induction of tgfβ and p may lead to the tumor suppressor property of egr (baron et al., ) . p , as a tumor suppressor, has also been implicated in other functions that play important roles in disease and health (fuhrman et al., ). p -dependent antiviral defense has been widely reported (takaoka et al., ; shin-ya et al., ; muñoz-fontela et al., ) . such as, p serves as an antiviral protein during influenza a virus infection by enhancing host innate and adaptive immune responses (muñoz-fontela et al., ) . egr directly induces the transcription of p (liu et al., ) , whether egr is also involved in host antiviral responses remains unknown. in this study, we determined that egr revealed an antiviral function against fmdv, which indicated that egr is implicated in host antiviral response. egr can be upregulated upon viral infection by epstein-barr virus, mouse hepatitis virus (mhv), veev, ev , rabies viruses and japanese encephalitis virus infections (saha and rangarajan, ; cai et al., ; kim et al., ; song et al., ; baer et al., ) . however, egr expression is related to viral pathogenesis during veev, mhv and ev replication. all these studies were performed using mouse or human cells, and most of these viruses can cause central nervous system (cns) diseases. in this study we investigated the function of porcine egr and showed the antiviral role of porcine egr against fmdv. fmdv infection does not cause any cns disease. whether the difference of species or tissue tropism resulted in the different role of egr in different virus infections remain unknown. however, egr has been suggested to participate in ifn-γ-stat pathway in t cells (shin et al., ) . t-bet is a th -specific transcription factor that is directly involved in t cells differentiation (djuretic et al., ) . egr regulates t-bet expression by binding to the promoter of t-bet and induces t-bet transcription (shin et al., ) . t-bet plays a vital role in innate immunity, and lacking of t-bet expression increases host susceptibility to inflammatory disease (garrett et al., ) . this implies a regulatory role of egr in innate immunity. besides, overexpression of egr downregulates nfκb inhibitor (kim et al., ) , which also implies a potential role of egr in innate immunity. in this study, we determined that egr is implicated in innate immunity during fmdv infection. a higher egr expression was observed in sap mutant fmdv-infected cells comparing with that in the wildtype fmdv-infected cells. it has been determined that sap mutant fmdv infection resulted in stronger type i ifn signaling than wildtype fmdv infection (de los santos et al., ) . we found that sap mutant fmdv infection triggered higher expression of ifn-β and isgs than wildtype fmdv infection. this result was similar as the result reported by de los santos et al. ( ) previously. whether the higher expression of egr correlated with the higher expression of ifn-β and isgs was therefore investigated. overexpression of egr significantly activated type i ifn signaling and ifn-β and isgs expression. knockdown of egr considerably impaired sev-induced ifn-β and isgs expression. type i ifn blocking antibodies obviously abrogated the inhibitory effects of egr on fmdv propagation. this suggested egr is implicated in type i ifn pathway activation. a link between egr and type i ifn pathway was reported for the first time. further investigation of egr -mediated enhancive effect showed that egr promoted activation of the type i ifn signaling during fmdv infection. overexpression of egr upregulated tbk phosphorylation during fmdv infection. tbk phosphorylation enhanced type i ifn signaling and strengthened antiviral activity which subsequently suppressed fmdv replication. egr is a transcription factor; however, it does not induce tbk expression. the interaction between egr and various adaptors of type i ifn signaling pathway was not observed by performing coimmunoprecipitation assay. the role of the transcriptional activity of egr for its antiviral function against fmdv was also evaluated. suppression of the transcriptional activity of egr did not affect its antiviral effect. egr might enhance type i ifn signaling independent of its transcriptional activity. how does egr promote tbk phosphorylation is not clear. several phosphatases have been identified as regulator of phosphorylation of tbk (gabhann et al., ; zhao, ) . the regulation of egr on these phosphatases should be studied in future, and the detailed mechanism of egr to promote tbk phosphorylation should be further investigated. in addition, the effect of egr on the adaption of other upstream molecules of tbk should also be exploited. in summary, we present the first investigation of egr in regulation of type i ifn signaling during fmdv infection. we determined that egr showed an antiviral function against fmdv. egr promoted activation of the type i ifn signaling during fmdv infection and resulted in the decreased replication of fmdv. egr suppressed fmdv replication independent of its transcriptional activity. these findings identify an important role of egr in enhancement of type i ifn signaling during fmdv infection. however, the exact mechanism for egr to promote type i ifn signaling should be investigated in future to gain deeper understanding of egr -mediated functions. venezuelan equine encephalitis virus induces apoptosis through the unfolded protein response activation of egr the transcription factor egr is a direct regulator of multiple tumor suppressors including tgfβ , pten, p and fibronectin: egr is a potential target of gene therapy for prostate cancer two novel members of the ablim protein family, ablim- and - , associate with stars and directly bind f-actin review of the foot and mouth disease situation in north africa and the risk of 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targeting the host antiviral response epidemiological patterns of foot-and-mouth disease worldwide planning for the progressive control of foot-and-mouth disease worldwide common host genes are activated in mouse brain by japanese encephalitis and rabies viruses t-bet expression is regulated by egr -mediated signaling in activated t cells intracellular interferon triggers jak/stat signaling cascade and induces p -dependent antiviral protection a regulatory element in the human interleukin gene promoter is a binding site for the zinc finger proteins sp and egr- foot-and-mouth disease in europe. fmd is economically the most important disease of farm animals. its re-emergence in europe is likely to have consequences that go beyond severe alterations of livestock production and trade early growth response- facilitates enterovirus replication by direct binding to the viral genome rna integration of interferon-|α|/|β| signalling to p responses in tumour suppression and antiviral defence retinoids inhibit measles virus through a type i ifn-dependent bystander effect progress and prospect of the technologies to control foot-andmouth disease and its pathogen characteristics worldwide negative regulation of tbk -mediated antiviral immunity genetic characterization of a new pandemic southeast asia topotype strain of serotype o foot-and-mouth disease virus isolated in china during the erassociated protein zdhhc is a positive regulator of dna virus-triggered, mita/sting-dependent innate immune signaling nonstructural protein of influenza a virus interacts with human guanylate-binding protein to antagonize antiviral activity foot-andmouth disease virus viroporin b antagonizes rig-i mediated antiviral effects by inhibition of its protein expression comparative proteomic analysis of wild-type and sap domain mutant foot-and-mouth disease virus-infected porcine cells identifies the ubiquitin-activating enzyme ube required for virus replication zz, xd, and hz conceived the study and wrote the manuscript. zz, xd, pl, xz, fy, and wc performed the experiments. ht, kz, and xl collected the data, analyzed the data, and revised the manuscript. the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © zhu, du, li, zhang, yang, cao, tian, zhang, liu and zheng. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- - qrg lqu authors: wang, yingchen; dong, tuo; qi, guiyun; qu, lixin; liang, wei; qi, binbin; zhang, zhe; shang, lei; gao, hong; du, xiqiao; lu, bing; guo, yan; liu, zhenwei; yu, huisong; cui, qi; wang, xiaocen; li, ye; guo, weiyuan; qu, zhangyi title: prevalence of common respiratory viral infections and identification of adenovirus in hospitalized adults in harbin, china to date: - - journal: front microbiol doi: . /fmicb. . sha: doc_id: cord_uid: qrg lqu background: respiratory infections pose a great challenge in global health, and the prevalence of viral infection in adult patients has been poorly understood in northeast china. harbin is one of the major cities in northeast china, and more than half of any given year in harbin is occupied by winter. to reveal the viral etiology and seasonality in adult patients from harbin, a -year consecutive survey was conducted in harbin, china. methods: from january to december , specimens were obtained from adult patients admitted to the second affiliated hospital of harbin medical university with lower respiratory tract infections. sputum samples were examined by direct immunofluorescence assays to detect seven common respiratory viruses, including influenza virus (type a and b), parainfluenza virus (type to ), respiratory syncytial virus and adenovirus. adenovirus positive samples were seeded onto a cells to isolate viral strains. phylogenetic analysis was conducted on the highly variable region of adenoviral hexon gene. results: a total of , hospitalized adult patients with lower respiratory tract infections were enrolled, in which patients ( . %) were detected as having at least one viral infection. the co-infection rate in this study was . % ( / ). the dominant viral pathogen from to was parainfluenza virus, with a detection rate of . %, followed by influenza virus, respiratory syncytial virus and adenovirus. based on the climate seasons determined by daily average temperature, the highest overall viral detection rate was detected in spring ( . %, / ), followed by winter ( . %, / ), autumn ( . %, / ) and summer ( . %, / ). adenovirus type strains with slight variations were isolated from positive cases, which were closely related to the gb strain from the united states, as well as the harbin b strain isolated locally. conclusion: this study demonstrated that common respiratory viruses were partially responsible for hospitalized lower respiratory tract infections in adult patients from harbin, china, with parainfluenza virus as the dominant viral pathogen. climate seasons could be rational indicators for the seasonality analysis of airborne viral infections. future surveillance on viral mutations would be necessary to reveal the evolutionary history of respiratory viruses. background: respiratory infections pose a great challenge in global health, and the prevalence of viral infection in adult patients has been poorly understood in northeast china. harbin is one of the major cities in northeast china, and more than half of any given year in harbin is occupied by winter. to reveal the viral etiology and seasonality in adult patients from harbin, a -year consecutive survey was conducted in harbin, china. methods: from january to december , specimens were obtained from adult patients admitted to the second affiliated hospital of harbin medical university with lower respiratory tract infections. sputum samples were examined by direct immunofluorescence assays to detect seven common respiratory viruses, including influenza virus (type a and b), parainfluenza virus (type to ), respiratory syncytial virus and adenovirus. adenovirus positive samples were seeded onto a cells to isolate viral strains. phylogenetic analysis was conducted on the highly variable region of adenoviral hexon gene. results: a total of , hospitalized adult patients with lower respiratory tract infections were enrolled, in which patients ( . %) were detected as having at least one viral infection. the co-infection rate in this study was . % ( / ). the dominant viral pathogen from to was parainfluenza virus, with a detection rate of . %, followed by influenza virus, respiratory syncytial virus and adenovirus. based on the climate seasons determined by daily average temperature, the highest overall viral detection rate was detected in spring ( . %, / ), followed by winter ( . %, / ), autumn ( . %, / ) and summer ( . %, / ). adenovirus type strains with slight variations were isolated from positive cases, which were closely related to the gb strain from the united states, as well as the harbin b strain isolated locally. lower respiratory tract infections are a persistent public health problem, causing more than two million deaths per year worldwide, with a rate of deaths per , population (gbd causes of death collaborators, . the morbidity and mortality of respiratory infections could be even worse in developing countries, including china. viral infections played an important role in pediatric lower respiratory tract infections, and the corresponding common viral pathogens were influenza a and b virus (iav and ibv), parainfluenza virus (piv, type to ), respiratory syncytial virus (rsv) and human adenovirus (adv) (pavia, ) . these seven viruses were also common in respiratory infections of the adult population in shandong province, china (liu et al., ) . to the best of our knowledge, there were no commercial vaccines available for most of these viruses, except for influenza a and b viruses (lambkin-williams et al., ) . the etiology of respiratory infection in the adult population has been overlooked, at least in china, although there are plenty of reports on the epidemiology of respiratory viral infection in the pediatric population (wang et al., ) . china is a vast country with notable variations in climate characteristics among different regions. due to the differences in socioeconomic, geographic or climate factors, the epidemiological features of viral infection in the respiratory tract show dramatic variation among different study populations (sloan et al., ; lu et al., ; he et al., ; rehder et al., ) . harbin is one of the major metropolises in northeast china, hosting more than eight million people. the winter in harbin lasts for more than half a year, while the summer is short, resulting in the length of four seasons being unequal. viral respiratory infections in the pediatric population from harbin were reported by the authors' team in (zhang et al., ) , in which the adult population was not included. a clear picture of the viral etiology in hospitalized adults with respiratory infections ought to be critical for both public health policy makers and clinical practitioners. phylogenetic history of several respiratory viruses isolated from china in recent years have been well established, including the influenza virus yang et al., ) , the piv (pan et al., ) and the rsv zou et al., ) . the information about adenoviral evolutionary history in china was limited to outbreak reports and novel strains identification (lu et al., ; li et al., ) , leaving a gap for circulating stains among adult patients. human adenovirus is one of the common pathogens in respiratory infections and could be divided into seven species (ismail et al., ) . the most recent reported human adenovirus type is type in species d, which was isolated from japan (hashimoto et al., ) . human adenovirus species b type (hadv b ) was reported to be responsible for adult pneumonia outbreaks in beijing china during with the genome identity as . %, comparing to the prototype strain of hadv b in (cheng et al., ) . adenoviral infections in children from harbin were found to be adv species b, as reported by the authors, while the viral type among adult patients is still poorly understood. the adenoviral virion was composed of capsomeres, containing hexons and pentons, while each penton was anchored by one fiber (or two fibers in several types) (nemerow et al., ) . the knob region in adenoviral fiber was responsible for cellular attachment through host receptors including car, cd , dsg- and sialic acids (baker et al., ; lenman et al., ) . the rgd loop of penton could be recognized by the host cell's integrin (veesler et al., ) . the hexon protein is the major neutralizing antigen of adenoviruses (madisch et al., ) , while the hyper variable region (hvr) in hexon protein hosts its type specific epitopes reported by the authors' team (yuan et al., ) . experiments on chimeric adenoviral capsids showed that the replacement of hvrs in hexon could alter the viral serotypes (qiu et al., ; tian et al., ) . the hexon specific cd + and cd + t cells have been proved to be an effective treatment for human adenoviral infections in clinical settings (zandvliet et al., ; feucht et al., ) . recent reports indicated that antibodies against fiber knob or penton base may also be involved in the neutralization of adenoviruses tomita et al., ) . to the best of our knowledge, the hypervariable region of adenoviral hexon protein plays an essential role in viral type-specific immunity. in this report, the prevalence of common viruses in the lower respiratory tract infection of hospitalized adult patients from harbin, china was explored in hopes of revealing the clinical and pathogenic features of respiratory viruses. adenoviral strains were isolated from positive cases to identify potential molecular variations within its hypervariable regions. from january to december , sputum samples were collected from , adult patients hospitalized for lower respiratory tract infections in the second affiliated hospital of harbin medical university. all patients enrolled in this study lived in urban and suburban areas of harbin without travel histories within months before admission and sampling. all patients were older than years and suffering from at least one complaint, including fever with body temperature ≥ • c, cough, expectoration, hemoptysis, chest tightness (chest pain), or dyspnea. the sputum samples were expectorated spontaneously into sterile containers and delivered to the laboratory within h, tested immediately or stored at − • c prior to use. patients' clinical data, including symptoms, history of illness, clinical diagnoses and laboratory test results were surveyed through the hospital's health information system. this study had been approved by the ethical review committees of harbin medical university in accordance with the declaration of helsinki. both informed and written consents were obtained from each participant who provided specimens. the presence of common respiratory viruses, including piv type to , influenza virus a and b virus (iav and ibv), rsv, and human adv was determined by the d ultra tm dfa respiratory virus screening kit (diagnostic hybrids, inc., athens, oh, united states). virus isolation for the adv-positive specimens was performed using the a cell line (provided by the cancer hospital of harbin medical university) following the protocol described previously (smith et al., ; huang et al., ) . in brief, cells seeded with clinical samples were incubated at • c for days. if there was no observed cytopathic effect, two additional blind passages of the culture were performed to avoid false negative results. the cultures with adv-like cytopathic effects were passaged again to confirm viral presence. the viral dna of human adv was extracted from infected cells using the axyprep multisource genomic dna miniprep kit (axygen biosciences) according to the manufacturer's instructions and eluted in µl elution buffer. the highly variable region in the adenoviral hexon gene was amplified using the following primer pairs: forward, -caattcactcgctccta- and reverse, -gtggaaaggcacataacg- . all primers were synthesized by comate bioscience co., ltd. pcr was performed with the platinum pcr supermix (invitrogen) following the manufacturer's instructions. a total reaction volume of µl contained µl of × pcr buffer (takara bio premix ex taq tm ), nm of each primer, nm of each dntps, µl of each dna sample, . µl taq enzyme (takara bio premix ex taq tm ), and sterile water. the cycling conditions were min at • c, followed by cycles of s at • c, s at • c, and s at • c, with a final -min extension at • c. dna extracted from the a cell culture and sterile water were used as negative controls. the amplicons were bi-directionally sequenced using the sanger sequencing method with an abi prism genetic analyzer (comate bioscience co., ltd., jilin, china). the daily average air temperature from january st, to december st, in harbin city was kindly provided by the harbin meteorological observatory. historical temperature datasets covering - in harbin were accessible from the china meteorological data service center. climate seasons were determined based on the smoothed daily average temperature curve according to the chinese national standard no: qx/t - (administration, ) . in brief, spring starts when the daily average temperature permanently rose above • c, while summer comes when it rose above • c permanently. the autumn starts when the daily temperature drops to • c permanently and the winter begins at • c. "permanently" means the daily temperature has remained above or below the threshold for at least five consecutive days. the details can be found in the supplementary table s . statistical analysis was conducted using the r language (version . . ). the prevalence (detection percentage) of viruses was calculated by dividing the sum of positive cases by the number of cases in total. chi-square tests or fisher's exact tests were selected for comparing the cross tables of categorical variables. the wilcoxon rank-sum or kruskal-wallis tests were chosen for continuous variable comparisons as appropriate. datasets were visualized by an excel spreadsheet. the quality of sequencing data was checked manually via chromas software version . (technelysium pty ltd., south brisbane, qld, australia), before contigs for each isolate were assembled using ugene (protsyuk et al., ) software suite (version . ) guided by the published hexon gene sequence of human adenoviruses. similar strains to the isolates in this report were found from the genbank database via the blast search method (boratyn et al., ) . fifty-eight nucleotide sequences of the hexon gene covering all seven species of human adv were selected as the reference in this study. before the tree building process, multiple sequences were aligned by muscle (edgar, ) software (version . ). the unrooted neighbor-joining tree was reconstructed using the kimura parameters distance, whose robustness was tested by the bootstrap method with , replications implemented in mega (kumar et al., ) software (version . . ). the parsimony tree was also built on the same dataset. in total, , hospitalized adult patients with respiratory infection were enrolled from january to december in harbin city. among these patients, were male and were female, as shown in bronchiectasis, with asthma and with other infections. some clinical signs were more frequent among cases with certain diagnoses. in all, ( . %) pneumonia cases complained of fever, while only ( . %) cases with bronchitis had fever (p < . ). cough was the chief complaint of the bronchial infections (p < . ), including bronchitis ( . %), copd ( . %) and asthma ( . %). out of , enrolled patients and were diagnosed with chronic diseases, including chronic respiratory disease, cancer, diabetes mellitus, chronic cardiac diseases, cerebrovascular disease, chronic kidney disease, and other chronic diseases lasting more than year. among the , total patients, ( . %) cases were determined to be positive with at least one of seven viruses, including influenza virus (a and b), piv (type , , and ), rsv and adv. one hundred and forty patients were infected with only one virus as the single infection, and patients were co-infected with more than one virus. double infections were observed in cases, while triple infection was found in six cases. as shown in table , the most frequent single infection was piv ( cases) and the major double infection was combined influenza and parainfluenza viruses ( cases). the details were accessible from the supplementary table s . the median age of the viral positive group was (iqr - ) and the negative group was (iqr - ). the age difference between the viral infection positive and negative group was not statistically significant (p = . ). the viral detection rate varied from . % ( / ) in the middle age group to . % ( / ) in the senior citizen group, but the difference was not statistically significant (p = . ). there was no significant difference in the viral infections between genders (p = . ). the virus detection rate among the study years varied significantly (p = . ) from . % ( / ) in to . % ( / ) in , as shown in table . the detection rate was not equal among calendar seasons (p = . ) or climate seasons (p = . ). based on the climate seasons, the highest overall detection rate was in spring ( . %, / ), followed by winter ( . %, / ), autumn ( . %, / ), and summer ( . %, / ). for individual virus positive samples, certain viruses were more frequently detected in spring than in other seasons, including influenza virus ( . %, / ), piv ( . %, / ), and rsv ( . %, / ), as shown in table . adv was more frequently detected in winter, with a detection rate of . % ( / ). piv was the dominant viral pathogen during the study period, accounting for . % ( / ) of the viral infection cases in , . % ( / ) in , . % ( / ) in , and . % ( / ) in . as shown in table , viral co-infections were more not equally distributed among seasons, with the highest ratio in spring at . % ( / , p = . ). flu piv rsv adv cases total overall detected respiratory viral positive cases were plotted against daily average air temperature in figure . the low temperature over days was a strong indicator for respiratory viral infections. positive cases were rare in summer, which began late in june and ended in mid of august. one exception was found in the summer in which was related to a sudden drop of air temperature from . • c on th july to . • c on th july . the overall percentage of viral infection was significantly connected with clinical diagnoses (p = . ), as shown in table . the highest detection rate was observed in the pneumonia group ( . %, / ), followed by bronchitis ( . %, / ), while the lowest detection rate was found in the asthma group ( %, / ). chest tightness was the only symptom that showed statistically significant difference (p = . ) between the positive ( . %, / ) and negative ( . %, / ) group of viral infections. the respiratory viral infection did not prolong hospital stay statistically (p = . ), although the median days in the hospital for the positive viral infection group was (iqr - ), which was shorter than in the negative group (iqr - ). overall, the viral detection rate was relatively lower in the chronic diseases group ( . % vs. . %), with statistical significance (p = . ). detection rates varied among different chronic diseases, but the difference was not significant (p = . ). the viral co-infection ratio among different diagnosed groups was not significant (p = . ). two strains of human adv were isolated from two patients diagnosed with pneumonia, respectively. the harbin b strain ( came from a male patient aged whose samples were collected in december . the other strain, named harbin a, was isolated from a male patient aged whose samples were collected in january . based on the evolutionary tree reconstruction of the hexon gene sequencing data, both strains isolated in this study were identified as human adv species b type . these two strains were nearly identical, with the closest evolutionary distance to the strain gb (genbank: ay ; atcc: vr- ) from the united states as well as the harbin b strain from a previous local epidemic, as shown in figure . the parsimony tree was similar in topology with neighborjoining tree (figure not shown in the article). multiple sequence alignments indicated that, compared with the gb strain, the harbin a and harbin b strain shared a variable site in the nd codon (gb numbering) of the hexon gene hosting an a to c mutation, resulting in threonine (t) to proline (p) mutation of the amino acid sequences. the harbin b strain had a threonine on the same site in the hexon protein encoded by the codon acu, which was identical to the harbin a and harbin b strain. adult populations with respiratory infections bear heavy burdens of hospitalization due to severe respiratory illness, especially for senior citizens (muller-pebody et al., ) . the constantly changing nature of viral pathogens has made the surveillance of these infections critical to public health authorities (zou et al., ; li et al., ) . the key indicators for viral infections from this report and recent published surveys in china has been summarized in table . the overall detection rate of viral infection among hospitalized adult patients in this report is . %, which was consistent with the result of . % in the age group above years old by a national survey from to in china (feng et al., ) . the dominant viral pathogen throughout this study period was piv, with a detection rate of . %, followed by influenza virus. the national survey on hospitalized patients with respiratory infection in china from to showed that influenza virus had the highest detection rate of . % in adult patients. the detection rate of respiratory viruses in the over years age group was . % in shandong province of china (liu et al., ) and was dominated by influenza virus ( . %) from to . from to , the respiratory viral detection rate in hospitalized patients above years old within beijing and shandong china was reported to be . %, in which influenza virus had the highest frequency of . % (yu et al., ) . compared with recent surveys in beijing, shandong and other regions in china, this report showed that the piv dominated epidemic seasons from to in adult patients in harbin, which should be important for preparing future epidemic control strategies in public health. the viral infection rate within asthma subgroup of this report was %, and it was one patient with rsv among asthma patients in total as shown in table . asthma is related to viral infection, while the detection rates varies among different pathogens. a recently published meta-analysis showed that the mean prevalence of rsv, influenza virus, piv and adv was . %, %, . %, and . %, respectively (zheng et al., ) . the apparently low detection rate within asthma group in this report could be possibly caused by the sampling bias due to the limited subgroup size. air temperature was a major meteorological variable associated with respiratory viral infection (chan et al., ) . harbin city is located at degrees north latitude with an annual average temperature of . degrees celsius. climate seasons are not in equal length in harbin, according to historical statistics from to . the longest season in harbin is winter, which starts on the th of october and lasts days, followed by spring on the rd of april with days. starting on the rd of june, summers in harbin are relatively short at days, while autumn is days and is the shortest season, starting on the th of august. in routine epidemiological analysis, months in a year were usually divided into four seasons equally, which is not the case in harbin and possibly compromises contagious disease seasonality prediction. based on climate seasons defined by the daily average air temperature, influenza, parainfluenza and rsv detected in this report all presented significant seasonal peaks in spring, although similar phenomena were absent within the calendar seasons. sputum samples were acceptable both in viral culture and antigen detection in respiratory infections compared with nasopharyngeal swabs and aspirates (covalciuc et al., ) , although they were not frequently collected in recent surveys on lower respiratory tract infected adult patients from china. detection of respiratory virus in sputum samples has been proven effective on a small scale, either by the immunofluorescence method (honkinen et al., ) or the pcr method (branche et al., ) . this report provided a medium scale test on the validity or effectiveness of respiratory viral detection from sputum samples with , cases. in this report, two nearly identical strains were isolated from pneumonia patients in harbin, possessing high similarity with the local strain harbin b and the strain gb. the harbin b strain was isolated from a pediatric patient in harbin (zhang et al., ) . adv strain gb was named vr- in the atcc database, which was isolated from an adult patient with common cold in maryland, united states in (huebner et al., ) . the similarity of these strains across asian and american continents through several decades may result from the globalization movement in modern china. it could also be explained by another hypothesis based on the evolution history of adenoviruses. on the geological scale, homo sapiens inherit human adenoviruses from our ancestors within the hominidae, resulting in an evolutionary rate of human adv as low as . e- substitution per site per year (hoppe et al., ) . considering its kbp genome, we would have to wait a millennium (or years exactly) before a single site mutation within an adenoviral genome occurred naturally. there were a few limitations to this study. first, the years of investigation on a single city in this present report was relatively short and much more localized than other long-term or national surveillance, which may lead to a bias of the viral etiology. second, the enrolled population in this study consisted of adult patients only, while the samples from pediatric patients were not collected simultaneously. the comparison of the viral prevalence status between adult and pediatric populations in this study was difficult. third, the bacterial culture results of the clinical samples were not included in this report, which prevents us from drawing a comprehensive picture on the pathogens associated with lower respiratory tract infections. in this report, . % of lower respiratory tract infections in hospitalized adults from harbin were found to be related with common respiratory viruses. the piv dominated viral infection from to with detection rates of . %. climate seasons based on daily temperature records were found to be reliable in seasonality analysis. adv type strains with slight variations were isolated from positive cases. further surveillance would be important to continuously monitoring the viral etiology in adult patients both local and abroad. zq, yw, and td designed this study and prepared the manuscript. gq, yg, and wg collected the specimens. yw, td, and yg conducted the epidemiological investigation. td, lq, wl, bq, zz, ls, hg, xd, bl, zl, hy, qc, xw, and yl performed the laboratory experiments. yw and td analyze the data and made the figures and tables. all authors reviewed the manuscript. division of climatic season designer oncolytic adenovirus: coming of age blast: a more efficient report with usability improvements detection of respiratory viruses in sputum from adults by use of automated multiplex pcr hospitalization incidence, mortality, and seasonality of common respiratory viruses over a period of years in a developed subtropical city comparative genomic analysis of re-emergent human adenovirus type pathogens associated with adult severe community-acquired pneumonia reveals conserved genomes and capsid proteins comparison of four clinical specimen types for detection of influenza a and b viruses by optical immunoassay (flu oia test) and cell culture methods muscle: multiple sequence alignment with high accuracy and high throughput viral etiologies of 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with viral clearance after stem cell transplantation as treatment for adenovirus infection respiratory viruses in hospitalized children with acute lower respiratory tract infections in harbin regional, age and respiratory-secretion-specific prevalence of respiratory viruses associated with asthma exacerbation: a literature review evolution and transmission of respiratory syncytial group a (rsv-a) viruses in guangdong we gratefully acknowledge the harbin meteorological observatory and the china meteorological data service center for providing air temperature datasets in this report. the supplementary material for this article can be found online at: https://www.frontiersin.org/articles/ . /fmicb. . /full#supplementary-material key: cord- -wbawzbhz authors: matsushima, yuki; mizukoshi, fuminori; sakon, naomi; doan, yen hai; ueki, yo; ogawa, yasutaka; motoya, takumi; tsukagoshi, hiroyuki; nakamura, noriko; shigemoto, naoki; yoshitomi, hideaki; okamoto-nakagawa, reiko; suzuki, rieko; tsutsui, rika; terasoma, fumio; takahashi, tomoko; sadamasu, kenji; shimizu, hideaki; okabe, nobuhiko; nagasawa, koo; aso, jumpei; ishii, haruyuki; kuroda, makoto; ryo, akihide; katayama, kazuhiko; kimura, hirokazu title: evolutionary analysis of the vp and rna-dependent rna polymerase regions of human norovirus gii.p -gii. in – date: - - journal: front microbiol doi: . /fmicb. . sha: doc_id: cord_uid: wbawzbhz human norovirus (hunov) gii.p -gii. (kawasaki variant) reportedly emerged in and caused gastroenteritis outbreaks worldwide. to clarify the evolution of both vp and rna-dependent rna polymerase (rdrp) regions of gii.p -gii. , we analyzed both global and novel japanese strains detected during – . time-scaled phylogenetic trees revealed that the ancestral gii. vp region diverged around , while the ancestral gii.p rdrp region diverged around . the evolutionary rates of the vp and rdrp regions were estimated at ~ . × (− ) and ~ . × (− ) substitutions/site/year, respectively. the phylogenetic distances of the vp region exhibited no overlaps between intra-cluster and inter-cluster peaks in the gii. strains, whereas those of the rdrp region exhibited a unimodal distribution in the gii.p strains. conformational epitope positions in the vp protein of the gii.p -gii. strains were similar, although some substitutions, insertions and deletions had occurred. strains belonging to the same cluster also harbored substitutions around the binding sites for the histo-blood group antigens of the vp protein. moreover, some amino acid substitutions were estimated to be near the interface between monomers and the active site of the rdrp protein. these results suggest that the gii.p -gii. virus has produced variants with the potential to alter viral antigenicity, host-binding capability, and replication property over the past years. human norovirus (hunov) is a major causative pathogen of acute viral gastroenteritis (de graaf et al., ) . although hunov is most prevalent during autumn and winter in the northern hemisphere, the virus can be detected throughout the year (ahmed et al., ) . a previous report suggested that approximately million people annually suffer from gastroenteritis due to hunov infection worldwide (kirk et al., ) . molecular epidemiological studies have indicated that distinct gii. variants have recurrently emerged every - years in the past decade to cause pandemics of gastroenteritis in all aged individuals (bull et al., ) . furthermore, gii.p -gii. (kawasaki variant) has suddenly been prevalent since in various countries including japan, china, south korea, italy, romania, argentina, brazil, and the usa (chan et al., ; fu et al., ; matsushima et al., ; medici et al., ; dang thanh et al., ; dinu et al., ; cannon et al., ; degiuseppe et al., ; silva et al., ) . however, the epidemiology of this virus over a long period of time is unclear at present. hunov belongs to the genus norovirus of the family caliciviridae, and it is further genetically classified into three genogroups, gi, gii, and giv. genetic analyses have shown that hunov gi and gii can be further classified into and genotypes, respectively (kroneman et al., ) . of these, many genotypes have been associated with gastroenteritis outbreaks throughout the world (hoa tran et al., ) . the genomes of noroviruses contain three open reading frames and encodes six non-structural proteins, including the rna-dependent rna polymerase and the two capsid proteins vp and vp (de graaf et al., ) . molecular evolutionary analysis based on advanced bioinformatics technologies is a powerful tool to better understand not only the phylogeny of pathogens, but also their antigenicity (bok et al., ; siebenga et al., ; boon et al., ; lu et al., ; parra et al., ; tohma et al., ; nagasawa et al., ) . furthermore, next-generation sequencing technologies are also useful for the comprehensive analysis of various virus genomes (quiñones-mateu et al., ) . in this study, we used both these methodologies to conduct a detailed molecular evolutionary analysis of the vp and rdrp regions of gii.p -gii. strains detected in various countries. a total of strains of gii.p -gii. detected in miyagi ( strains), kanagawa ( samples), saitama ( samples), ibaraki ( strains), gunma ( strains), aichi ( strains), hiroshima ( strains), tochigi ( strains), fukuoka ( strains), yamaguchi ( strains), and aomori ( strain) prefectures from to were sequenced in this study. fecal samples were collected from patients with acute gastroenteritis associated with hunov infection under compliance with the food sanitation law and the law concerning the prevention of infections and medical care for patients of infections of japan. informed consent was obtained from all participants, which was acquired from the subjects or their legally acceptable representatives for sample donation. the personal data of the patients was anonymized. to perform extraneous study (this study) and due to the lack of written informed consent, this study obtained ethical approval from the research and ethical committees for the use of human subjects of the national institute of infectious diseases, tokyo, japan (no. ). all methods were conducted in accordance with the approved guidelines. information on the samples is given in table s . rna was extracted from % suspensions of fecal samples in phosphate buffered saline using a qiaamp viral rna mini kit (qiagen, hilden, germany). the extracted rna was subjected to sequencing as described below. sequencing was performed with sanger and next-generation sequencers. for sanger sequencing, a reverse transcriptionpolymerase chain reaction (rt-pcr) was first performed for min at • c and then min at • c, followed by a total of cycles of s at • c, s at • c and s at • c, and then a final extension of min at • c using specific primers for the vp and rdrp regions and a primescript ii high fidelity one step rt-pcr kit (takara, shiga, japan; table s ). cycle sequencing was performed for min at • c, followed by a total of cycles of s at • c, s at • c and min at • c using a bigdye terminator v . cycle sequencing kit (applied biosystems, carlsbad, california, usa). the dna sequences were analyzed using a genetic analyser (applied biosystems). full-length nucleotide sequences of the vp and rdrp regions were acquired using the primer walking method. next-generation sequencing was conducted as described previously (dennis et al., ; ide et al., ) . data analysis was performed using clc genomics workbench v . . (qiagen). contigs were assembled from the obtained sequence reads by de novo assembly. hunov genotypes were determined using the norovirus genotyping tool (version . ) and the human calicivirus typing tool (kroneman et al., ) . all full-length nucleotide sequences of the vp and rdrp regions of gii. , including information on sample collection years and no mixed nucleotides, were obtained from genbank (accessed on august, ). for the gii.p -gii. genotype, only sequences with information on sample collection years and months were used in this study. moreover, nine sequences associated with some recent outbreaks of gii.p -gii. were combined with those of the new japanese strains above. to construct time-scaled phylogenetic tree, we added representative vp sequences of all gii genotypes, including porcine nov gii (gii. , gii. , and gii. ) and other hunov gii genotypes ( strains), as well as an outgroup strain of hunov gi genotype (gi. ) to the dataset of the vp region (resulting in a total of strains). the representative rdrp sequences of the gii genotypes, including porcine nov (gii.p and gii.p ) and other hunov gii genotypes ( strains), as well as an outgroup strain of hunov gi genotype (gi.p ), were appended to produce an rdrp dataset consisting of strains ( table s ). the constructed datasets were aligned with mafft software (katoh and standley, ) . phylogenetic trees with molecular clocks were generated by the bayesian markov chain monte carlo (mcmc) method using the beast package v . . (bouckaert et al., ) . best substitution models (gtr+Γ +i for both vp and rdrp) were determined for the constructed datasets ( strains for vp and strains for the rdrp) by comparison of the bayesian information criterion (bic) values using jmodeltest software (guindon and gascuel, ; darriba et al., ) . appropriate clock and tree prior models (relaxed clock exponential and coalescent exponential population tree prior for both vp and rdrp) were selected by path-sampling/stepping stone-sampling marginal-likelihood estimation using the beast package (baele et al., ) . the mcmc runs were conducted with chain lengths of , , steps with sampling every , steps for vp , and with chain lengths of , , steps with sampling every , steps for rdrp. the analyzed data was evaluated by the effective sample sizes (ess) values using tracer software, and values of or more were accepted. maximum clade credibility trees were generated by discarding the first % of trees (burn-in) using treeannotator v . . in the beast package. the time-scaled phylogenetic trees were visualized by figtree v . . software. branch reliability was supported by highest posterior densities (hpds) of %. moreover, the evolutionary rates of gii.p -gii. strains were estimated as described above. the analyzed parameters with substitution, clock, and tree prior models are shown in table . in this study, the evolutionary rates of overall clusters in the gii.p -gii. strains were calculated since the rate of a cluster could transition of effective population sizes was estimated by a bayesian skyline plot using the beast package v . . . appropriate substitution models were selected based on comparison of the bic values in the dataset, including strains of the kawasaki variant in the vp region. the clock models were appropriately selected on the basis of path-sampling/stepping stone-sampling marginal-likelihood estimation. the mcmc was run on chain lengths of , , steps with sampling every , steps. after evaluation based on the ess values, the bayesian skyline plot was generated by tracer software. phylogenetic trees were created using the maximum likelihood method in mega software (kumar et al., ) . best substitution models (gtr+Γ +i for vp and k +Γ for rdrp) were selected using the jmodeltest . branch reliability was supported by , replications of bootstrap values. phylogenetic distances between gii. strains were calculated by patristic software (fourment and gibbs, ) . were generated based on homology modeling using the modeller software v . (webb and sali, ) . three crystal structures for vp (pdbid: ihm, f m and lkc) and one for rdrp (pdbid: sh ) were used as the templates based on basic local alignment search tool (blast) analyses of the proteins. amino acid sequences of the templates and the target strains were aligned using mafftash software (standley et al., ; katoh et al., ). the constructed structures were minimized using the gromos , implemented by swiss pdb viewer v . and evaluated by ramachandran plots via the rampage server, which showed the favored regions of . % ± . (vp ) and . % ± . (rdrp), the allowed regions of . % ± . (vp ) and . % ± . (rdrp), and the outlier regions of . % ± . (vp ) and . % ± . (rdrp) (mean ± sd) of all residues in each structure (guex and peitsch, ; lovell et al., ) . the final models were modified and colored using chimera v . (pettersen et al., ) . conformational epitopes on the vp dimer structures of the gii. strains were predicted using discotope . , epces, and epsvr programmes (liang et al., (liang et al., , kringelum et al., ) . cut-off values were set at − . for the discotope . and for the epces and epsvr in order to encompass epitopes numbers of strains substitution models clock models tree prior models evolutionary rates ( % hpd intervals) on the vp of gii. identified by a previous in vitro study (lindesmith et al., ) . consensus sites identified by more than one of the three tools and regions with two or more closely apposed residues of the sites on the vp dimer structures were determined to be conformational epitopes. positive selection sites in the vp region of the gii. strains were predicted using the fast, unconstrained bayesian approximation (fubar) and mixed effects model of evolution (meme) algorithms on the datamonkey server (pond and frost, ; delport et al., ; murrell et al., murrell et al., , weaver et al., ) . generally, fubar hypothesizes constant selection pressure at each site for the entire phylogeny and utilizes a bayesian approach to estimate non-synonymous (dn) and synonymous (ds) substitution rates at each site. meme is used to detect positive selection sites under a proportion of branches. significance levels were set at posterior probabilities of > . for fubar and p < . for meme. transmission links between gii.p -gii. strains were analyzed using a median joining network with popart software (bandelt et al., ; leigh and bryant, ) . we constructed a dataset that covered nucleotide sequence lengths from the start position of rdrp to the terminal position of vp (resulting in a total of strains). the dataset was then processed to detect recombination between the rdrp and vp sequences based on seven primary exploratory recombination signal detection methods (rdp, geneconv, bootscan, maxchi, chimera, siscan, and seq) using rdp software (martin et al., ) and the threshold of the p-value for significance was set at . . recombination regions were assigned when they were identified by more than four of the seven methods; however, this criterion resulted in the identification of no recombinant sequences in the dataset. the median joining networks were analyzed using an epsilon value of zero. time-scaled phylogeny of the vp and rdrp regions in the gii.p -gii. strains we constructed time-scaled phylogenetic trees using the mcmc method based on the full-length of the vp ( strains) and rdrp ( strains) regions in the gii. strains detected in the various countries (figures , ) . the mcmc tree for the vp region estimated that the common ancestor of the gii. , gii. , and gii. diverged in september, ( % hpd january, -february, . a common ancestor of the gii. strains diverged in november, ( % hpd september, -november, ) and further diverged into seven clusters by around october, . of these, the gii.p -gii. strains belonged to clusters and (figure ) . the gii. strains in clusters , , , and (no information on the rdrp sequence in cluster ) were composed of the distinct rdrp genotypes, including gii.p , gii.p , gii.p , and gii.pe, respectively (figure and table s ). most strains of the gii.p -gii. belonged to the cluster (the kawasaki type). the common ancestor of the cluster diverged in october, ( % hpd may, -august, , while that of cluster (the kawasaki type) emerged in july, ( % hpd july, -september, ) (figure ) . moreover, analyses of phylogenetic distances exhibited no overlap between intra-and inter-cluster peaks at a value of . ( figure a ).with respect to the rdrp region (figure ) , the common ancestor of the gii.p and gii.p diverged in january, ( % hpd december, -may, ). subsequently, the common ancestor of the gii.p diverged in october, ( % hpd may, -april, and then further into two clusters by around august, . the gii.p -gii. strains were contained in clusters and in the rdrp region, which was compatible with the classification in the vp region. most strains of the gii.p -gii. also belonged to cluster (the kawasaki type). the common ancestor of cluster diverged in august, ( % hpd december, -january, ), while that of cluster (the kawasaki type) did so in april, ( % hpd september, -october, . furthermore, phylogenetic distances overlapped between the intra-and inter-cluster peaks ( figure b) . these results suggest that the gii.p -gii. strains emerged and formed two variants in approximately the past years. phylodynamics of gii.p -gii. strains in the vp region we estimated the transition of population sizes of gii.p -gii. in the vp region using a bayesian skyline plot. the population of this region increased sharply in around and remained constant from after an immediate reduction (figure ) . these results suggest that this was compatible between the fluctuation of the plot and actually epidemiological gii. prevalence. conformational epitopes and selective pressures in the vp protein were analyzed in order to assess the possibility of antigenicity changes at pre and post emergence of gii.p -gii. based on the time-scaled phylogeny for the vp region in figure . as a result, common epitopes among strains of clusters , (gii.p -gii. clusters) and [a closely related cluster (gii.p -gii. strain) to the gii.p -gii. clusters] were found in the shell domain and the protruding (p ) domain. although the amino acid epitope positions were similar among the strains, many amino acid substitutions were identified ( figure and table ). substitutions in epitopes were also identified in strains belonging to the same cluster. of these substitution residues, amino acids (aa) , aa , aa , aa , and aa are located close to the histo-blood group antigen figure | that comprise the dimer structures is colored gray (chain a) and dim gray (chain b). the predicted epitope regions of the strains are circled in black and the amino acids of the epitopes with no substitutions are colored blue for the shell domain and cyan for the p domain. positive selection sites are colored yellow with the positions under the order of alignments. orange circles represent the hbga binding sites on the structures. amino acid substitutions on the epitopes, non-epitopes, and positive selection sites within the clusters are colored green, magenta, and red, respectively. and arg leu/glu/pro). of these sites, aa and aa were at predicted epitopes ( figure ) . these results indicate that gii.p -gii. could evolve with changes in antigenicity and binding affinity to hbgas. to assess the possibility of changes of rna replication properties at pre and post emergence of gii.p -gii. based on the timescaled phylogeny for the rdrp region in figure , we mapped the amino acid substitutions between gii.p strains and other rdrp genotypes onto three-dimensional structures of the rdrp protein (figure ) . a total of amino acid substitutions were identified among the gii.p and gii.p strains (accession number; fj ). of these, the substitutions at aa , aa , aa , and aa were located close to the interface between monomers, while substitutions at aa , aa , aa , aa , and aa were located close to the active site ( figure a) . moreover, a total of amino acid substitutions were estimated among the gii.p and gii.p (a closely related genotype to gii.p ) strains (accession number; kj ). the substitution at aa was located adjacent to the interface between monomers, whereas the substitutions at aa , aa , aa , and aa were located proximal to the active site ( figure s ). for intra-gii.p genotype, phe leu and lys arg substitutions were found around the active site in the strains of cluster ( strains) ( figure b ). thirty one substitutions were also found in the strains belonging to the cluster ( strains), and a ser asn substitution was located proximally at the interface between monomers. ile val, ile val, asp glu, ile val, thr ala, val ile, asn ser, val met, gly ser, and phe leu substitutions were located close to the active sites ( figure c ). links in circulation of prevalent gii.p -gii. (cluster ) in japanese regions and in other countries were analyzed using a median joining network. the strains analyzed in this study were divided into four clusters. strains belonging to cluster were mainly found from japan, while those belonging to clusters and were found in various countries including japan, china, hong kong, taiwan, and australia. the strains in cluster were detected only in miyagi prefecture in japan. notably, the network contained the key strains that were linked to the many other strains (figure ) . these results suggest that the gii.p -gii. strains form a broad network and that some strains are associated with the prevalence of the virus. in this study, we demonstrated the molecular evolution of both the vp and rdrp regions of gii.p -gii. since the emergence of the virus. first, a mcmc time-scaled evolutionary phylogenetic tree based on gii. vp nucleotide sequences showed that all the present strains grouped into a total of seven clusters (figure ) . trees also indicated that the recently emerged gii.p -gii. strains uniquely formed clusters (clusters and ) based on both the vp and rdrp regions. the common ancestor of the vp region of the gii.p -gii. viruses diverged first, followed by divergence of the rdrp region after a severalyear delay (figures , ) . previous reports have also suggested a gap in the divergence of gii.p -gii. strains (mizukoshi et al., ; nagasawa et al., ) . to resolve this issue, we analyzed the phylogenetic distances of the vp and rdrp regions and found that the distance of the vp region was longer than that of rdrp, indicating that the genetic diversity of vp in gii.p -gii. is larger than that of rdrp (figure ) . however, the evolutionary rates for the overall cluster in gii.p -gii. were similar between the vp and rdrp regions (table ) . thus, we also calculated the rates of the strains belonging to the cluster , which resulted in reduction of the value in the rdrp region, but not in the vp region (data not shown). these results suggest that the differences of divergent times and genetic distances between these regions may be associated with those in the changes of the evolutionary rates. furthermore, we analyzed the time-scaled mcmc phylogenetic trees by adding sequences of gii. for the vp , sequences of gii.p for the rdrp and data of not only collection years but also months, which produced confidence intervals ( % hpd values) that were smaller in our analyses than in an earlier report (sang and yang, ) . thus, more precise collection data, as well as the use of a large number of virus strains, may facilitate the construction of more precise mcmc phylogeny. the evolutionary rates of the vp and rdrp regions in the gii.p -gii. were ∼ . and ∼ . × − substitutions/site/year, respectively ( table ) . the rate for gii. is likely similar to that for gii. in the vp region, but lower than that of gii. (bok et al., ; siebenga et al., ; mizukoshi et al., ; motoya et al., ) . furthermore, the rate for gii.p is likely analogous to that for gii.p , but lower than that of gii.p ozaki et al., ) . interestingly, it has been suggested that gii.p -gii. virus prevalence is associated with the rapid evolution of the vp regions with a high ratio of non-synonymous amino acid substitutions to synonymous substitutions (bull et al., ; parra et al., ) . previous reports have also suggested that the rates of non-synonymous amino substitutions in the vp protein differ between gii. and gii. , with gii. being greater than gii. (mori et al., ) ; therefore, the rates of antigenicity change between gii. and gii. viruses may be distinct. in contrast, it has been estimated that the number of negative selection sites in gii.p rdrp are larger than that in gii.p (ozaki et al., ) , and thus variations in nucleotide substitution in gii.p rdrp may be restricted compared to gii.p . however, despite such restrictions, we observed a lower evolutionary rate in gii.p than gii.p . this may be due to differences in replication activities and/or in replication errors of the rdrp protein between gii.p and gii.p . additional in vitro studies may be needed to address these possibilities. we also assessed the phylodynamics of gii.p -gii. and found that the genome population sizes of gii.p -gii. increased rapidly during - and then decreased rapidly thereafter (figure ) . these fluctuations may be associated with epidemics of the newly emerged hunov and the acquisition of herd immunity. for example, the fluctuations in the phylodynamics of the gii. virus could be associated with the emergence of a variant virus with altered antigenicity and the acquisition of herd immunity to the vp capsid protein of that virus lindesmith et al., ; motoya et al., ) . thus, it is possible that the phylodynamic fluctuations in gii.p -gii. identified in this study are associated with epidemics in various regions and subsequent acquisition of herd immunity (chan et al., ; fu et al., ; matsushima et al., ; dang thanh et al., ) . these results suggest that continuous phylodynamic analysis may provide early notice of other hunov epidemics and repeat prevalence of hunov, including gii. . we additionally found amino acid substitutions in the conformational epitopes of the vp capsid protein of gii.p -gii. ( figure and table ). such substitutions were also identified around the hbga binding sites. such evolutionary substitutions may induce both changes in antigenicity and in hbga binding ability (tan et al., ; chen et al., ; de rougemont et al., ; lindesmith et al., ; debbink et al., ; jin et al., ) . indeed, lindesmith et al. showed that some amino acid substitutions (corresponding to aa -aa in our alignments) are located adjacent of the hbga binding sites and result in changes in the antigenicity of the gii. capsid protein (lindesmith et al., ) . jin et al. also reported changes to the antigenicities and hbga binding affinities of gii. capsid proteins belonging to different phylogenetic clusters (jin et al., ) . moreover, an amino acid substitution (aa ) located adjacent the hbga binding sites was identified as a possible positive selection site. previous reports have suggested that aa and aa are associated with the hbga binding ability (he et al., ; koromyslova et al., ) ; however, to the best of our knowledge, there have been no reports regarding possible roles of the aa substitution in antigenicity and the hbga binding capability, which is a subject for possible further study. we also showed that gii.p -gii. contains amino acid substitutions in residues adjacent to the rdrp active sites and the contact surfaces between rdrp monomers (figure ) . ng et al. showed that the amino acids around the active sites regulate viral genome replication (ng et al., ) . moreover, substitutions around the monomer the residues of active sites for rna replication are colored red. the purple quadrilaterals highlight the region of the active site cleft. figure | a median joining network for the major epidemic cluster of gii.p -gii. strains based on nucleotide sequences from the rdrp to vp ( bp). the detected areas for japanese strains used in the analysis are shown with the number of strains on a japanese map. the filled circles represent viral haplotypes, including collection years and months for the strains. the sizes of the circles represent the number of strains within the haplotype. red arrows indicate strains associated with the production of many haplotypes. contact surfaces of rdrp affect the stability of the dimer and its rna binding abilities . thus, the similar substitutions found in the present study warrant additional investigation into changes in the properties of the rdrp protein. previous studies have also revealed key amino acids associated with the efficiency of hunov genome replication (bull et al., ; eden et al., ) . of these, the threonine residue at aa is phosphorylated by a host factor, akt, and is involved in producing high replication rates (eden et al., ) . this residue is located around the interface between monomers in the rdrp structure. the rdrp protein of gii.p contains a threonine residue at the position, while that of gii.p contains an asparagine (data not shown). this may suggest that the enzyme activity of rdrp differs between the gii.p and gii.p genotypes. we constructed a genome network of the gii.p -gii. strains examined in this study and found four major clusters (figure ) . notably, the strains belonging to clusters and were detected exclusively in japan, while the strains belonging to clusters and were detected from various countries. moreover, the two haplotype strains found in japan might give rise to many variant types. however, we could not determine specific amino acid substitutions in the strains belonging to clusters and , which contained the haplotype strains (data not shown). thus, these haplotype strains may exhibit no phenotypes acquiring high infectivity due to the mutations, although the reason for this remains unclear at present. moreover, the strains belonging to cluster had unique amino acid substitutions in p , p , protease, rdrp, and vp proteins (data not shown). these viruses were found only in miyagi prefecture in japan, during short periods and was never detected in other areas, perhaps because these mutations are deleterious to the propagations of the virus. as a limitation, the results of the present analyses may partially be affected by selection bias introduced in the collection of the strains. in conclusion, the gii.p -gii. virus has evolved differently to gii.p -gii. . however, this virus may have the potential to alter its antigenicity, host-binding capability (i.e., hbga) and genome replication efficiency. such changes could recurrently generate variants of gii. with the potential to produce pandemics such as those caused by gii. variant strains. thus, additional and continuous evolutionary analyses of this genotype should be needed in the future. the datasets generated for this study can be found in the genbank and the accession numbers are as follow; lc -lc , lc , lc , lc -lc , lc -lc , lc -lc . the studies involving human participants were reviewed and approved by the research and ethical committees for the use of human subjects of the national 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conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited we would like to thank enago for the english language review. the supplementary material for this article can be found online at: https://www.frontiersin.org/articles/ . /fmicb. . /full#supplementary-material key: cord- - e u authors: paploski, igor adolfo dexheimer; corzo, cesar; rovira, albert; murtaugh, michael p.; sanhueza, juan manuel; vilalta, carles; schroeder, declan c.; vanderwaal, kimberly title: temporal dynamics of co-circulating lineages of porcine reproductive and respiratory syndrome virus date: - - journal: front microbiol doi: . /fmicb. . sha: doc_id: cord_uid: e u porcine reproductive and respiratory syndrome virus (prrsv) is the most important endemic pathogen in the u.s. swine industry. despite control efforts involving improved biosecurity and different vaccination protocols, the virus continues to circulate and evolve. one of the foremost challenges in its control is high levels of genetic and antigenic diversity. here, we quantify the co-circulation, emergence and sequential turnover of multiple prrsv lineages in a single swine-producing region in the united states over a span of years ( – ). by classifying over , prrsv sequences (open-reading frame ) into phylogenetic lineages and sub-lineages, we document the ongoing diversification and temporal dynamics of the prrsv population, including the rapid emergence of a novel sub-lineage that appeared to be absent globally pre- . in addition, lineage was the most prevalent lineage from to , but its occurrence fell to . % of all sequences identified per year after , coinciding with the emergence or re-emergence of lineage as the dominant lineage. the sequential dominance of different lineages, as well as three different sub-lineages within lineage , is consistent with the immune-mediated selection hypothesis for the sequential turnover in the dominant lineage. as host populations build immunity through natural infection or vaccination toward the most common variant, this dominant (sub-) lineage may be replaced by an emerging variant to which the population is more susceptible. an analysis of patterns of non- synonymous and synonymous mutations revealed evidence of positive selection on immunologically important regions of the genome, further supporting the potential that immune-mediated selection shapes the evolutionary and epidemiological dynamics for this virus. this has important implications for patterns of emergence and re-emergence of genetic variants of prrsv that have negative impacts on the swine industry. constant surveillance on prrsv occurrence is crucial to a better understanding of the epidemiological and evolutionary dynamics of co-circulating viral lineages. further studies utilizing whole genome sequencing and exploring the extent of cross-immunity between heterologous prrs viruses could shed further light on prrsv immunological response and aid in developing strategies that might be able to diminish disease impact. porcine reproductive and respiratory syndrome virus (prrsv), the etiological agent of prrs, is one of the most important endemic viruses affecting the swine industry in the united states (holtkamp et al., ) and globally (stadejek et al., ; vanderwaal and deen, ) . the economic impact of the disease in the united states has been estimated at $ million annually (holtkamp et al., ) . clinical signs in affected farms vary by viral variant and according to the farm's production stage (e.g., breeding or growing herd), herd management, immune status, and other factors (goldberg et al., ) . premature farrowing can occur in - % of sows in an affected farm, and up to % of piglets are stillborn during an outbreak (christianson and joo, ) . piglets may be born with low weight and can present with lethargy and anorexia, which can lead to a mortality of more than % among piglets (pejsak et al., ) . prrsvinfected pigs are also susceptible to secondary infections leading to poor average daily gain and feed conversion, further increasing production loss (solano et al., ; xu et al., ) . up to % of united states breeding herds experience outbreaks annually (tousignant et al., a) and control of the disease in the united states, europe, and globally is challenging due to high levels of antigenic variability and its rapidly expanding genetic diversity (frossard et al., ; brar et al., ; guo et al., ; smith et al., ) . porcine reproductive and respiratory syndrome virus was first recognized almost simultaneously in europe (wensvoort et al., ) and north america (collins et al., ) in the late s and early s, but genetic differences suggested a much earlier evolutionary divergence between the north american and european viral types. thus, prrsv is divided into two major phylogenetic clades, prrsv type (more prevalent in europe) and type (more prevalent in north america) (shi et al., a,b; stadejek et al., ) . within each clade, high levels of genetic and antigenic diversity exist and cross-protection is only partial (roberts, ; kim et al., ; correas et al., ) . genetic similarities between prrsv isolates have been used as a tool to understand disease transmission and epidemiology (kapur et al., ; wesley et al., ) , and several different strategies have been used for classifying isolates of prrsv into epidemiologically meaningful groups. for prrsv type , the most commonly used classification system is based on restriction fragment length polymorphisms (rflp) and sequencing, both of which are typically based on the open reading frame (orf ) portion of its genome (kapur et al., ; wesley et al., ) . the orf gene encodes for the major envelope protein (gp ), which plays a role in inducing virus neutralizing antibodies and cross-protection among prrsv variants (dea et al., ; kim et al., ) . rflps have been broadly adopted by the u.s. swine industry despite shortcomings, such as the fact that the genetic relationship between different rflp types is unclear, the potential for two distantly related viruses to share the same rflp type, and the instability of rflp-typing when assessing isolates related to each other by as few as animal passages (cha et al., ) . in , a classification system based on the phylogenetic relatedness of the orf portion of the virus's genome was proposed (shi et al., a,b ). this classification system aggregates isolates into phylogenetic lineages based on the ancestral relationships and genetic distance among isolates. using this system, nine different lineages were described within prrsv type , each of which was estimated to have diverged between and (shi et al., b) . phylogeny-based classification of organisms is seen as the most powerful and robust instrument for distinguishing between variants of a viral population (hungnes et al., ) and has been used in the study of other viral diseases (liu et al., ) . phylogeny-based classification of prrsv, rather than rflp profiling, is expected to provide fewer ambiguities and more insight into the evolutionary relatedness amongst different variants. while the existence of prrsv lineages is well established, the dynamics of their cocirculation within a given region has not been well documented. vaccination is often used as a tool to mitigate clinical impact and viral shedding (holtkamp et al., ) . although specific practices vary across farms, gilts are typically vaccinated before entering the herd, and sometimes the sow herd is mass vaccinated during the year. most commercial prrsv vaccines currently sold in the united states are considered "modified live vaccines" (mlv), which means that the vaccine is an attenuated live virus. vaccines against prrsv show different degrees of protection against homologous and heterologous challenges (cano et al., ; díaz et al., ; geldhof et al., ) ; the exact definition of what constitutes a homologous or heterologous challenge is often not clear, especially taking into consideration the genetic diversity existing within prrsv type (shi et al., b) . five major prrsv vaccines are commercialized in the united states, each developed using a different wild prrsv isolate (lineages , , , and , with the lineage vaccine being the most widely used historically). porcine reproductive and respiratory syndrome virus is known to possess a high mutation rate (hanada et al., ; brar et al., ) . genetic mutations for prrsv are thought to result from rna polymerase errors (murtaugh et al., ) and from the lack of proofreading (kappes and faaberg, ) . coupled to that, genetic recombination events can contribute to prrsv diversity (forsberg et al., ) . thus, the emergence of new variants of prrsv is expected to occur potentially through both mutation and recombination. viral variants can quickly emerge in animals (goldberg et al., ) even after inoculation with a single variant (chang et al., ) . thus, the viral population within an animal can be referred to as a viral cloud or swarm (lauring and andino, ) , which suggests that mutation has a considerable impact in virus diversification even on short time scales. in addition, it is assumed that the immune response removes genetic variants of the virus that it recognizes with high specificity, potentially creating selection pressure favoring antigenically divergent prrsv variants (murtaugh et al., ) . hypervariable portions of the viral genome may be subject to immune selective pressure (chen et al., ) ; variation in proteins coded by those sites may play a role in evasion of host immune defenses (ansari et al., ; darwich et al., ) . prrsv vaccines are known to diminish the severity of clinical signs once an infection occurs, but not to prevent an infection from occurring (lyoo, ) . at the population scale, it can be expected that most animals have some level of immunity because of the high prevalence of natural infection and widespread use of vaccine. this creates the potential for immune-mediated selection to be a driver of prrsv diversification and evolution (murtaugh et al., ) . the identification of point mutations that are undergoing positive selective pressure is often interpreted as evidence of increased evolutionary fitness (kryazhimskiy and plotkin, ) . one way to identify such sites is to evaluate dn/ds ratios, which measure the rate at which substitutions at non-synonymous sites (dn) occur relative to substitutions in synonymous sites (ds). substitutions in synonymous sites are thought to be mostly neutral, but a higher occurrence of substitutions in nonsynonymous sites can be interpreted as evidence of selective processes that favor changes in the protein sequence (kosakovsky pond and frost, ) . positive selective pressure in sites that code for epitopes recognized by the host immune system are of special interest, because they suggest that the origin of such selective pressure, if present, could be driven by the host immune response. the rapid evolution of prrsv coupled with the periodic emergence of new and sometimes more virulent viral variants creates a need to continually update our knowledge on circulating prrsv variants. reports that show the waxing and waning of different viral types in the whole north america (shi et al., b) are helpful when understanding continent-wide status of prrsv lineages. however, understanding viral dynamics on a regional scale could provide important insights into local evolutionary and ecological dynamics of prrsv, including an improved understanding of how often new variants emerge or re-emerge within the region. here, we describe the temporal dynamics of prrsv occurrence in a swine-dense region of the united states, characterizing these patterns according to orf genetic lineages and sub-lineages. we quantify the contemporary occurrence of each lineage, investigate the temporal dynamics and turnover of lineages, identify emerging sub-lineages, and examine evolutionary patterns for evidence of positive selective pressures. monitoring project (mshmp) were used for this analysis. briefly, mshmp is an ongoing voluntary producer-driven nation-wide monitoring program for endemic swine diseases that affect the u.s. swine industry. based at the university of minnesota (umn), this program collects weekly reports on the infection status of sow farms from participating swine-producing companies, veterinary practices, and regional control programs, which serves to capture the occurrence of infectious diseases in the country (tousignant et al., a,b; perez et al., ) . infection status data classifies farms into the following categories (holtkamp et al., ) : status : positive-unstable, status : positive-stable, either through use of live virus inoculation ( lvi) or use of vaccines ( vx); status : provisional negative; and status : negative. the main difference between positive-unstable (status ) and positive-stable (status vx or lvi) is that unstable herds have an active clinical outbreak and are weaning prrsv rt-pcr positive piglets. in contrast, prrsv may be still present in positive-stable herds (through use of field virus inoculation or modified live vaccine) but clinical disease is controlled and piglets weaned from such farms are prrsv-negative as a result of herd immunity, decreased shedding, and maternal antibodies (holtkamp et al., ) . mshmp collects farm-level data from approximately . million sows, which represents approximately . % of the united states breeding herd population (national agricultural statistics service [nass] , agricultural statistics board, and united states deparment of agriculture [usda], ). specific production systems (companies involved in pig production) participating in the project also share the orf prrsv sequences identified on their farms as part of routine veterinary management. for example, samples may be submitted by veterinary practitioners to determine if circulating prrsv on the farm is the same or different from the vaccine virus or a previous variant present on the farm. for this analysis, we analyzed , sequences reported between and from mshmp participants located in a relatively isolated swine-dense region in the united states with an approximate area of thousand square kilometers. production systems operating in this region account for ∼ % of the united states sow population. approximately % of farms within this region participate in mshmp and in this project in particular. sequences used in this study came mostly from sow ( . % of sequences), nursery ( . %) and finisher farms ( . %), followed by boar stud farms ( . %) and sequences without a description of their origin ( . %). sequences shared with us by project participants were sequenced according to standardized protocols adopted by laboratories at sdsu (animal disease research and diagnostic laboratory et al., ), isu (zhang et al., ) and eurofins genomics. of the orf gene sequences used in this analysis, seven had fewer than nucleotides. these were deemed incomplete and were excluded from further analysis. we also included orf gene sequences previously classified into nine different genetic lineages (shi et al., a,b) and added these to the collection of mshmp sequences. these sequences, assembled from a database of sequences that spanned from to , were used as guides to classify the mshmp sequences into the previously described genetic lineages, and will be referred to here as "anchor" sequences. we also obtained the orf gene sequences for five vaccines (ingelvac prrsv atp -genbank id dq . , ingelvac prrsv mlv -genbank id af . (both from boehringer ingelheim), fostera prrsv from zoetis -genbank id kp . , prime pac prrsv rr from merck -genbank id dq . , and prevacent, from elanco -genbank id ku . ). the ingelvac prrsv atp and fostera vaccines use isolates belonging to lineage , while ingelvac prrsv mlv uses a lineage isolate, prime pac a lineage isolate and prevacent a lineage isolate. we also obtained two prrsv prototypes (lelystad -genbank id nc_ . , and vr -genbank id ef . , which represent the prototypical european type and north american type viruses, respectively). the sequence dataset used here is sequences were aligned using the muscle algorithm implemented in aliview (larsson, ) using default settings. the alignment was then examined for the presence of recombinants using the recombinant detection program version (martin et al., ) , followed by removal of potential recombinants. in addition, duplicated sequences (with % nucleotide similarity) were identified and set aside for the allocation of sequences into lineages. the aligned and cleaned dataset was imported into mega (kumar et al., ) , where the genetic pairwise distance was measured as a percentage nucleotide difference. using stata (statacorp, ), each of the mshmp sequences were assigned to the lineage that had the smallest genetic distance to an anchor. after sequences were classified into lineages, the duplicated sequences were allocated to their respective lineage group according to the sequence with % similarity that was kept in the lineage classification process. a flow-chart of these steps can be seen in figure . a maximum likelihood phylogenetic tree illustrating genetic relatedness of sequences was constructed based on , bootstraps, adopting the tamura-nei model for substitution of amino acids (tamura and nei, ; kumar et al., ) . clusterpicker software was used to further stratify the most abundant lineage into sub-lineages (ragonnet-cronin et al., ) , in a matter that seemed consistent with the tree main branches while still returning epidemiological meaningful sublineages. the phylogenetic tree was then colored according to the lineage classification and source of sequences (anchor versus mshmp) using microreact (argimón et al., ) . traditional bootstrap support is estimated based on resampling and replication, which tends to yield low support particularly on deep branches and in large trees with hundreds or thousands of sequences (lemoine et al., ) . branch support on the phylogenetic tree thus was evaluated using the bootstrap support by the transfer method (lemoine et al., ) . this method circumvents issues of traditional bootstrapping by assigning a gradual "transfer" index to each clade within the tree rather than a binary presence/absence index for the presence of a clade in each bootstrap (i.e., a clade is considered absent in the bootstrap replicate if the sequences found within the clade is different by even a single member). temporal changes in the frequency of different lineages was tabulated by quarter of the year. graphs representing the relative frequency of prrsv lineages over time were constructed using stata . the frequency with which each lineage occurred over different years was compared using trend analysis for proportions (using the ptrend command) in stata (statacorp, ) . for this test only, lineages with fewer than sequences overall were grouped. the ratio of synonymous to non-synonymous mutations (dn/ds) for all sites in the orf gene region was calculated using the single-likelihood ancestor counting protocol (kosakovsky pond and frost, ) , implemented on the datamonkey webserver . because the analysis can only be performed on sequences at a time, the analysis was repeated on ten random subsets of sequences (after removal of % identical sequences). sites were considered under positive selective pressure if the p-value associated with a higher rate of non-synonymous versus synonymous mutations was smaller than . . the dn/ds (re-scaled for branch length) of all sites from different runs were averaged and the percentage of runs in which each codon was identified as under significant positive selection was calculated. after removal of the seven inadequately sized and two recombinant sequences from the mshmp data, the remaining , mshmp sequences were classified in five different lineages. . % ( , sequences) were classified as lineage , . % ( ) as lineage , . % ( ) as lineage , . % ( ) as lineage , and . % ( ) as lineage . a group of . % ( ) of the mshmp sequences were genetically closer to the european prototype (lelystad) reference, and were thus classified as type prrsv sequences. lineage was further separated into five sub-lineages (a to e). out of the total , sequences in lineage , . % ( ) were classified in lineage a, . % ( ) in lineage b, . % ( ) in lineage c, . % ( ) in lineage d and . % ( ) in lineage e. the phylogenetic tree with all sequences used in the analysis can be seen on figure . using the booster method (lemoine et al., ) , branch support on main branches (lineages and sub-lineages) was above %. the within-and between-lineage nucleotide pairwise genetic distance is shown in table . in general, between lineage/sub-lineage distances are higher than within lineage variation. the distances between sublineages of lineage seem to be smaller between them than between other lineages. broad tree topology was similar when the tree was constructed using nucleotides or amino acids alignment (supplementary figure s ) . on average, the total number of sequences reported to mshmp increased by each year (supplementary table s ), and there was a clear seasonal pattern (figure b ). the first quarter of each year (january -march) was the one with highest number of sequences reported in all but year. the relative frequency of each lineage changed through time ( figure a and supplementary table s ), and specific patterns are noteworthy. first, the absolute and relative occurrence of lineage decreased over time from . % ( sequences) in to < % ( sequences) in the years - . as lineage occurrence to determine whether changes in sampling effort across time impacted general patterns observed here, we repeated the analysis five times, each time randomly sampling orf sequences per quarter. general patterns of lineage occurrence did not change, suggesting that patterns of lineage occurrence were not affected by sampling effort in each quarter (supplementary figure s ) . the visual patterns and turnover of lineages apparent in figure a were shown to be statistically significant. the increase in the frequency of lineages a, , , and type (p < . ) was significant, and changes in the grouped frequency of other lineages (a sum of lineages d, e, and , p = . ) was also significant, but with a difficult interpretation since this is an aggregate of several uncommon lineages. lineages b and c increased in frequency and then decreased (p < . ). lineage frequency decreased over time (p-value < . ), while lineage occurrence remained unchanged (p-value = . ). a total of sites were identified as under positive selection in at least one single-likelihood ancestor counting run (figure ) . some sites were identified as under positive selection in all runs, while others were only identified in some runs. those identified in all runs (with the largest p-value across all runs), were sites (p-value = . ), (p-value = . ), (pvalue < . ), (p-value < . ), (p-value < . ), (p-value < . ), (p-value = . ), and (p-value = . ). a list of all sites identified as under positive selection in at least one run can be found in the caption of figure . most of the sites positively selected were located in the first third of the prrsv orf . the infection status of farms part of mshmp in the studied area over the study time span is shown in figure . this data show two periods in which vaccine usage increased, the first one in mid- , and a second in approximately mid- . not all farms that reported its status to mshmp contributed to sequences to this analysis. we documented the circulation, emergence and sequential turnover of multiple prrsv lineages in a single united states swine-producing region over a span of years ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . by classifying over , prrsv orf contemporary sequences into phylogenetic lineages based on pre- data (shi et al., a,b) , we illustrated the continual diversification and temporal dynamics of the prrsv population. through further stratifying lineage into three main sub-lineages, we also describe the rapid emergence of a sub-lineage ( a), which was absent in the pre- analysis even though that dataset was based on > sequences from across the world (including the region in which we collected our samples) (shi et al., b) . we also identified sites within prrsv orf gene and resultant orf protein that showed evidence of positive selective pressure, indicating that non-synonymous mutations that lead to amino acid changes in the protein at these sites are favored. from to , lineage was the most prevalent genetic group observed in our dataset. shi et al., a,b showed that lineage was rapidly increasing in genetic diversity, which is a proxy for the effective population size of the virus, from to , and reached a peak from to . our data suggests that, at least for our study region, the occurrence of lineage peaked pre- , after which it rapidly declined and was replaced mostly by lineage variants. from to , three different major sub-lineages within lineage emerged, two of those being the most prevalent lineage in certain years ( c from to , a from to ). the emergence of sublineage a, beginning in and peaking in was perceived by veterinarians in the studied area as being a noteworthy event coinciding with the spread of the - - rflp-type. in our dataset, . % of the sequences belonging to the a sub-lineage were rflp-typed as - - (followed by . % of sequences with rflp - - and less than % of - - , - - , - - , - - and several others with less than % -see supplementary table s ) . while the failure to achieve consistent and reliable prrsv control and prevention through vaccination demonstrates gaps in our understanding of prrsv immunology (murtaugh, ) , based on current understanding, prrsv vaccines are expected to better protect against wild viral variants that have a higher degree of similarity to the original parental isolate used for vaccine development (cano et al., ; díaz et al., ; geldhof et al., ) . despite our limited understanding of heterologous cross-protection for prrsv, the emergence and sequential dominance of different variants leading to lineages and sub-lineages is consistent with the theory of multi-strain dynamics (gupta et al., ; kucharski et al., ) . immune responses, whether originating from human interventions or accumulation of immunity toward wild variants, can exert selective pressure that can ultimately lead to the emergence of new pathogen sub-populations (gupta et al., ) . as a virus evolves, immune responses generated against a past variant are expected to become less effective, resulting in a highly complex system, with different lineages interacting through the partial cross-immunity that they generate in the host population (gupta et al., ; kucharski et al., ) . theory predicts that due to frequency-dependent selection amongst co-circulating viral variants, rare antigenic variants are expected to spread more widely in the host population but then subsequently decline as herd immunity rises. such dynamics have been more thoroughly understood for influenza a (webster et al., ; mccullers et al., ; ferguson et al., ; nelson et al., ) and hiv (mcmichael et al., ) . for prrsv, recent research demonstrates that antibodies can exert a strong selective pressure to viral pathogens by targeting specific viral sub-populations, while allowing for the establishment of other sub-populations (wang, ) . when comparing prrsv genetic diversity before and after vaccine adoption in south korea, prrsv vaccination was suggested to increase viral genetic heterogeneity and the emergence of new glycosylation sites in viral populations (kwon et al., ) . however, the extent in which prrsv immunity, whether from natural infection or vaccination, can potentially drive the evolution of the virus in the field remains largely unanswered. our data does show a dominance of non-vaccine related lineages over time, which leads to speculation that these lineages have partially escaped the immunity induced by commercial vaccines or natural infection by variants in other lineages. prrsv vaccines do not protect against infection (scortti et al., ) , but diminish clinical signs and improve animal performance (cano et al., ) . since our project did not evaluate clinical signs of animals, it is difficult to assess the effects of vaccination in that regard. however, despite high region-wide vaccine usage from onward (figure ) , lineage a spread widely in the studied region, suggesting that vaccination and other biosecurity measures were insufficient to limit the transmission of lineage a. lineages shown (figure ) and discussed here and elsewhere are based on phylogenetic relationships in the orf region, and might not be predictive of cross-protection and immunological responses developed by hosts when faced with viruses belonging to different lineages. despite that, the lineage classification protocol used in this study did reveal temporal patterns consistent with what is expected based on epidemiological theory related to the spread of disease in immunologically naive populations. for example, epidemic-shaped curves of occurrence of different prrsv populations were seen, a pattern consistent with the spread of new pathogens (or subtypes) within a naive population. new (sub-) lineages may potentially be able to become the dominant prrsv in the population if they are sufficiently immunologically distinct to overcome herd immunity, and herds with different levels of immunity induced by pre-exposure protocols or natural infections might create selective pressure that changes how fast a new viral variant is selected in that population. for prrsv, it is apparent that protection against homologous prrsv is more robust than against heterologous variants, though the definition of what constitutes a heterologous virus is highly variable (cano et al., ; díaz et al., ; geldhof et al., ) . at the same time, genetic distance has not been shown to correlate with cross-protection, perhaps because pairwise nucleotide identity fails to capture key mutations that impact cross-protection. studies that further explore the immunological cross-reactivity among prrsv lineages are needed. with the data available in this study, it was not possible to investigate the occurrence of specific lineages with vaccination use and more precisely to which vaccine each farm/system used or to which virus was circulating previously on a specific farm. mshmp data of farms from systems that contributed sequences to this paper ( figure ) show two periods in which vaccine usage increased. the first increase in mid- , and a second in approximately mid- . the second spike in vaccine usage coincided with when lineage a began spreading in the study area. it is possible that this second spike in vaccine usage was a reaction to the shift in circulating lineages (more specifically, to the emergence of lineage a prrsv). it is also possible that the increased use of vaccines onward (shown on figure ) and the occurrence of lineages b and c (shown on figure ) immunologically selected sequences in a manner that allowed for the emergence of lineage a in . by mid- , a proportion of farms began using live virus inoculation (lvi). this strategy refers to the use of controlled exposure in gilts through inoculation with live virus isolated from recent clinical outbreak(s) at the farm (desrosiers and boutin, ) . the rationale is that by exposing gilts to virus found in a farm, gilts will mount "homologous" immunity to that specific wildtype virus and contribute to herd immunity and thus stability. according to veterinarians in the area, the increased use of lvi was due to the circulating virus being "different enough" from the viruses used in commercial vaccines. the practice of lvi in the systems here reported began primarily in (figure ). it is difficult to assess the impact that lvi might have on immunologically selecting for specific viral populations within specific lineages, especially with the aggregated data used in this analysis. while the inability of vaccination to control the spread of prrsv lends credence to immunological selection as a driver of prrsv diversification (murtaugh et al., ) , the impacts that immune-driven selection could have on long term prrsv evolution remain unknown. recording exposure procedures (lvi or vaccine use) within farms is crucial when trying to interpret longitudinal patterns of occurrence of prrsv. in future research aimed at more robustly testing hypotheses about immunity as a driver of evolutionary change, this crucial information would allow for investigation of frequencies in which specific lineages occur in farms pre-and post-vaccine/lvi adoption. within orf , we found sites under positive selective pressure within or near two hypervariable regions (figure ; hanada et al., ; delisle et al., ) located near the principal neutralizing epitope (pne). the pne is located between amino acids - and forms an ectodomain which triggers antibodies development during prrsv infection (plagemann et al., ; hanada et al., ) . the flanking hypervariable regions can be linked to the development of an immune response that block accessibility of antibodies to the pne (popescu et al., ) , including n-linked glycosylation sites such as n , n , and n (ansari et al., ) . in general terms, glycosylation may modulate protein-protein interactions, whether these proteins involve the humoral or cellular immune response of the host (lisowska, ) . in prrsv, there is evidence that these glycosylation sites play a role in glycan shielding, which is an important mechanism by which the virus evades neutralizing immune responses (vu et al., ) . while our findings do not explicitly explain the change in lineage, it does raise one hypothesis of the mechanism behind such change. further studies on how specific portions for the genome, both within orf and the whole genome, modulate immune recognition and possibly selective pressure are needed. we also consistently identified positive selective pressure within the pne region, specifically for amino acid . the identification of positive selective pressure in this region suggests that viral variants with different amino acid composition in that region may experience higher fitness and thus are favored. since this region seems to be the primary binding site of neutralizing antibodies developed during prrsv infection (plagemann et al., ; kim et al., ) , this suggests that the reason for such selective pressure could be immune in nature. such a scenario has been considered as a possible explanation for long-term evolution of rna viruses (domingo et al., ; pérez-sautu et al., ) . additional in vitro research is necessary to further clarify the immunological importance of sites identified in our analysis. however, our results suggest the plausibility of a scenario where prrsv variants with mutations in key immunological regions are able to evade immune responses and thus persist and spread within host populations with partial immunity (figure ) . further studies to investigate the role of an incomplete immunity on the evolution of prrsv are required. other mechanisms that might change the ability of the virus to infect hosts have also been proposed. non-muscle myosin heavy chain (myh ) is a molecule that has been shown to be an essential host factor for prrsv infection (gao et al., ) . myh interacts with prrsv glycoprotein (coded for by orf ), changing cell susceptibility to infection. further studies that investigate the contribution that molecules such as myh have on the infection of different orf prrsv variants are needed. additionally, non-neutralizing antibodies can delay the induction of neutralizing antibodies (ostrowski et al., ) in prrsv infection. indeed, the mean level and duration of viremia in pigs was greater among animal injected with sub-neutralizing prrsv-specific igg antibodies (yoon et al., ) , suggesting the existence of an antibody-dependent enhancement (ade) effect in prrsv. the extent in which prior exposures to the virus can elicit such effect, and how this may relate to emergence of new viral variants, also remains uncertain. as an epidemiologic study relying on secondary data generated at the population level, this study has several limitations. our sequence data were generated by different production systems that differ in number of farms, number of samples submitted, management practices, and health monitoring protocols. because of that, information may be incomplete and interpretation of data might not always be straightforward. for example, the reason for sample collection (clinical outbreak or routine monitoring), sample composition (single versus pool of animals) and type of sample (serum or tissues) is not always clear. the lack of a denominator (total amount of animals sampled in a farm, total number of farms tested) does not allow for the calculation of risk indicators for disease occurrence. data contribution by each system also varies with time. however, restricting the data to only the periods in which all systems contributed to the dataset would limit our ability to visualize long-term trends. additionally, the production system that was responsible for % of all sequences was present in the study for the entire study period. therefore, we believe that biases introduced by this issue were likely small and would not have changed the conclusions of our work. in this united states region, systems that participate in the mshmp represent approximately % of the swine farms. the remaining % of farms belong to smaller systems in the area or independent farmers. by having data from systems that represent the vast majority of farms in this region, we expect our data to be reasonably representative of prrsv occurrence in the region as a whole. additionally, despite the shortcomings mentioned above, the usage of mshmp data allows us to work with data directly from the systems, which might suffer less bias toward diseased animals than usual veterinary diagnostics laboratories data do. another limitation of this analysis involves the data generation process for the sequences analyzed here. production systems usually collect samples and send them to different diagnostic laboratories. laboratory details on quality of sequence reads were not available. these sequences most likely represent a consensus of viral sub-populations present within the host (goldberg et al., ; lauring and andino, ) , but further information that could help in assessing the quality of the read and the variability of sub-populations is not available. the sequences used here are from the orf gene alone and may not fully represent evolutionary dynamics elsewhere in the genome, since the orf gene represents approximately % of the whole genome of prrsv. studies that further explore whole genome sequencing as a tool to understand prrsv epidemiological and evolutionary patterns are required. factors affecting prrsv dynamics in specific farms are not clearly understood. we show overall temporal dynamics of prrsv in a swine-producing region of the united states, however, we have limited farm-level information. thus, we have limited ability to track turnover of viral variants within farms, though we expect this to be influenced by management practices, such as the vaccination protocol adopted by farms, the movement figure | we hypothesize that prrsv evolution is partially driven by immune-mediated selective pressure. immune-mediated pressure (either within an animal or during transmission between animals/farms) selects for escapee viral variants (inset). over time, the selection of escapees may allow for emergence of a heterologous viral populations (i.e., strains, genetic groups, or lineages) which are able to spread within the host population. in scenarios in which some method of pre-exposure is adopted, prevalence of immunity against specific types of prrsv is high (often artificially through vaccination or live virus inoculation) despite high population turnover, possibly favoring the occurrence of immune-mediated selection. of animals and personnel to and between farms, the proximity to other swine producing farms, how neighboring farms manage their animals, etc. pig production in the u.s. swine industry is characterized by multi-site pig production, which refers to segregating the breeding herd from the growing herd such that animals in each stage of production are housed at separate locations. multi-site production results in the movement of animals between different production sites, which can be located in different states within the united states (valdes-donoso et al., ; kinsley et al., ) . the role of animal movement in shaping the temporal dynamics of prrsv lineages is outside the scope of this study, but is an area of active research. in addition, the commingling of animals from different sources, which might have been previously exposed to different viral populations, may allow for the introduction of viral types prevalent in other parts of the country and also exacerbate the potential for recombination of viral populations. still, in our dataset we found evidence for recombination in only two mshmp sequences. immune interaction between infections of differing prrsv isolates remains poorly understood in swine. the vast adoption of control protocols that rely on imperfect immune response aimed mostly at reducing severity of upcoming infections (such as pre-exposure protocols with commercial vaccines or with lvi) suggests that a better understanding of the cross-immunity generated by infection with different isolates of the virus would be valuable to the industry as a whole. prospective studies that obtain sera from sow farms under different pre-exposure regimens and follow the farms through time recording prrsv occurrence would provide valuable information of potential cross-immunity in field conditions. of interest also is the better understanding of how the spread different lineages/sublineages are related to epidemiological data, for example, animal movement data and farm proximity. this might allow for a better comprehension of drivers for prrsv transmission while allowing for the evaluation of the effectiveness of practices aimed at reducing prrsv risk (dead animals disposal, manure composting, filtering the air of farms, to name a few). this study reflects data from a single united states region, which possibly does not reflect prrsv diversity and temporal dynamics of the whole swine industry in the country (shi et al., b) . that being said, the data presented here reflects a substantial portion of the u.s. swine industry in a region that is relatively spatially discontinuous from other swine producing regions in the united states. in addition, the general pattern of emergence and turnover of different lineages over time observed here describe an evolutionary phenomenon that is expected to also occur in other united states regions. a better understanding of the natural history of prrsv can provide insights that can potentially aid in mitigating the impact of the emergence of new viral variants as well as serving as a basis for further work exploring the evolution of prrsv and the effect this has on disease control, management and impact on the industry. here, we describe the occurrence of prrsv over years in a single united states region. we identified the emergence and turnover of different lineages and sub-lineages in the commercial pig population. such rapid turnover in the dominant lineage through time suggests that temporal patterns of prrsv occurrence are characterized by multi-strain dynamics, where different prrsv variants potentially interact through immune-mediated competition or selection. however, cross-immunity between different prrsv lineages elicited by natural or intentional infection is not fully understood, which hinders the effectiveness of disease control. more research is needed on drivers of evolution and emergence of new sub-lineages in order for the industry to be able to predict, prevent, and mitigate the impacts of prrsv. ongoing surveillance for prrsv using molecular epidemiological methods is invaluable to characterize the evolution of the virus but also to identify recent and historical trends that help understanding the natural history of prrsv in the united states. the sequence dataset used here is available in genbank under the accessions numbers mn -mn . ip and kv analyzed, conceptualized, and designed the study. cc, js, cv, and kv contributed to acquisition of the data. ip, cc, ar, js, cv, ds, and kv interpreted the data. mm aided in early interpretation of data. all authors but mm were involved in drafting the manuscript and revising it critically for intellectual content and have given final approval of the version to be published. dna sequencing of prrsv using orf influence of n-linked glycosylation of porcine reproductive and respiratory syndrome virus gp on virus infectivity, antigenicity, and ability to induce neutralizing antibodies microreact: visualizing and sharing data for genomic epidemiology and phylogeography genomic evolution of porcine reproductive and respiratory syndrome virus (prrsv) isolates revealed by deep sequencing evolutionary diversification of type porcine reproductive and respiratory syndrome virus effect of vaccination with a modified-live porcine 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the united states temporal and spatial dynamics of porcine reproductive and respiratory syndrome virus infection in the united states using machine learning to predict swine movements within a regional program to improve control of infectious diseases in the us global trends in infectious diseases of swine role of animal movement and indirect contact among farms in transmission of porcine epidemic diarrhea virus immune evasion of porcine reproductive and respiratory syndrome virus through glycan shielding involves both glycoprotein as well as glycoprotein immunological selection as a driver of porcine reproductive and respiratory syndrome virus evolution and ecology of influenza a viruses mystery swine disease in the netherlands: the isolation of lelystad virus differentiation of a porcine reproductive and respiratory syndrome virus vaccine strain from north american field strains by restriction fragment length polymorphism analysis of orf secondary infection with streptococcus suis serotype increases the virulence of highly pathogenic porcine reproductive and respiratory syndrome virus in pigs antibody-dependent enhancement (ade) of porcine reproductive and respiratory syndrome virus (prrsv) infection in pigs high-throughput whole genome sequencing of porcine reproductive and respiratory syndrome virus from cell culture materials and clinical specimens using next-generation sequencing technology we gratefully thank the contributions that emily smith and andres perez made on early stages of the project. we would like to acknowledge the industry partners who contributed to data for this analysis and to shic and mshmp in general, especially to emily geary, involved in the mshmp data curation. the supplementary material for this article can be found online at: https://www.frontiersin.org/articles/ . /fmicb. . /full#supplementary-material key: cord- -rnjnxymc authors: kariithi, henry m.; İnce, İkbal agah; boeren, sjef; murungi, edwin k.; meki, irene k.; otieno, everlyne a.; nyanjom, steven r. g.; van oers, monique m.; vlak, just m.; abd-alla, adly m. m. title: comparative analysis of salivary gland proteomes of two glossina species that exhibit differential hytrosavirus pathologies date: - - journal: front microbiol doi: . /fmicb. . sha: doc_id: cord_uid: rnjnxymc glossina pallidipes salivary gland hypertrophy virus (gpsghv; family hytrosaviridae) is a dsdna virus exclusively pathogenic to tsetse flies (diptera; glossinidae). the kb gpsghv genome contains open reading frames and encodes more than confirmed proteins. the asymptomatic gpsghv infection in flies can convert to symptomatic infection that is characterized by overt salivary gland hypertrophy (sgh). flies with sgh show reduced general fitness and reproductive dysfunction. although the occurrence of sgh is an exception rather than the rule, g. pallidipes is thought to be the most susceptible to expression of overt sgh symptoms compared to other glossina species that are largely asymptomatic. although glossina salivary glands (sgs) play an essential role in gpsghv transmission, the functions of the salivary components during the virus infection are poorly understood. in this study, we used mass spectrometry to study sg proteomes of g. pallidipes and g. m. morsitans, two glossina model species that exhibit differential gpsghv pathologies (high and low incidence of sgh, respectively). a total of host proteins were identified, of which and proteins were significantly up- and down-regulated, respectively, in g. pallidipes compared to g. m. morsitans. whereas gpsghv proteins were detected in g. pallidipes f( ) progenies, only viral proteins were detected in g. m. morsitans. unlike in g. pallidipes, qpcr assay did not show any significant increase in virus titers in g. m. morsitans f( ) progenies, confirming that g. m. morsitans is less susceptible to gpsghv infection and replication compared to g. pallidipes. based on our results, we speculate that in the case of g. pallidipes, gpsghv employs a repertoire of host intracellular signaling pathways for successful infection. in the case of g. m. morsitans, antiviral responses appeared to be dominant. these results are useful for designing additional tools to investigate the glossina-gpsghv interactions. glossina pallidipes salivary gland hypertrophy virus (gpsghv; family hytrosaviridae) is a dsdna virus whose kb genome encodes more than confirmed proteins (abd-alla et al., , b kariithi et al., a) . the hytrosaviridae family consists of only one other member, the housefly musca domestica (diptera; muscidae) hytrosavirus (mdsghv; coler et al., ) . however, detection of hytrosavirus-like infection symptoms, i.e., the salivary gland hypertrophy syndrome (sgh) in the narcissus bulb fly merodon equestris (diptera; syrphidae; amargier et al., ) and in male accessory gland filaments of the parasitic wasp diachasmimorpha longicuadata (hymenoptera; braconidae; luo and zeng, ) implies that the hytrosaviridae potentially contains other members. the intrinsic properties of hytrosaviruses, i.e., covert chronic infection of adult stages without expression of detectable sgh symptoms, have probably hindered the discovery of other hytrosaviridae family members up until now. gpsghv is exclusively pathogenic to the tsetse fly (diptera; glossinidae), the vector of a group of neglected tropical diseases called the african trypanosomiases (mattioli et al., ) . research on gpsghv pathobiology has been hindered by a lack of an in vitro cell culture system to support the virus replication (abd-alla et al., a) . attempts to multiply gpsghv in alternative insect hosts such as m. domestica have so far been unsuccessful. the only available method to multiply gpsghv is via intra-hemocoelic injections of virus suspension in g. pallidipes (kariithi et al., b) . a mature gpsghv virion contains four distinct structural components (nucleocapsid core, tegument, envelope, and helical surface projections) composed of virally-encoded proteins . the gpsghv virion also contains hostderived cellular proteins: some are incorporated into the virus particles and may play roles in virus replication and transmission (kariithi et al., a,b) . in g. pallidipes, gpsghv is transmitted horizontally via saliva during feeding (abd-alla et al., ) and vertically (transovarial) via the fat body tracheal system and milk gland secretions (boucias et al., ) . gpsghv infection in laboratory colonies of g. pallidipes can either be asymptomatic or symptomatic with the former being the most rampant in laboratory colonies of this tsetse species (abd-alla et al., ) . however, the asymptomatic infection state can convert to a symptomatic state, leading to reproductive dysfunction and reduced fecundity in addition to sgh symptoms (abd-alla et al., ; lietze et al., ; boucias et al., ) . more than % of salivary gland (sg) proteins appear to be specifically expressed in g. pallidipes flies with overt sgh symptoms but not in asymptomatic flies (kariithi et al., ) . unlike in the laboratory tsetse fly colonies, gpsghv infection is mainly covert (latent) in wild g. pallidipes populations. occurrence of sgh symptoms have been reported in other glossina species such as g. m. morsitans (jura et al., ) and g. m. centralis (sang et al., ) . however, sgh symptoms are rare especially in species other than g. pallidipes. notably, even in g. pallidipes the occurrence of sgh symptoms is an exception rather than the rule (boucias et al., ) . the pathobiology of gpsghv in species other than g. pallidipes has not been so far investigated. whether naturally or artificially infected, the gpsghv infection rate is low, but males are more susceptible to infections compared to females (abd-alla et al., ; boucias et al., ) . after acquisition through a blood meal, gpsghv translocates to the sgs where it primarily replicates (garcia-maruniak et al., ) . in g. pallidipes, intra-hemocoelic gpsghv injection leads to significant increase in the viral titters in the whole fly, but the injected virus is not released via saliva during feeding and there is no development of overt sgh symptoms (boucias et al., ) . rather, sgh symptoms are overt in the f progenies of the infected mothers. it is yet to be confirmed in which host tissues gpsghv replicates after artificial injection. however, the current school of thought is that in naturally-infected g. pallidipes, the virus replicates in the male reproductive accessory glands (sang et al., ) and the gut (sang et al., ) but without any teratogenic effects. the pathological, morphological and ultrastructural effects of gpsghv infection in g. pallidipes sgs have been studied to considerable length (kariithi et al., (kariithi et al., , a guerra et al., ) . however, no such studies have been performed in other glossina species. further, the molecular basis for the differential gpsghv pathology in different glossina species is still unclear. here, we investigated gpsghv-induced modulation of total protein expression in the sgs of g. pallidipes and g. m. morsitans, with special emphasis on the host pathways that are potentially employed by the virus during infection. we hypothesized that gpsghv infection in glossina is under the control of host-and/or virus-encoded factors (proteins/peptides) whose interactions influence the expression or lack of overt sgh symptoms. we tested the hypothesis by comparing the sg proteomes of gpsghv-infected vs. mock-infected g. pallidipes and g. m. morsitans flies. the host (and viral) proteins identified in this study are potential targets for control of gpsghv infections in tsetse fly mass production facilities. for instance, antiviral strategies could be developed to block virus replication and egress (esfandiarei et al., ; cheshenko et al., ; chen et al., ) , prevent the establishment of virus replication complexes (saxena et al., ) and prevent development of cellular proliferation (guergnon et al., ) . such antiviral approaches are applicable in the control of virus infections in mass production of other insects. the g. m. morsitans and g. pallidipes flies used in this study were obtained from a colony maintained at the joint fao/iaea insect pest control laboratories (ipcl), seibersdorf, austria. for each treatment described below, groups of experimental flies were kept in holding cages (diameter of cm and height of cm) at a density of flies per cage and a mating ratio of : (male: female). the holding cages had netting on top and bottom for fly feeding and pupae collection, respectively. the experimental flies were reared at ± • c, - % relative humidity, h scotophase and fed on defibrinated bovine blood meals ( - min; times per week; feldmann, ) . the pupae from the sequential larviposition cycles were collected and incubated at • c until eclosion of the adult f progenies. for further analyses, male f progenies were selected from the fourth larviposition cycle (g ) based on available data that the incidence of sgh symptoms reaches % at the g (boucias et al., ) . it should be noted that males were used because they are significantly more susceptible to expression of sgh symptoms than the females (abd-alla et al., ) . to allow for development of sgh symptoms, the selected f male progenies were reared for weeks (equivalent to blood meals) under the same insectaria conditions and handled as the parents. all the treatments described here were replicated at least three times. to prepare the gpsghv inoculum, one intact pair of sgs displaying overt sgh symptoms were dissected from an adult ( -day old) male g. pallidipes fly and stored in ml of icecold sterile saline (ph . ). the sgs were then homogenized and clarified by brief centrifugation ( × g; min; • ) to remove tissue debris. the supernatants were sterilized by passing through a . -µm filter unit and the virus titters present in the filtrate were estimated by a quantitative polymerase chain reaction (qpcr) as described by abd-alla et al. ( a) . by this qpcr method, an average of × virus copies were estimated to be present in a µl aliquot of the virus preparation and was used for tsetse fly injections. for infections, teneral (newly eclosed; nonfed) female g. m. morsitans and g. pallidipes flies were artificially (intra-hemocoelic) injected with the virus preparations using a protocol described by boucias et al. ( ) . briefly, the female flies selected from the colony as described above were inoculated with either µl of the virus inoculum or µl of filter-sterilized pbs (mock infections). following the injections, the females were mated with asymptomatic males; these females were then separated from the males and subsequently maintained in the insectary until they produced the f progenies as described above. detection of viral dna in infected g. pallidipes and g. m. morsitans flies to confirm gpsghv infections, the week-old f male progenies produced by the mock-and virus-infected mothers were screened using a diagnostic pcr protocol described by abd-alla et al. ( ) . for this, genomic dna was extracted from one intermediate excised leg of individual flies using dneasy tissue kit (qiagen inc., valencia, ca). pcr amplifications were performed using primers and conditions previously described (abd-alla et al., , b , and the pcr products analyzed on % agarose gels. flies were considered to be non-infected, moderately-infected or highly infected if there were no visible bands or showed faint bands or thick bands, respectively, on agarose gels as previously described (abd-alla et al., ) . the actual virus copy numbers and the virus density levels were determined using qpcr essentially as described by abd-alla et al. ( b) . for virus density levels, the qpcr data were normalized against the tsetse β-tubulin gene . the samples with high virus infections (based on the agarose gels) were subsequently used for mass spectrometry measurements as virus-infected samples. this cut off was especially used in the case of g. m. morsitans flies, which do not usually show overt sgh symptoms. for g. pallidipes, samples with overt sgh symptoms (corresponding to samples with high infections from the agarose gels) were selected for mass spectrometry measurements. for negative controls, samples from -week-old male flies were selected from the mock-infected fly groups (confirmed to be pcr-negative). ten flies from each of the replicated treatments described above were selected for subsequent sg dissections. to prepare protein extracts, sgs from the f progenies described above were dissected days after the flies had their last blood meals to allow for full digestions (abd-alla et al., a) . from each of the selected flies, intact pairs of sgs were individually dissected, during which the occurrence of sgh symptoms was assessed. the dissected sgs were preserved (at • c) in µl sterile saline complemented with protease inhibitors (roche diagnostics, germany). then, each of the pool of pairs of sgs were homogenized using a glass/teflon homogenizer and ultrasonicated (sonifier cell disruptor, branson, ct, usa) as described by kariithi et al. ( a) . the homogenates were freeze-thawed and clarified three times by centrifugation ( × g; min; • c). the supernatants were pooled and the proteins quantified using bca protein assay (bio-rad) according to manufacturer's instructions. then, equal quantities ( ng) of the proteins (from each of the pooled sgs per each of the three replicates) were electrophoresed using % sds-page gels (invitrogen) as described by green and sambrook ( ) . the gels were stained with colloidal cbb stain (nupage novex; invitrogen). the middle sections of entire gel lanes were excised, and each of the gel lanes was divided into eight slices (equal portions) each of which was cut into approximately mm pieces. to prepare peptides for mass spectrometry measurements, the gel slices containing the sg proteins were subjected to in-gel trypsin digestions as previously described (kariithi et al., a) . briefly, after washing the gel pieces with mm ammonium bicarbonate (abc) buffer and abc buffer/ % (vol/vol) acetonitrile (acn), proteins were reduced and alkylated using dithiothreitol and iodoacetamide, followed by washing with abc/abc-acn buffers and trypsin digestions. tryptic peptides were then analyzed by liquid chromatography-tandem mass spectrometry (lc-ms/ms; lu et al., ) . to identify the sg proteins, the ms/ms spectra obtained from the lc-ms/ms measurements were searched against a tsetse fly database, a gpsghv database, a contaminant database containing sequences of common contaminants, and a decoy database constructed by reversing all protein sequences downloaded from uniprot. the ms searches were performed using maxquant v . . . (cox and mann, ) and andromeda as the database search engine (cox et al., ) . a maximum false discovery rate (fdr) of less than . was set at the peptide and protein levels. maxquant search parameters included variable oxidation of m, fixed carboxamidomethylation of c, and extra variable modifications for de-amidation of n and q. "label-free quantification" (lfq) and "match between runs" (set to min) options were enabled. de-amidated peptides were allowed to be used for protein quantification. to quantify the sg proteins, the resulting maxquant protein list was filtered to show only those proteins with a minimum of two peptides matching the same protein, of which at least one peptide was unique and unmodified. all other quantification settings were set at default. any hits to the decoy sequences and hits with modified peptides only were deleted from the list of protein/peptides groups. to easily compare abundances of the same proteins between the controls and virus-infected samples, logarithms (log ) of normalized lfqs were used. logarithms of the total intensity corrected for the number of measurable peptides (i.e., intensity based absolute quantization; ibaq) were used to compare the levels of different proteins from the same sample (mocks vs. gpsghv-infected; schwanhausser et al., ) . proteins were considered to be up-or down-regulated when their log protein abundance ratios were larger or smaller than zero, respectively. proteins were considered significantly upregulated when their log protein abundance ratios were larger than six. gene ontology annotation of the identified proteins were created using blast go v . . (conesa et al., ) . analyses of the protein motif/domain were performed using various bioinformatics softwares, including smart (schultz et al., ) and interproscan (zdobnov and apweiler, ) . we first analyzed gpsghv replication in the f progenies from virus-infected mothers. we detected varying levels of gpsghv infections in g. m. morsitans flies: non-infected, moderately infected and highly infected as evidenced by the absence of bands, faint bands and thick bands, respectively, in the agarose gels presented in the figure a . the different gpsghv infection levels we obtained for g. m. morsitans in figure were comparable to the results obtained for g. pallidipes, as well as results from our previous studies in g. pallidipes (compare with figure in abd-alla et al., ) . qpcr analysis of the samples in the "highly infected" category revealed high virus genome copies in g. m. morsitans ( figure b) . when dissected however, none of these f progenies from gpsghv-infected g. m. morsitans mothers showed any sgh symptoms. this is unlike in the g. pallidipes f progenies, which revealed % prevalence of sgh symptoms (data not shown), a result which was in agreement with our recent report (boucias et al., ) . notably, the gpsghv density levels in f progenies of g. pallidipes were significantly higher (p = . ) compared to the virus-infected g. m. morsitans f progenies ( figure c) . importantly, the virus density levels in both the mock-and the virus-infected g. m. morsitans f progenies were comparable to those of the mock-infected g. pallidipes (see figure c ). we then performed mass spectrometry on the sg protein extracts from the f progenies of g. m. morsitans flies with high viral titters and the g. pallidipes flies with overt sgh symptoms. analyses of the gpsghv-infected g. m. morsitans and g. pallidipes proteomes compared to their mock-infected counterparts resulted in unique peptides that mapped to non-redundant proteins. of these proteins, . % (n = ) were host (glossina)-specific, while . % (n = ) and . % (n = ) were from gpsghv and bacterial endosymbionts (wigglesworthia glossinidia and sodalis glossinidius), respectively. we then filtered out proteins with single and modified peptides, which resulted in proteins, of which , , and proteins were from the host, gpsghv and w. glossinidia, respectively. all the identified proteins are detailed in supplementary material (tables s -s ). when we compared the lc-ms/ms data sets obtained from the gpsghv-infected to mock-infected sgs, we found clear differential protein expression patterns in response to the virus infections in g. pallidipes and g. m. morsitans vs. their respective mock-infected controls (compare figure and figure ). from these two figures, gpsghv infection had more drastic effects in the protein expression in the sgs of g. pallidipes than that of the g. m. morsitans. in the figure , gpsghv infection in g. m. morsitans had little overall effects on the host's sg protein expression patterns, i.e., majority of the proteins (confidently identified by ≥ unique peptides per protein) were aligned around the y-axis (circled). on the other hand, a cohort of sg proteins were detectable in the proteome of gpsghv infected g. pallidipes, but were hardly detectable in the proteome of the mock-infected flies (see circled proteins in figure ) . notably, in contrast to the g. m. morsitans sg proteins (figure ), only few proteins were not significantly affected by gpsghv-infection in g. pallidipes (see proteins along the y-axis in figure ). overall, comparing the gpsghv-infected flies to their mockinfected counterparts, the majority of the host's sg proteins in g. m. morsitans had less than -fold up-or down-regulation compared to the proteome of g. pallidipes (see dotted red lines in figures , ) . we then made a more comprehensive comparison of the gpsghv-induced protein modulation by generating a log-log plot of the abundance distribution ratios of g. m. morsitans and g. pallidipes sg proteins. by combining the proteomics datasets obtained from the g. m. morsitans and g. pallidipes frontiers in microbiology | www.frontiersin.org , ) . shown are non-infected samples (no bands), moderately-infected samples (faint bands) and highly infected samples (thick bands). m is molecular marker. the pcr amplifications were performed using genomic dna extracted from single intermediate legs excised from -week old male f progenies produced by the control (mock), or virus-infected (gpsghv) flies. determination of the gpsghv copy numbers and the virus density levels by qpcr are shown in (b,c), respectively. for determination of virus copy numbers (b), -fold serially diluted viral dna (targeting odv-e gene) were used as internal standards as described by abd-alla et al., a . for determination of the virus expression levels, qpcr data were normalized using a tsetse fly housekeeping gene (β-tubulin). viral density levels in the virus-infected g. pallidipes progenies were significantly higher (p = . ) than the levels in the virus-infected g. m. morsitans flies. the values in the parentheses (c) indicate the virus density levels. letters a and b represent significant differences between the samples (i.e., there was no significant difference between samples labeled a, while a and b were significantly different). sg proteomes (figures , , respectively), the identified host proteins fell into two broad categories. the first category consisted of proteins that were down-regulated or up-regulated in either or both g. m. morsitans and g. pallidipes proteomes. the second category consisted of the proteins that were detectable in one of the two glossina species and in not the other. these categories are presented in the figure . proteins in the first category consisted of four groups: first, compared to mock-infected controls, a total of proteins were up-regulated in the gpsghv-infected g. pallidipes sg proteome but were down-regulated in g. m. morsitans ( figure a ; table s ). of the proteins, showed more than -fold upregulation in g. pallidipes proteome and they were all downregulated in virus-infected g. m. morsitans (table ) . second, proteins were up-regulated in both gpsghv-infected g. m. morsitans and g. pallidipes sg proteomes compared to their mock-infected counterparts ( figure b ; table s ). third, compared to the mock-infected controls, proteins were downregulated in the virus-infected g. pallidipes but up-regulated g. m. morsitans ( figure c ; table s ), nine of which were upregulated ≥ -fold in g. m. morsitans compared to the g. pallidipes sg proteomes (table ) . lastly, nine proteins were down-regulated in both virus-infected g. m. morsitans and g. pallidipes as measured from their proteomes ( figure d ; table s ). similarly, the proteins that were detectable in the proteome of one of the two gpsghv-infected tsetse species and not in the other fell into two main groups. first, compared to mockinfected flies, proteins were detectable in the virus-infected g. m. morsitans sg proteome but not in that of g. pallidipes (figure , x-axis), . % (n = ) of which were up-regulated (table s ) . second, proteins were detectable in g. pallidipes but not in g. m. morsitans (figure , y-axis), . % of which were up-regulated (table s ) . a closer look at the proteomics data indicate that majority of the heavily modulated proteins in the sg proteome of g. pallidipes appear to be spread over a wide range of host pathways. these pathways included, among others: atp/ubiquitin-dependent and s proteasome (ups) pathways, integrin-liked kinase pathway, transketolase pathway, hippo signaling pathway and diverse signaling pathways ( table ). in the case of g. m. morsitans, proteins potentially involved in the host's antiviral defenses appear to be quite dominant. some of the antiviral defense-related systems included induction of innate immune response via reactive oxygen species (virus degradation), the ubiquitin/ s proteasome system, v-atpase system (virus degradation via acidification of endosomes and or/lysosomes), the phagocytic engulfment system (to clear virus infection) and adaptive mitochondrial-mediated immune responses (interfere with production of progeny virus) ( table ) . frontiers in microbiology | www.frontiersin.org whereas only five gpsghv proteins (encoded by orfs sghv , sghv , sghv , sghv and sghv ) were detectable in g. m. morsitans, a total of proteins were detected in the sg proteome of g. pallidipes (see figures - ; table s ). these proteomics results reflects the findings we obtained from the qpcr assays described above. it should be noted that sghv is the most abundant of all gpsghv proteins; sghv is a high-molecular weight ( kda) viral protein, while sghv is a one of the major viral envelop proteins (kariithi et al., a) . the high abundance and large size, respectively, of these virion proteins possibly explains their detection in g. m. morsitans. further, proteins encoded by the orfs sghv and sghv have not been detected in our previous proteomic studies in g. pallidipes, and were in the current study only detectable in the g. m. morsitans proteome but not in the g. pallidipes proteome (figure ) . the failure to detect other viral proteins in g. m. morsitans does not necessarily imply complete absence of these proteins. rather, their abundances could have been too low or their detection could have been masked by the highly abundant host proteins. all the nine wigglesworthia proteins were in the detected in the sg proteome of g. m. morsitans (table s ) , probably because unlike the g. m. morsitans, the genome of g. pallidipes is not yet available. whereas four of the nine wigglesworthia proteins were up-regulated in the virus-infected compared to mock-infected flies, five were down-regulated (figure ) . the up-regulated proteins included transcription repair coupling factor, chaperone protein dnak, chaperone protein htpg, and imp-cyclohydrolase. down-regulated proteins included protein tola, dna polymerase i, exonuclease v, bifunctional enzyme glmu and a potassium transporter protein. since wigglesworthia is housed within the host's bacteriocytes (pais et al., ) , the detection of wigglesworthia proteins in the g. m. morsitans sgs suggest that these proteins likely "leak" into the hemolymph, potentially during bacteriocytes turnover, and eventually move from the hemocoel to the sgs. hytrosaviruses replicate primarily in the sg tissue of their insects hosts (garcia-maruniak et al., ) . as such, one would expect that the virus-induced modulation of the sg microenvironment (i.e., morphological and biochemical/functional features of the tissue) results in the expression of various proteins/peptides specifically to the advantage of viral replication and dissemination. whereas gpsghv in g. pallidipes occurs in both asymptomatic and symptomatic infection states, the virus infection in other glossina species is almost always asymptomatic. we have previously demonstrated the dynamics of the development of virus-induced sgh symptoms (abd-alla et al., and trans-generational transmission of the virus in g. pallidipes (boucias et al., ) . however, the case of gpsghv infection in other glossina species has not been investigated. importantly, we have so far not observed any overt sgh symptoms in the g. morsitans colony maintained at the ipcl seibersdorf, which was used as the source of the experimental flies we have described here. in the current study, the effects of gpsghv infections in g. m. morsitans were remarkably different from previous studies. for instance, virus injection into newly larvipositioned third-instar larvae of two morsitans groups resulted in varying prevalence of sgh-like symptoms in developed adults, which ranged from . % (jura et al., ) to . % (kokwaro et al., ) in g. m. morsitans, and up to % in g. m. centralis (sang et al., ) . in our case, we did not observe any sgh symptoms in g. m. morsitans, potentially because whereas we injected the virus into newly eclosed adults, the researchers in the previous studies injected the virus suspensions into larvae. we therefore conclude that the injected virus is capable of infecting and replicating during ontogeny on the sgs during pupation (as evidenced from the previous studies). further, the injected virus appears figure | abundance distribution ratios of g. pallidipes sg proteins. the figure depicts the distribution of proteins detected in the sg proteome of g. pallidipes infected by gpsghv compared to the mock-infected controls. the host proteins detected by two or more peptides per protein are shown in blue and red, respectively, while the gpsghv proteins are shown in green. the proteins that were up-regulated and down-regulated in gpsghv-infected sg are shown on the right and left sides of the y-axis, respectively. the large blue circle depicts proteins that were detectable in the sg proteome of gpsghv-infected but not in the proteome of mock-infected g. pallidipes. proteins which were not significantly modulated are depicted along the y-axis. the dotted red lines represent -fold protein regulation. ibaq denotes intensity-based absolute quantification. incapable of infecting and inducing overt sgh symptoms in fully differentiated sg cells in adults (as evidenced from our study). notably, the observed high virus titters in g. m. morsitans could represent dna replication but may not represent production of infectious viral particles. the comparable virus titters between the virus-infected g. m. morsitans and the mockinfected g. pallidipes suggest that the virus may be undergoing only partial replication in adult cells to maintain steady-state titters throughout the adulthood. this is in agreement with previous studies in g. pallidipes whereby, utilizing a diagnostic pcr, abd-alla et al. ( ) detected gpsghv pcr positives in % of colonized g. pallidipes that did not exhibit overt sgh symptoms. taken together, the analyses of gpsghv loads (by agarose gels), virus density levels (by qpcr), and protein expression (by lc-ms/ms) imply that either g. m. morsitans, at least in the sgs, is far less permissive to virus replication or that the virus undergoes limited replication in g. m. morsitans (whereby only a subset of genes are expressed) compared to g. pallidipes. potentially, unlike in g. pallidipes where sgh symptoms can be overt, we are of the opinion that gpsghv infection is entirely latent in g. m. morsitans as previously proposed by kariithi et al. ( b) . potentially, the difference in gpsghv replication, i.e., partial replication or latency in g. m. morsitans vs. active replication in g. pallidipes, explains the differences in the repertoire of proteins detected in the sg proteomes of the two glossina species. the clear gpsghv-induced differential modulation of sg protein expression in glossina raises the question of what host pathways are potentially globally regulated to facilitate successful virus infection. it is well known that for cellular entry and induction of pathogenesis, many viruses manipulate key host signaling pathways that globally regulate many cellular processes (diehl and schaal, ) . so far, we have not been able to elucidate the precise mechanism(s) through which gpsghv induces overt sgh symptoms, mainly because of a lack of cell culture system to support the virus multiplication (arif and pavlik, ) . therefore, the precise mechanisms of gpsghv infection (cellular attachment, entry, intracellular trafficking, replication, maturation, and egress) in glossina remains elusive. in an attempt to unravel the pathobiology of gpsghv, we draw inferences from other virus-host systems that have been studied so far. of the proteins we identified in this study, proteins that showed significant differential expression patterns in virusinfected flies are particularly interesting since they potentially reflect involvement in gpsghv pathogenesis. in this regard, the proteins that were significantly up-regulated in the sg proteome of g. pallidipes but down-regulated in that of g. morsitans (table ) are interesting to focus on. also important were the nine proteins found to be up-regulated in proteome of g. morsitans but down-regulated in the proteome of g. pallidipes (table ) . notably, our annotation of the nine proteins revealed that these proteins may be involved in pathways related to the host's antiviral responses to virus infection (see table and the references therein). in the following sections, we briefly discuss the potential roles of the proteins stipulated in tables , with regard to viral entry into host cells, intracellular trafficking, and evasion of host's immune response, replication/translation and cellular proliferation. for entry, some viruses attach to host cell receptors thus inducing conformational changes that cause fusion of the viral envelope with the host's plasma membrane (thorley et al., ) . this is followed by delivery of the viral nucleocapsids into the cellular cytoplasm and uncoating of the viral genome. integrinlinked kinases (ilks), which were up-regulated in g. pallidipes (d tmu ; table ), have been implicated in viral cellular entry. for instance, kaposi's sarcoma-associated herpesvirus (kshv) envelop glycoprotein b (gb) hijacks ilks to induce the fak-src-pi k-rhogtpase signaling pathway (naranatt et al., ; sharma-walia et al., ) . similar to the kshv gb envelope protein, the gpsghv sghv protein, which was detected in the current study (table s ) , contains an arginyl-glycyl-aspartic acid (rgd) motif that may interact with the host's ilks. pending experimental validations, gpsghv potentially employs an entry mechanism similar to kshv . following cellular entry, a critical phase in viral pathogenesis is intracellular trafficking of viral nucleocapsids, a process that requires intricate signaling. one of the key pathway components targeted by several viruses is the gtpase rab protein. notably, this protein was found to be up-regulated in g. pallidipes (d tma ; table ) , unlike in g. m. morsitans. gtpases regulate membrane trafficking, particularly in the formation, motility and docking of vesicles (zerial and mcbride, ) . some viruses activate gtpase-mediated pathways to facilitate their intracellular trafficking (chien et al., ) . for instance, in the absence of rab a/b, herpes simplex virus (hsv- ) was unable to traffic from the er to cytoplasmic viral assembly complexes, leading to a build-up of un-enveloped viral particles in the cell cytoplasm (zenner et al., ) . gtpases were indeed found to be up-regulated in shrimps infected with whispovirus (wu and zhang, ) , another large invertebrate dsdna virus like gpsghv. it is tempting to postulate that gpsghv up-regulates gtpases for intracellular trafficking in g. pallidipes, especially because the virus genome shares at least putative homologs with the above-mentioned large dsdna viruses, including hsv and whispoviruses (abd-alla et al., ) . apart from the knowledge that the host's sg is the primary replication organ for gpsghv (garcia-maruniak et al., ) , and that the virus is transmitted from the infected mother to the progeny via the milk gland secretions (boucias et al., ) , the precise virus replication and dissemination mechanisms are unknown. by comparing our data with the data available from other virus-host systems, it is possible to postulate theories on gpsghv replication and dissemination in glossina. some viruses modulate the mitochondrial transport machinery to provide energy necessary for replication, especially for the viruses whose genomes are a+t-rich (ohta and nishiyama, ; anand and tikoo, ) . the gpsghv genome is a+t-rich ( %; abd-alla et al., ) , implying that virus-modulation of glossina mitochondrial transport machinery is a good possibility. the mitochondrial oxoglutarate/malate carrier (ogc) protein is important for the tricarboxylic acid cycle (tca), gluconeogenesis and nitrogen metabolism (cappello et al., ) . ogc is reportedly upregulated as an adaptive response to prevent mitochondrial injury (ripoli et al., ) . thus, the up-regulation of ogc (d tm ) in g. pallidipes (table ) may be gpsghv-induced when robust virus replication occurs, especially in the event of overt sgh symptoms. another host protein targeted by viruses to facilitate replication is the transketolase (tktl)- (d tm ). we found tktl protein to be up-regulated more than -fold in g. pallidipes (see table ) unlike in g. m. morsitans. tktl provides a link between the glycolytic, pentose-phosphate, and nucleotide synthesis pathways (brault et al., ) . during active virus replication when rapid dna synthesis is required, carbohydrate molecules are channeled to the dna synthesis machinery through the tktl pathway, a process of utmost importance in proliferating tissues (chen et al., ) . this is of particular interest in this case of induction of the sgh symptoms in g. pallidipes, especially because sgh is mainly due to cell proliferation (guerra et al., ) . since the tktl pathway allows synthesis of ribose without the need of oxygen, gpsghv may highjack the tktl pathway to circumvent the need for oxygen (noch and khalili, ) , thus allowing rapid gpsghv genome replication. another host protein involved in viral replication is proteasome α- (d tp ; table ), a key protein in the atp/ubiquitin-dependent non-lysosomal proteolytic pathway. for instance, the interaction of proteasome α-subunit psma with hepatitis c virus (hcv) led to an inhibition of host protease activity and thus stimulated transcription trans-activation by hcv (krüger et al., ) . several other host proteins involved in viral replication that were detected in the current study included eif m (d tmn ; cheshenko et al., ) , molecular chaperones (e.g., hsp ; d ts ; kariithi et al., ) and s proteasome regulatory complex proteins (d tnj ; verchot, ; see table ). the hypothetical conserved protein (d trx ; table ) is % identical to the m. domestica anoxia up-regulated like protein and its expression in our case is virus-induced. mutuel et al. ( ) reported a significant induction of reactive oxygen species (ros) in the tracheal and fat body systems of lepidopteran insects early in infection with junonia coenia densovirus. the authors made this observation prior to viral replication before any detectable disease symptoms. interestingly, decrease of ros induction positively correlated with exponential phase of viral infection. it has been proposed that gpsghv may replicate in the host's fat bodies, and that the host's tracheal system provides a conduit for the virus transmission (kariithi, ) . it is likely that this and perhaps other similar proteins play roles during gpsghv replication. taken together, our data provide potential targets for future investigations of how gpsghv replicates and is disseminated in the host. upon successful cellular entry, viruses must evade the host's immune responses, a process for which the host's ubiquitin/proteasome system (ups) has significant roles. in the current study, we detected the main components of the ups, i.e., e ligase (d tnj ) and s proteasome (d trz ) ( table ) . the ups is essential for persistent infection of some viruses. for instance, plant rna viruses in the family luteoviridae (genera poleroviruses and enamoviruses) encode viral suppressors of rna silencing (vsrs) that hijack the ups components to promote degradation of key components of the host's rnainterference (rnai) system, thereby promoting virus replication (verchot, ) . dna viruses are known to be under host rnai surveillance. these include invertebrate iridoviruses (bronkhorst et al., ; kemp et al., ) , baculoviruses (jayachandran et al., ) , densoviruses (ma et al., ) , whispoviruses (huang and zhang, ) , and plant viruses (blevins et al., ) . as stated above, gpsghv infections are frequently observed, but remain asymptomatic and seldom result in sgh symptoms both in nature and in laboratory-bred glossina species. although the questions of how gpsghv infection progresses from a covert asymptomatic infection to an overt symptomatic infection are yet to be answered, we speculate that the virus is under host rnai surveillance, hence components of the host's ups systems form ideal candidates for further studies. another group of host antiviral defense proteins detected in this study were the v-atpases (d tli ; d tsc ; d tlr ; and d tlb ; table ), whose activity leads to acidification of intracellular compartments, necessary for multiple cellular processes (jefferies et al., ) . recently, lu et al. ( ) reported that over-expression of v-atpase in bombyx mori nucleopolyhedrovirus (bmnpv)-infected cells significantly inhibited viral proliferation. potentially, the acidification of endosomes and lysosomes by v-atpase renders these organelles competent for viral degradation. it is therefore not surprising that in the current study, v-atpase were up-regulated in the sg proteome of g. m. morsitans as opposed to that of g. pallidipes ( table ) as the former appear to be less permissive to gpsghv replication compared to the latter. similar to the v-atpase, mitochondrial atp synthase was down-regulated during white spot syndrome virus (wssv) infection in shrimps (wang et al., ) . the ubiquinol-cytochrome c reductase iron-sulfur subunit (rieske subunit/bc ) detected in the current study (d tr ; table ) was demonstrated to be up-regulated during the infection of anopheles gambiae by plasmodium falciparum (marie et al., ) . the up-regulation of bc in these two cases could have been due to the presence of the parasites in the mosquito, which could be a response involved in parasite resistance. the α-and β-subunits of mitochondrial processing peptidase (mpp; d trb ; table ) are homologous to the core and core proteins of the bc complex (braun and schmitz, ) . mpp and bc complex appear to have similar pathogen-induced modulation patterns, and their up-regulation in g. m. morsitans may be an adaptive antiviral host resistant response. it is still not clear how gpsghv induces cellular proliferation in the host's sg tissue. however, other studies have demonstrated that some viruses induce cellular proliferation via modulation of specific signaling pathways. protein phosphatase a (pp a; d tln ; table ) is critical in the regulation of cell proliferation, signal transduction, cytoskeletal dynamics, and apoptosis (seshacharyulu et al., ) . some viral proteins such as the small t antigen of sv specifically target and directly interact with and displace pp a's scaffolding b subunit thereby inducing cellular proliferation (guergnon et al., ) . to activate intracellular signaling pathways, some viruses use various approaches to hijack the g-protein-coupled receptors (gpcrs; d tpg ; table ), leading to enhancement of viral pathogenesis (sodhi et al., ; lin et al., ) . other viruses such as kshv encode potent and constitutively active gpcr homologs that modulate cellular proliferation (kirshner et al., ) . hypothetically, some gpsghv envelop proteins (table s ) could interact with host proteins to trigger signaling pathways resulting in hyperplasia as has been reported in other viruses such as the fowl poxvirus (afonso et al., ) . it is interesting to experimentally validate whether these virus and/or host proteins are actually involved in the development of sgh in glossina. an important step during assembly of viruses is processing of viral mrnas, which in some cases involve the host transacting splicing factors such as serine/arginine-rich proteins (srps; akopian et al., ) . to ensure production of their own protein diversity, adenoviruses, hsv- , influenza a viruses (iav) and hiv manipulate mrna splicing by phosphorylating srps (estmer-nilsson et al., ; sciabica et al., ; fukuhara et al., ; dubois et al., ) . therefore, it not surprising that in the current study, srp (d tn ) was up-regulated in gpsghv-infected g. pallidipes (active viral replication), but down-regulated in g. m. morsitans (less permissive to viral replication) ( table ) . however, it is currently unknown how gpsghv mrnas are processed. another host protein involved in viral assembly is tailless-complex polypeptide protein- (tcp- ; d tp and d tmk ; table ). for instance, tcp- has been implicated in the assembly of hepatitis b/c virus capsids (lingappa et al., ; inoue et al., ) , while annexins (d trs and d tme ; table ) are involved in the hiv- assembly in lipid rafts (harrist et al., ; saxena et al., ) . other proteins that may be involved in viral assembly include the s proteasome non-atpase regulatory subunit (atpase ; d tn ), which was down-regulated as seen in the sg proteome of g. pallidipes (table ) . potentially, this protein may regulate (by blocking) degradation of viral proteins. a gene similar to atpase was found to be down-regulated more than -fold in the rice stripe virus (rsv) infected small brown plant hopper, laodelphax striatellus (lee et al., ) . induction of the sgh symptoms is possibly a reflection of active production of viable progeny virus particles. during active virus progeny production, enveloped viruses are known to depend on the endoplasmic reticulum (er) for maturation of viral envelope glycoproteins, and proteins involved in the formation of replication complexes, assembly, envelopment and genome packaging (medigeshi et al., ; scheel and rice, ) . this imposes a tremendous protein load in the er, leading to er stress. consequently, er stress results in the induction of the unfolded protein response (upr), an evolutionary conserved prosurvival pathway that signals the nucleus to induce the expression of various chaperones (walter and ron, ) . in some cases, interaction between the induced chaperones and viral proteins is critical for processing of viral proteins and assembly of mature virions. during prolonged and overwhelming er stress, upr switches from being prosurvival to proaptotic (szegezdi et al., ) . prolonged virus-induced er stress/upr responses modulate a variety of signaling pathways that contribute to viral pathogenesis (fung and liu, ) , and may lead to cellular proliferation and hypertrophy. the up-regulation of upr-associated proteins and several molecular chaperones in flies with sgh symptoms (see table ) implicates the upr/er stress machinery in the development of overt sgh symptoms in g. pallidipes. as discussed above, the tktl pathway may also be involved in the expression of overt sgh symptoms in g. pallidipes. the current data provide potential targets for development of rationally designed antiviral strategies in large tsetse fly rearings for sterile insect technique. the data presented in this study provide hints as to why g. m. morsitans is much less susceptible host to gpsghv infection compared to g. pallidipes. the known and/or putative functions inferred from sequence similarity analyses revealed that the differentially modulated proteins we have identified are potentially involved in various aspects of gpsghv pathogenesis. specifically, and like in many other viruses, gpsghv appears to deploy a repertoire of strategies to exploit the host intracellular signaling pathways for replication, especially in g. pallidipes. in the case of g. m. morsitans, host proteins involved in antiviral defense systems appeared to be dominant. some of the pathways that appear to be targets of the virus include the upr and tktl pathways, implicating their involvement in expression of overt sgh symptoms in g. pallidipes. the proteins involved in these pathways deserve further functional (experimental) validations to understand the relevance of the differences in their expression patterns. the current study is a critical baseline data in a new coordinated research project (crp) initiated by iaea, aimed at gaining a deeper knowledge of the glossina/symbiont/gpsghv tripartite interactions and how these interactions affect trypanosoma parasite transmission (van den abbeele et al., ) . we have designed rnai bioassays to further investigate how asymptomatic gpsghv infection is maintained in glossina. candidate proteins experimentally validated as essential for efficient gpsghv pathogenesis are ideal targets for developing rationally designed antiviral strategies to control the virus infections in tsetse mass rearing facilities. in a larger view, our data are important for future studies on molecular and biochemical routes employed by members of the new entrants into the family of insect viruses, the hytrosaviridae. hk,İİ, jv, mv, and aa participated in the design of the study. aa and im set up the bioassays. hk andİİ processed and quantified the salivary gland proteins. sb performed the lc-ms/ms measurements. sb and hk analyzed the proteomics data sets. hk and em annotated/characterized the proteins. hk wrote the manuscript. jv, mv, aa,İİ, em, eo, and sn contributed in writing the paper, providing critical comments and suggestions. all the authors read and approved the final manuscript. the supplementary material for this article can be found online at: http://journal.frontiersin.org/article/ . /fmicb. . development of a non-destructive pcr method for detection of the salivary gland hypertrophy virus (sghv) in tsetse flies quantitative pcr analysis of the salivary gland hypertrophy virus (gpsghv) in a laboratory colony of glossina pallidipes genome analysis of a glossina pallidipes salivary gland hypertrophy virus reveals a novel large double-stranded circular dna virus managing hytrosavirus infections in glossina pallidipes colonies: feeding regime affects the prevalence of salivary gland hypertrophy syndrome hytrosaviridae: a proposal for classification and nomenclature of a new insect virus family dynamics of the salivary gland hypertrophy virus in laboratory colonies of glossina pallidipes (diptera: glossinidae) tsetse salivary gland hypertrophy virus: hope or hindrance for tsetse control? universal primers for rapid detection of hytrosaviruses the genome of fowlpox virus signal recognition particle: an essential protein-targeting machine ubiquitin and plant viruses, let's play together! plant physiol mise en evidence et purification d'un virus dans la proliferation monstrueuse glandulaire d'insectes. 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regulation discovery of the genes in response to white spot syndrome virus (wssv) infection in fenneropenaeus chinensis through cdna microarray intercommunity effects on microbiome and gpsghv density regulation in tsetse flies characterization of a rab gtpase up-regulated in the shrimp peneaus japonicus by virus infection interproscan-an integration platform for the signature-recognition methods in interpro analysis of rab gtpase-activating proteins indicates that rab a/b and rab are important for herpes simplex virus secondary envelopment rab proteins as membrane organizers the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © kariithi, ince, boeren, murungi, meki, otieno, nyanjom, van oers, vlak and abd-alla. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- -fz ucwwm authors: freundt, eric c.; drappier, melissa; michiels, thomas title: innate immune detection of cardioviruses and viral disruption of interferon signaling date: - - journal: front microbiol doi: . /fmicb. . sha: doc_id: cord_uid: fz ucwwm cardioviruses are members of the picornaviridae family and infect a variety of mammals, from mice to humans. replication of cardioviruses produces double stranded rna that is detected by helicases in the rig-i-like receptor family and leads to a signaling cascade to produce type i interferon. like other viruses within picornaviridae, however, cardioviruses have evolved several mechanisms to inhibit interferon production. in this review, we summarize recent findings that have uncovered several proteins enabling efficient detection of cardiovirus dsrna and discuss which cell types may be most important for interferon production in vivo. additionally, we describe how cardiovirus proteins l, c and l(∗) disrupt interferon production and antagonize the antiviral activity of interferon effector molecules. picornaviridae is an important family of single-stranded, positive-polarity rna viruses that includes > genera with over species (zell et al., ) . within picornaviridae, the genus cardiovirus includes encephalomyocarditis virus (emcv), theiler's murine encephalomyelitis virus (tmev) and saffold viruses (safv). although emcv has been described as a potential zoonotic agent, safvs are the only cardioviruses known to regularly infect humans, with the vast majority of people showing evidence of infection (zoll et al., ; carocci and bakkali-kassimi, ) . emcv has been found to infect over host species and contains one serotype, mengo virus, which was isolated in in the mengo district of uganda (dick et al., ) . tmev was discovered in by max theiler and is found in wild mice and rats worldwide. tmev can cause different diseases, depending on the virus strain and host genetics, ranging from fatal encephalitis to a chronic demyelinating disease that has served as a model for multiple sclerosis (brahic et al., ) . the genome of cardioviruses is approximately . - . kb and contains and untranslated regions (figure ) . translation of the genome gives rise to a polyprotein that is cleaved by the c protease, leading to the production of proteins. two additional proteins, l * and b * , are expressed from alternate open reading frames. l * is only expressed by tmev and is important for infection of macrophages, persistence of the virus in mice and inhibiting rnase l (van eyll and michiels, ; sorgeloos et al., ) , b * results from a frameshifting mechanism conserved in cardioviruses that regulates the ratio of structural and non-structural proteins translated over time. protein b * itself is only thought to be important for replication of emcv, as mutants that abolish its expression had a small plaque phenotype. b * in tmev and safv is unlikely to act as a protein as it is predicted to encode a peptide of amino acids (loughran et al., ) . in this review, we focus on the ways in which cardioviruses trigger the innate immune response and the efficient mechanisms that they have evolved to suppress these signaling pathways. we consider which cell types may be most important for production of interferon (ifn) in vivo, and also describe how cardioviruses disrupt the functions of interferon effectors. double-stranded rna (dsrna) is a necessary product of picornavirus replication, as positive-stranded genome is copied to produce a negative-stranded, full-length template, which is in turn used to produce additional genomes. dsrna is recognized by several sensor proteins within the cell and triggers a signal transduction pathway that results in transcription of the type i ifn (ifn-α/β) genes, as well as ifn-λ. in the endosome, dsrna is detected by toll-like receptor (tlr ), which signals through the adaptor protein trif to activate ifn regulatory factor (irf- ) and nuclear factor kappa b (nf-κb) (yamamoto et al., ) . cytoplasmic dsrna is detected by the rig-i-like receptor (rlr) family of proteins, which includes rig-i (retinoic acid-induced gene i) and mda (melanoma differentiation-associated gene ). upon recognition of dsrna, these proteins undergo a conformational change that exposes n-terminal caspase activation and recruitment domains (cards). the rlrs are then capable of stimulating the mitochondrial antiviral signaling (mavs) protein, also known as ips- , cardif, and visa, which in turn activates tank-binding kinase- (tbk ), inducible i-κb kinase (ikk-ε) and irf- , which then translocates to the nucleus to facilitate transcription of ifn genes (reviewed in gebhardt et al., ) . although rig-i and mda both detect dsrna within the cytosol, their functions are non-redundant. rig-i recognizes relatively short dsrna species (< kb) with ppp or pp, which are produced in certain virus infections (hornung et al., ; pichlmair et al., ) . in contrast, mda recognizes long dsrna, which is present during picornavirus replication (kato et al., ; pichlmair et al., ) . thus, while rig-i is activated during infection with flaviviruses, paramyxoviruses, influenza, and others, mda is responsible for detection of picornaviruses (gitlin et al., ; kato et al., ; pichlmair et al., ; wang et al., ; feng et al., ) . the importance of mda for control of cardioviruses was demonstrated in mda -deficient mice, which failed to control emcv infection and did not efficiently produce ifn (gitlin et al., ; kato et al., ) . in addition to rig-like helicases described above, which activate the mavs pathway, another ifn-inducible rna helicase, moloney leukemia virus homolog (mov ) was reported to enhance ifn induction (cuevas et al., ) . mov expression in hek cells restricted emcv replication. interestingly, mov acts through irf- activation, in a rlr and mavs-independent way and signals require ikk-ε but not tbk . such mavs-independent pathways are likely not critical for global ifn production in emcv infected mice, given the major impact of mda or mavs deficiency in mice, but they may be important in specific cell types or in conditions where the other pathways may be less potent. laboratory of genetics and physiology (lgp ), also known as dhx , is also a member of the rlr family but lacks a card domain (yoneyama et al., ) . given its structural similarity and lack of a card domain, lgp was initially thought to negatively regulate dsrna recognition by rig-i, as its overexpression limited ifn induction by sendai virus and newcastle disease virus (rothenfusser et al., ) . however, although the negative effect of lgp on rig-i remained controversial, later studies have established that lgp acts as a co-activator of mda . mice deficient for lgp were impaired in responding to rna ligands for mda or to emcv infection (venkataraman et al., ; satoh et al., ) . lgp was also shown to increase the rate of mda interaction with rna and downstream signaling by facilitating the formation of numerous, shorter mda filaments (bruns et al., ) . thus, it appears that lgp can act as both a positive and negative regulator of rlr signaling, with the outcome likely dependent on the concentration of lgp (bruns and horvath, ) . however, recombinant mda was directly activated as measured by an atp hydrolysis assay by the replicative form of dsrna coxsackievirus b , showing that lgp is not essential for activation of mda in vitro by dsrna (feng et al., ) . both lgp and mda are important for detecting cardiovirus replication. mefs deficient for either protein produce lower amounts of ifn-β when infected with emcv (deddouche et al., ) . intriguingly, lgp may enhance activation of mda during emcv infection by binding to rna complementary to the leader (l) gene and forming a complex with mda . this rna sequence from l was also shown to be a potent activator of mda in the absence of virus infection (deddouche et al., ) . although dsrna can bind and activate recombinant mda in the absence of other proteins, it is likely that additional partners are required for efficient activation of mda in vivo. for example, mda is phosphorylated in resting cells, and requires dephosporylation by pp α/γ (wies et al., ; takashima et al., ) . additional proteins that participate in recognition of cardiovirus dsrna have recently been described, including a study showing that tar rna binding protein (trbp) interacts with lgp in a yeast two-hybrid screen (komuro et al., ) . lgp was found to interact with trbp in co-immunoprecipitation experiments and depletion of trbp by sirna reduced interferon production induced by tmev and emcv. moreover, trbp enhanced ifn induction by tmev and emcv when overexpressed. depletion of trbp did not affect induction of ifn by sendai virus, which is recognized by rig-i. this study establishes that trbp participates in detection of cardiovirus dsrna by lgp /mda but the mechanism and its importance in vivo remain to be elucidated. as trbp is a component of the rnai machinery (chendrimada et al., ; haase et al., ) , other molecules involved in rnai were assessed for their role in dsrna detection and protein activator of pkr (pact) was found to also participate in activation of ifn signaling by cardioviruses. when pact was depleted by sirna, interferon production was decreased during infection of both tmev and emcv. overexpression of pact also increased ifn activation when lgp was co-expressed with mda . intriguingly, single-stranded tmev genome enhanced the association of lgp and pact, which suggests that a secondary structure in the tmev genome might facilitate this interaction (miyamoto and komuro, ) . in a separate report, pact was shown to be required for induction of ifn by emcv but not sendai virus. this study also demonstrated that pact and mda were recruited to dsrna (poly(i:c)) but not single stranded rna, and that pact expression increased the amount of mda oligomerization (lui et al., ) . both trbp and pact have also been reported to bind to double stranded rna-dependent protein kinase (pkr), and pact can bind rig-i (park et al., ; patel and sen, ; kok et al., ) . at the present time, it is not clear if these interactions are important for mediating recognition of cardioviruses. yet another partner in detecting dsrna in cardiovirus infection was recently uncovered. a cdna screen to identify genes involved in regulating ifn signaling revealed that dhx expression increased transcription of an ifn-β reporter plasmid in response to high molecular weight (hmw) poly(i:c) (zhu et al., ) . depletion of dhx resulted in decreased phosphorylation of tbk and irf- in response to hmw poly(i:c) and emcv as well as decreased production of ifn-β. the authors also show that dhx binds to mda but not mavs or rig-i, and binding could only be detected when mda was activated by emcv or hmw poly(i:c). mechanistically, this study demonstrated that dhx mediated rna binding of mda by interacting with mda through its n-terminus and rna through its dexd and helicase domains, and that dhx promotes formation of mda filaments, which are required for activation (zhu et al., ) . dhx was also independently described to interact with rig-i (sugimoto et al., ) , although it may be of greater importance for activation of mda (zhu et al., ) . together, these studies show that multiple proteins facilitate recognition of dsrna by mda during cardiovirus infection (figure ). thus far, these proteins include lgp , dhx , pact and trbp. additional helicases are also likely to participate in rlr-dsrna complex formation but their activities remain to be clarified (oshiumi et al., ) . moreover, recent discoveries have identified a novel role for pkr in recognition of dsrna and activation of mda , which will be discussed below. as the number of proteins mediating mda activation grows, so does the number of questions about how this pathway functions. for example, what role does each of the proteins play and how do they work together mechanistically to activate mda ? do they play non-redundant roles, or do they function in the same way but in different cell types? resolving these questions will allow for deeper understanding of the first line of immune defense against rna viruses. upon recognition of dsrna, pkr controls virus infection by phosphorylating eukaryotic initiation factor (eif α), which inhibits translation (farrell et al., ) . in this way, an infected cell can suppress production of viral proteins. phosphorylation of eif α also leads to stress granule formation and induces autophagy, and both pathways are commonly observed during virus infection (paul and munz, ; poblete-duran et al., ) . not surprisingly, many viruses have evolved strategies to inhibit the activation or function of pkr (reviewed in garcia et al., ) , as evidenced by the fact that pkr-deficiency did not modify the survival time of emcv infected mice (yang et al., ) . in addition to its role in inhibiting translation, recent evidence has emerged to show that pkr participates in production of ifn. for example, pkr appears to be important for nuclear translocation of irf- following mda activation (pham et al., ) . pkr was found to bind to mda and this interaction was not disrupted by nuclease treatment, indicating that binding does not depend on the presence of rna. in pkr-deficient cells, emcv infection failed to induce irf- translocation to the nucleus. moreover, a constitutively active mutant of pkr induced ifn through mavs. the effect of pkr on induction of ifn required its catalytic activity but did not depend on phosphorylation of eif α (pham et al., ) . in a separate report, pkr was shown to be important for normal processing of ifn-β mrna, suggesting that pkr may function at multiple points in the ifn pathway (schulz et al., ) . additionally, activation of pkr by hmw poly(i:c) was shown to be inhibited in cells depleted of mda and mavs, suggesting that mavs influences activation of pkr. moreover, mavs and pkr were found to interact in co-ip experiments, and the interaction depended on the card domain in mavs and the dsrna binding domain of pkr (zhang et al., ) . together, these studies clearly indicate a role for pkr in mda -dependent ifn induction, although several mechanisms may be involved, which require clarification. the role of pkr in ifn production after cardiovirus infection remains to be resolved. in one study that examined mengo virus infection, knockdown of pkr led to decreased induction of ifn-β in hela cells, suggesting that pkr may play a role in the mda pathway during cardiovirus infection (langereis et al., ) . this observation fits with the model proposed by onomoto et al. ( ) , which was based on influenza virus infection and suggests that pkr triggers the formation of "antiviral stress granules" that serve as a recruitment platform for dsrna and rig-like helicases, thereby enhancing ifn production. pkr is, however, not essential for ifn production in cardiovirus-infected cells because mengo virus possessing a deletion in the zinc-finger of l, which abrogates its functions as an interferon antagonist, was found to induce interferon in the cells lacking pkr and rnase l (feng et al., ) . cardioviruses may also inhibit pkr through activity of the l protein, as stress granule formation was prevented by the l protein of mengo, tmev, and safv- during virus infection (borghese and michiels, ) . however, direct inhibition of pkr by l remains to be demonstrated. during infection, viral dsrna can also be released into the extracellular milieu, either non-specifically during lysis of infected cells or possibly intentionally by exocytosis to trigger innate immunity by uninfected cells. dsrna can then be endocytosed by neighboring cells and infiltrating immune cells and lead to ifn production. tlr recognizes dsrna in endosomes and signals through trif to activate irf- and nf-κb. the importance of tlr in context of cardiovirus infection may depend on the model of infection. for example, mice deficient for myd or tlr were not significantly more susceptible than wild-type mice to emcv infection . in a separate report, however, tlr -deficient mice had higher viral loads in the liver and heart and were more susceptible to infection (hardarson et al., ) . also, a study that evaluated the role of tlr in controlling a strain of emcv with tropism for β cells of the pancreas found that tlr protected mice from a fatal infection and that tlr -deficent mice produced less ifnβ early in infection ( and h post-infection). however, this deficiency was transient and mice lacking tlr produced levels of ifn-β equivalent to wild-type animals at h post-infection. in the same experiments, the authors demonstrated that mice lacking mda succumbed to the infection more rapidly than tlr -deficient mice and produced less ifn-β at h postinfection (mccartney et al., ) . finally, a recent report using intracerebral inoculation of the gdvii strain of tmev evaluated the importance of these molecules for control of virus replication and induction of ifn. trif-/-, myd -/-, and mice lacking both trif and myd showed wild-type levels of ifn induction, while mavs-deficient animals were slightly but significantly impaired. when trif, myd and mavs were all depleted, however, mice were unable to induce ifn. only mice lacking myd and trif, or mice lacking myd , trif and mavs showed increased titers of gdvii in the brain (pfefferkorn et al., ) . thus, both tlr and rlrs contribute to controlling virus replication in vivo, although the relative importance of these pathways may depend on the virus and route of inoculation. surprisingly, mda is also responsible for the vast majority of ifn produced from extracellular dsrna in vivo. mice deficient for mda produced substantially less ifn when administered polyi:c, whereas tlr -deficient mice responded like wild-type (gitlin et al., ) . these results raise the question of how dsrna taken up through endocytosis could gain access to the cytoplasm. the mechanism by which dsrna could be internalized and then access the cytoplasmic rlrs has been unresolved until a recent discovery identified sidt , the mammalian ortholog of the sid- dsrna transporter in caenorhabditis elegans, as a transporter of dsrna from the endosome to the cytoplasm. sidt was shown to be important for mediating detection of dsrna in the context of emcv infection in vivo. mice that were deficient for sidt failed to control replication, produced less ifn-β, and succumbed to infection (nguyen et al., ) . these data suggest that a crucial pathway for innate signaling in emcv infection is release of viral rna into the extracellular milieu, endocytosis, and subsequent transfer of viral rna to the cytoplasm to access mda . two proteins encoded by cardioviruses were shown to counteract ifn production in infected cells: protease c, which is responsible for the processing of the virus-encoded polyprotein, and the leader protein (l), which corresponds to the n-terminal peptide of the polyprotein. c is a cysteine proteinase with a trypsin-like serine protease fold, responsible for most cleavages occuring during the maturation of the viral polyprotein (pelham, ) . like c proteases of other picornaviruses that were shown to target critical factors involved in ifn induction such as rig-i (barral et al., ) , emcv c was reported to cleave traf family member-associated nf-kb activator (tank) in infected cells, thus disrupting the complex involving tbk , ikke and irf and limiting type i ifn production (huang et al., ) . likewise, emcv c was shown to target mov , an rna helicase that acts in a mavs-independent way, as a possible innate immune evasion mechanism (cuevas et al., ; figure ). l is a small, multifunctional protein of - amino acids expressed by all cardioviruses (figure ). l contains an n-terminal zinc finger motif (cys-his-cys-cys), an acidic domain, a serine/threonine rich domain, and a c-terminal theilo domain, which is present in safv and tmev but absent in emcv. l was shown to be dispensable for replication of tmev in cell culture but its loss inhibits spread in cells that have a functional interferon response, like l cells, and also impairs the viral persistence in vivo (van pesch et al., ) . l deletions in figure | interferon antagonism by the cardiovirus c protease. the c protease is responsible for cleavage of the cardiovirus polyprotein produced from translation of the genome. in addition, the cardiovirus c protease cleaves host proteins, such as tank and mov , to prevent the cell from producing ifn. frontiers in microbiology | www.frontiersin.org emcv prevent the virus from shutting off host protein synthesis and enable interferon production (zoll et al., (zoll et al., , . mengo virus containing a mutation in the zinc finger of l failed to inhibit ifn synthesis and its replication was inhibited during low moi infections in vitro. in mice lacking the ifn α/β receptor, the mutant virus behaved as wild-type, but in wild-type mice, replication of the l mutant virus was impaired and it failed to cause disease, demonstrating that activity of l is important for pathogenesis in vivo (hato et al., ) . mutations in the zinc finger domain or the theilo domain of tmev or safv l inhibit its ability to antagonize interferon signaling (ricour et al., ) . a critical step in production of ifn following detection of viral replication by mda and other molecules is nuclear translocation of irf- and nf-κb. these proteins enable transcription of the ifn genes to produce mrna, which must then be exported from the nucleus for translation. since picornaviruses do not replicate within the nucleus, many viruses within this family disrupt nucleocytoplasmic trafficking, which results in inhibition of ifn production and translocation of nuclear proteins to the cytosol to benefit viral replication (reviewed in yarbrough et al., ; flather and semler, ) . the mechanism of how l interferes with ifn production may be due to its abilities to disrupt nucleocytoplasmic trafficking, activation of irf- , and assembly of stress granules in infected cells (figure ) . each of these activities will be explored below. the l protein of cardioviruses perturbs the function of the nuclear pore complex (npc) (porter et al., ) . in mammals, the npc consists of approximately different proteins, called nucleoporins (nups) and enables transit across the nuclear membrane (gorlich and kutay, ; wente and rout, ) . while small molecules and ions are able to diffuse through the npc, molecules larger than approximately - kda require active transport, which is regulated by transport receptors called karyopherins (yarbrough et al., ) . transport into the nucleus requires a short amino acid motif, called a nuclear localization sequence (nls) that can interact with either the α or β subtypes of karyopherins, depending on the sequence of the protein's nls. binding and dissociation of nls-containing proteins by karyopherins is also regulated by small gtpase ran. in the cytosol, ran is bound to gdp and can bind cargo proteins. once in the nucleus, however, ran is converted to the gtp bound form by the ran guanine nucleotide exchange factor (rangef) and dissociates from cargo. export then requires a nuclear export sequence (nes) that binds to karyopherins bound to rangtp, and dissociation of this complex occurs in the cytoplasm when a ran gtpase-activating protein (rangap) hydrolyzes gtp to gdp. in this way, the rangdp/gtp gradient regulates directional transport into and out of the nucleus. localization of l to the nucleus depends on expression of a, which contains a nls in its c-terminus (groppo et al., ) . upon nuclear localization, l interacts with ran with high affinity and a is displaced as the binding sites for a and ran partially overlap (petty et al., ) . l from emcv, tmev, and safv induce hyper-phosphorylation of nups including nup and nup (ricour et al., ; ciomperlik et al., ) , likely by recruiting and activating a kinase, which may be facilitated by l binding of exportins crm and cas (ciomperlik et al., ) . chemical inhibition of erk and p was able to block l-mediated hyper-phosphorylation of nups (porter et al., ) . additionally, l of emcv is phosphorylated by casein kinase (ck ) and this phosphorylation is required for nup phosphorylation, although ck did not phosphorylate l of safv or tmev . it is possible, although it remains to be shown, that these kinases also play a role in inhibition of nucleocytoplasmic trafficking by l of tmev and safv. in addition to its role in disrupting nucleocytoplasmic trafficking, tmev and mengo l prevent production of type i ifn in infected cells by interfering with irf- dimerization and tmev l also prevents export of mrna from the nucleus (delhaye et al., ; ricour et al., ) . for both tmev and mengo virus, dimerization of irf- was impaired despite the protein having been phosphorylated. inactivation of irf- occurs despite reports that it accumulates in the nucleus of infected cells (delhaye et al., ) . these data suggest that dsrna is detected in cardiovirus infected cells leading to activation of mavs and downstream kinases, but that irf- is unable to induce ifn transcription. stress granules can form in cells during virus infection and often result from inhibition of translation following phosphorylation of eif α by pkr (white and lloyd, ) . the l protein of mengo, tmev, and safv- inhibits stress granule formation during infection and ectopic expression of l was able to prevent thapsigargin-and arsenite-induced stress granules (borghese and michiels, ) . stress granules formed during infection with viruses containing deletions in the zinc-finger domain or a mutation in the theilo domain of l, indicating that these motifs are also important for inhibition of stress granules (borghese and michiels, ) . while l inhibits nucleocytoplasmic trafficking, stress granule formation, and possibly pkr activation, it has not been possible to uncouple these events using l mutants. when one function of l is disrupted, all functions are simultaneously impaired. therefore, it remains possible that l inhibits ifn production by blocking pkr activation, by interfering with irf- dimerization or nucleocytoplasmic trafficking, or through a combination of these mechanisms (figure ) . the importance of these antiviral pathways in controlling infection is underscored by the variety of mechanisms that viruses have evolved to prevent their activity. for example, the l protein of foot-and-mouth disease virus (fmdv), a picornavirus in the genus aphthovirus, has proteolytic activity whereas the l protein of cardioviruses does not. despite the major differences in these proteins, they all still function to inhibit induction of ifn. fmdv l pro can cleave eif g (devaney et al., ; kirchweger et al., ; guarne et al., ) and therefore reduce translation of cellular mrnas, and can also perturb ifn transcription by cleaving nf-κb (de los santos et al., . however, in the once in the nucleus, l interacts with high affinity with ran gtpase, thus displacing a. the l-ran complex would activate a kinase and trigger nucleoporin hyper-phosphorylation, thereby leading to nuclear pore complex dismantling and to nucleocytoplasmic trafficking perturbation. on the other hand, l may inhibit pkr, thus preventing translation arrest through eif α phosphorylation and therefore block assembly of stress granules. inhibition of ifn gene transcription by l may result from irf- trafficking perturbation and/or from the absence of pkr-enhanced dsrna detection by mda . context of a chimeric mengo virus infection, fmdv l pro was less effective at inhibiting ifn induction in vitro and in vivo (hato et al., ) . similar convergent evolution is apparent when considering the a protein of picornaviruses. whereas a functions as a protease for most picornaviruses and cleaves mediators of type i interferon signaling, this activity is not present in cardioviruses. nevertheless, l still targets some of these same molecules for inactivation (agol and gmyl, ) . additionally, both a of enteroviruses and l of cardioviruses inhibit stress granule assembly (yang et al., ) . the functions of l appear to be sufficiently important to the virus so that it maintains high levels of l expression throughout infection. emcv and tmev undergo a frameshift during translation later in infection by a binding to a stem-loop structure in the genome (napthine et al., ) . this frameshift decreases expression of non-structural proteins bc- abcd by - % (finch et al., ) . a follow up study using metabolic labeling estimated the frameshifting to be - % efficient (ling and firth, ) . this mechanism may allow for cardioviruses, and perhaps other picornaviruses, to increase the translation of structural proteins later in infection. due to its position in the genome, however, l expression would remain high throughout infection despite it not having a structural role for virus assembly. thus, it may be important for cardioviruses to express sufficient levels of l to counteract the immune response throughout the replication cycle. with effective ways to inhibit the production of interferon during infection, control of cardioviruses likely depends on nearby uninfected cells to produce interferon. intriguingly, these pathways seem to also depend on mda , although tlr may also be important in certain cell types such as plasmacytoid dendritic cells (hornung et al., ) . as discussed, recent data suggest a model where viral dsrna is released, endocytosed, and then the rna is translocated to the cytosol where it is detected by mda . in the cns, astrocytes appear to be the primary producers of ifn-β for several neurotropic viruses that preferentially infect neurons, such as tmev and la crosse virus (kallfass et al., ; pfefferkorn et al., ) . using transgenic mice that expressed firefly luciferase under the control of the ifn-β promoter restricted to different cell types, the authors were able to determine that % of ifn-β production during a neurotropic tmev infection was from astrocytes, whereas only % was from neurons, which are the primary target of infection. in mice lacking mavs, ifn-β production was slightly but significantly reduced, suggesting that the rlr pathway is active during infection but may not be the only pathway activated by tmev in astrocytes. intriguingly, mice deficient for myd and trif did not show a significant decrease in ifn-β induction, although induction of ifn-β was completely abrogated in mice deficient for mavs, myd and trif. therefore, it appears that both rlr and tlr signaling are important for ifn-β production after tmev infection of the cns. astrocytes were also shown to be primary producers of ifn during infection with rabies virus and vesicular stomatitis virus. in the case of rabies virus, astrocytes are stimulated to produce ifn by an abortive infection. that the virus is unable to replicate fully may prevent expression of viral interferon antagonists and allow for robust production of ifn. how viral replication is prevented in these cells will be important to uncover and may lead to novel insights about viral control in vivo. it is likely that abortive infection of astrocytes occurs during infection by tmev. however, this remains to be demonstrated and viral rna may well be encountered by other means. mda is critical for induction of ifn against cardioviruses in the periphery as well. ex vivo, cells such as macrophages, conventional dendritic cells and fibroblasts depend on mavs for production of ifn in response to dsrna (sun et al., ) . similarly, mda was shown to be essential in these cells for type i ifn production after emcv infection, in contrast to pdcs which induce ifn production in a tlr-dependent fashion (gitlin et al., ; kato et al., ) . after emcv infection of mice, some ifn is produced through tlrs, likely by pdcs, but most ifn was produced by mda activation (gitlin et al., ; kato et al., ) . levels of ifn-i were strongly decreased in the serum of mda -deficient mice infected by emcv. whereas mda expression strongly influenced survival in response to infection, the effect of myd depletion had a modest effect and loss of trif or rig-i did not affect survival . thus, mda is essential for controlling emcv infection in the periphery. interferon secreted by infected cells binds to its receptor on surrounding cells, activating a signaling cascade that leads to expression of hundreds of interferon-stimulated genes (isgs). two of these isgs, pkr and oligoadenylate synthetases (oas) are part of the best-characterized interferon effector pathways. as described earlier, pkr is likely antagonized by the l protein, as l inhibits pkr-induced stress granule assembly. moreover, a recent study reported increased sumo conjugation figure | inhibition of rnase l activation by l * tmev l * binds rnase l ankyrin repeats and (numbered) through a direct protein-protein interaction, thereby preventing association of - a with rnase l monomers and the consequent dimerization and activation of the enzyme. of pkr in emcv-infected cells, which dampens pkr activation and promotes caspase-dependent pkr degradation (maarifi et al., ) . oligoadenylate synthetases are enzymes responsible for rnase l activation. cardioviruses have evolved two strategies to interfere with the oas-rnase l pathway. in an infected cell, oas are activated by dsrna and produce - oligoadenylates ( - a). binding of two - a molecules to the ankyrin domain of the latent endoribonuclease rnase l triggers its dimerization and activation (figure ) . active rnase l then cleaves viral and cellular ssrna leading to decreased viral replication and ultimately to apoptosis of the cell. interestingly, rna fragments generated by rnase l can amplify ifn production in a rig-i, mda and mavs-dependent way (malathi et al., ) . rnase l targets both viral and cellular mrna but is also predicted to cleave the genome of ssrna viruses, as reported for emcv (li et al., ) . in addition to -phosphodiesterases and phosphatases that tightly regulate the system by degrading - a within minutes of their synthesis, rnase l activity can be negatively regulated by the rnase l inhibitor (rli/abce) (bisbal et al., ) . rli/abce expression is induced by emcv and correlates with rnase l inhibition (martinand et al., ) . accordingly, overexpression of rli/abce inhibited the action of ifn against emcv (bisbal et al., ) . rnase l inhibition by emcv-induced rli is, however, partial as rnase l antiviral activity against emcv was demonstrated in vitro using dominant negative rnase l and oas overexpression (chebath et al., ; zhou et al., ) and in vivo, in rnase l-deficient mice, which presented increased emcv infection and mortality compared to wild-type mice (zhou et al., ) . the l * protein of tmev was found to potently inhibit rnase l through a direct protein-protein interaction (sorgeloos et al., ) . mechanistically, l * binds to rnase l ankyrin repeats and , thereby preventing - a binding to the enzyme and further activation steps (drappier et al., ; figure ). in wild-type macrophages, replication of l * -mutant was significantly impaired as compared to that of the wild-type virus (sorgeloos et al., ) . in contrast, l * -mutant and wildtype viruses replicated to the same level in rnase l-deficient primary peritoneal macrophages. moreover, l * was shown to be active in vivo in the context of mhv chimeric viruses; l * could substitute for another viral rnase l inhibitor, namely the ns phosphodiesterase of mhv, in the liver of infected mice (drappier et al., ) . the fact that the virus devotes one of its proteins to rnase l antagonism highlights the importance of this antiviral pathway against tmev. interestingly rnase l inhibition by l * is highly species-specific; l * of a mouse tmev strain inhibits mouse rnase l but not its orthologs from other species including rat (sorgeloos et al., ; drappier et al., ) . accordingly, l * of a rat tmev strain inhibits rat but not mouse rnase l. theiler's murine encephalomyelitis virus is the only cardiovirus expressing l * , and thus the only cardiovirus known to directly inhibit rnase l. this could stem from its tropism for macrophages, which are the main tmev target during the chronic phase of infection and in which the oas-rnase l system is particularly active (zhao et al., ) . however, macrophages were reported to play important roles in emcv pathogenesis, including for viral replication and dissemination in piglets (papaioannou et al., ) and as reservoir cells for emcv persistence in rats (psalla et al., ) . since emcv is sensitive to rnase l activity, it is possible that another emcv protein will have developed some rnase l antagonistic activity, which might be identified using the appropriate host-pathogen context. interactions between safvs and rnase l have yet to be described, but it is also likely that these viruses have evolved ways of inhibiting this pathway. given the many ways that picornaviruses inhibit interferon production and signaling in infected cells, it is not surprising that the most important producers of ifn would be uninfected or abortively infected cells. indeed, cardioviruses efficiently block ifn in infected cells but loss of mda in mice causes them to be more susceptible to virus infection. these data indicate that detection of cytoplasmic dsrna by mda occurs in cells that are not productively infected (gitlin et al., ) and the recent finding that sidt mediates this process opens many new exciting areas of research (nguyen et al., ) . which molecules might be important for release of viral rna? is there a role for exosomes in this process? how might these pathways be stimulated pharmacologically? preventing release of viral rna and subsequent detection by uninfected cells may represent selective pressure favoring non-lytic release. given the exquisite genetic malleability in response to natural selection displayed by viruses, it is likely that viruses will have evolved mechanisms of inhibiting detection of viral rna by uninfected cells, perhaps by restricting dsrna release or by secreting proteins that inhibit rna transport into uninfected cells. it will be exciting to see how discoveries unfold in this area of research. several recent studies involving cardioviruses have revealed a more complicated picture regarding initial detection of replicating rna and induction of ifn. while it is clear that the helicases lgp , dhx , pact and trbp work in concert with mda for detection of dsrna, it remains to be determined how these molecules coordinate and interact and whether they function in a cell-type specific manner. it will also be important to resolve the way in which pkr functions to activate ifn signaling. future studies in this area will likely have broad relevance for innate detection of viruses. finally, the mechanisms by which l disrupts nucleocytoplasmic trafficking, stress granule formation, and interferon production clearly require further clarification. mutational analysis of l has revealed that these activities are tightly coupled, suggesting that the l interacts with protein(s) that can serve as a common node in each of these pathways. as ifn signaling and stress granules are important for a variety of viral pathogens, answers to these questions may provide broadly relevant insight into host-pathogen interactions. ef, md, and tm wrote the manuscript and approved its final version. ef was supported by a david delo research grant. md was supported by action de recherches concertée (arc). research in the tm lab was supported by the belgian fund for medical research (frsm, pdr # t. . ) and by eos joint program of fonds de la recherche scientifique -fnrs and fonds wetenschapelijk onderzoek -vlaanderen -fwo (eos id: ). viral security proteins: counteracting host defences rig-i is cleaved during picornavirus infection encephalomyocarditis virus leader is phosphorylated by ck and syk as a requirement for subsequent phosphorylation of cellular nucleoporins cloning and characterization of a rnase l inhibitor. a new component of the interferon-regulated - a pathway the leader protein of cardioviruses inhibits stress granule assembly the genetics of the persistent infection and demyelinating disease caused by theiler's virus lgp synergy with mda in rlrmediated rna recognition and antiviral signaling the innate immune sensor lgp activates antiviral signaling by regulating mda -rna interaction and filament assembly the encephalomyocarditis virus constitutive expression of ( '- ') oligo a synthetase confers resistance to picornavirus infection trbp recruits the dicer complex to ago for microrna processing and gene silencing three cardiovirus leader proteins equivalently inhibit four different nucleocytoplasmic trafficking pathways cardiovirus leader proteins bind exportins: implications for virus replication and nucleocytoplasmic trafficking inhibition mov provides antiviral activity against rna viruses by enhancing rig-i-mavs-independent ifn induction the leader proteinase of foot-and-mouth disease virus inhibits the induction of beta interferon mrna and blocks the host innate immune response degradation of nuclear factor kappa b during foot-and-mouth disease virus infection a conserved domain in the leader proteinase of foot-and-mouth disease virus is required for proper subcellular localization and function identification of an lgp -associated mda agonist in picornavirus-infected cells the leader protein of theiler's virus interferes with nucleocytoplasmic trafficking of cellular proteins leader protein of foot-and-mouth disease virus is required for cleavage of the p component of the cap-binding protein complex mengo encephalomyelitis; a hitherto unknown virus affecting man a novel mechanism of rnase l inhibition: theiler's virus l * protein prevents - a from binding to rnase l phosphorylation of initiation factor elf- and the control of reticulocyte protein synthesis mda detects the double-stranded rna replicative form in picornavirus-infected cells characterization of ribosomal frameshifting in theiler's murine encephalomyelitis virus picornaviruses and nuclear functions: targeting a cellular compartment distinct from the replication site of a positivestrand rna virus the dsrna protein kinase pkr: virus and cell control discrimination of self and non-self ribonucleic acids essential role of mda- in type i ifn responses to polyriboinosinic:polyribocytidylic acid and encephalomyocarditis picornavirus transport between the cell nucleus and the cytoplasm mutational analysis of the emcv a protein identifies a nuclear localization signal and an eif e binding site structure of the foot-and-mouth disease virus leader protease: a papainlike fold adapted for self-processing and eif g recognition trbp, a regulator of cellular pkr and hiv- virus expression, interacts with dicer and functions in rna silencing toll-like receptor is an essential component of the innate stress response in virus-induced cardiac injury the mengovirus leader protein blocks interferon-alpha/beta gene transcription and inhibits activation of interferon regulatory factor differential ifn-alpha/beta production suppressing capacities of the leader proteins of mengovirus and foot-and-mouth disease virus '-triphosphate rna is the ligand for rig-i encephalomyocarditis virus c protease attenuates type i interferon production through disrupting the tank-tbk -ikkepsilon-irf complex visualizing production of beta interferon by astrocytes and microglia in brain of la crosse virus-infected mice length-dependent recognition of double-stranded ribonucleic acids by retinoic acid-inducible gene-i and melanoma differentiation-associated gene differential roles of mda and rig-i helicases in the recognition of rna viruses foot-and-mouth disease virus leader proteinase: purification of the lb form and determination of its cleavage site on eif- gamma the double-stranded rna-binding protein pact functions as a cellular activator of rig-i to facilitate innate antiviral response the tar-rna binding protein is required for immunoresponses triggered by cardiovirus infection mda localizes to stress granules, but this localization is not required for the induction of type i interferon rnase l mediates the antiviral effect of interferon through a selective reduction in viral rna during encephalomyocarditis virus infection an analysis by metabolic labelling of the encephalomyocarditis virus ribosomal frameshifting efficiency and stimulators ribosomal frameshifting into an overlapping gene in the b-encoding region of the cardiovirus genome pact facilitates rna-induced activation of mda by promoting mda oligomerization differential effects of sumo and sumo on pkr activation and stability small self-rna generated by rnase l amplifies antiviral innate immunity rnase l inhibitor is induced during human immunodeficiency virus type infection and down regulates the - a/rnase l pathway in human t cells rna sensor-induced type i ifn prevents diabetes caused by a beta cell-tropic virus in mice pact is required for mda -mediated immunoresponses triggered by cardiovirus infection via interaction with lgp protein-directed ribosomal frameshifting temporally regulates gene expression sidt transports extracellular dsrna into the cytoplasm for innate immune recognition critical role of an antiviral stress granule containing rig-i and pkr in viral detection and innate immunity accessory factors of cytoplasmic viral rna sensors required for antiviral innate immune response pathogenesis of encephalomyocarditis virus (emcv) infection in piglets during the viraemia phase: a histopathological, immunohistochemical and virological study tar rna-binding protein is an inhibitor of the interferon-induced protein kinase pkr pact, a protein activator of the interferoninduced protein kinase, pkr autophagy and mammalian viruses: roles in immune response, viral replication, and beyond translation of encephalomyocarditis virus rna in vitro yields an active proteolytic processing enzyme binding interactions between the encephalomyocarditis virus leader and protein a abortively infected astrocytes appear to represent the main source of interferon beta in the virus-infected brain pkr transduces mda -dependent signals for type i ifn induction rig-i-mediated antiviral responses to single-stranded rna bearing '-phosphates activation of mda requires higher-order rna structures generated during virus infection who regulates whom? an overview of rna granules and viral infections a picornavirus protein interacts with ran-gtpase and disrupts nucleocytoplasmic transport nucleoporin phosphorylation triggered by the encephalomyocarditis virus leader protein is mediated by mitogen-activated protein kinases pathogenesis of experimental encephalomyocarditis: a histopathological, immunohistochemical and virological study in rats inhibition of mrna export and dimerization of interferon regulatory factor by theiler's virus leader protein the rna helicase lgp inhibits tlrindependent sensing of viral replication by retinoic acid-inducible gene-i lgp is a positive regulator of rig-i-and mda -mediated antiviral responses protein kinase r contributes to immunity against specific viruses by regulating interferon mrna integrity evasion of antiviral innate immunity by theiler's virus l * protein through direct inhibition of rnase l helicase proteins dhx and rig-i cosense cytosolic nucleic acids in the human airway system the specific and essential role of mavs in antiviral innate immune responses riok -mediated phosphorylation of mda interferes with its assembly and attenuates the innate immune response influence of the theiler's virus l * protein on macrophage infection, viral persistence, and neurovirulence the leader protein of theiler's virus inhibits immediate-early alpha/beta interferon production loss of dexd/h box rna helicase lgp manifests disparate antiviral responses mda and mavs mediate type i interferon responses to coxsackie b virus the nuclear pore complex and nuclear transport regulation of stress granules in virus systems dephosphorylation of the rna sensors rig-i and mda by the phosphatase pp is essential for innate immune signaling role of adaptor trif in the myd -independent toll-like receptor signaling pathway picornavirus a protease regulates stress granule formation to facilitate viral translation deficient signaling in mice devoid of double-stranded rna-dependent protein kinase viral subversion of nucleocytoplasmic trafficking shared and unique functions of the dexd/h-box helicases rig-i, mda , and lgp in antiviral innate immunity ictv virus taxonomy profile: picornaviridae ips- plays an essential role in dsrna-induced stress granule formation by interacting with pkr and promoting its activation antagonism of the interferon-induced oas-rnase l pathway by murine coronavirus ns protein is required for virus replication and liver pathology interferon action and apoptosis are defective in mice devoid of ' , '-oligoadenylate-dependent rnase l impact of rnase l overexpression on viral and cellular growth and death dhx functions as an rna co-sensor for mda -mediated emcv-specific antiviral immunity saffold virus, a human theiler'slike cardiovirus, is ubiquitous and causes infection early in life mengovirus leader is involved in the inhibition of host cell protein synthesis the mengovirus leader protein suppresses alpha/beta interferon production by inhibition of the iron/ferritin-mediated activation of nf-kappa b key: cord- -hp ky q authors: minich, jeremiah j.; power, cecilia; melanson, michaela; knight, rob; webber, claire; rough, kirsten; bott, nathan j.; nowak, barbara; allen, eric e. title: the southern bluefin tuna mucosal microbiome is influenced by husbandry method, net pen location, and anti-parasite treatment date: - - journal: front microbiol doi: . /fmicb. . sha: doc_id: cord_uid: hp ky q aquaculture is the fastest growing primary industry worldwide. marine finfish culture in open ocean net pens, or pontoons, is one of the largest growth areas and is currently the only way to rear high value fish such as bluefin tuna. ranching involves catching wild juveniles, stocking in floating net pens and fattening for to months. tuna experience several parasite-induced disease challenges in culture that can be mitigated by application of praziquantel (pzq) as a therapeutic. in this study, we characterized the microbiome of ranched southern bluefin tuna, thunnus maccoyii, across four anatomic sites (gill, skin, digesta, and anterior kidney) and evaluated environmental and pathological factors that influence microbiome composition, including the impact of pzq treatment on microbiome stability. southern bluefin tuna gill, skin, and digesta microbiome communities are unique and potentially influenced by husbandry practices, location of pontoon growout pens, and treatment with the antiparasitic pzq. there was no significant relationship between the fish mucosal microbiome and incidence or abundance of adult blood fluke in the heart or fluke egg density in the gill. an enhanced understanding of microbiome diversity and function in high-value farmed fish species such as bluefin tuna is needed to optimize fish health and improve aquaculture yield. comparison of the bluefin tuna microbiome to other fish species, including seriola lalandi (yellowtail kingfish), a common farmed species from australia, and scomber japonicus (pacific mackerel), a wild caught scombrid relative of tuna, showed the two scombrids had more similar microbial communities compared to other families. the finding that mucosal microbial communities are more similar in phylogenetically related fish species exposes an opportunity to develop mackerel as a model for tuna microbiome and parasite research. aquaculture is the fastest growing primary industry worldwide. marine finfish culture in open ocean net pens, or pontoons, is one of the largest growth areas and is currently the only way to rear high value fish such as bluefin tuna. ranching involves catching wild juveniles, stocking in floating net pens and fattening for to months. tuna experience several parasite-induced disease challenges in culture that can be mitigated by application of praziquantel (pzq) as a therapeutic. in this study, we characterized the microbiome of ranched southern bluefin tuna, thunnus maccoyii, across four anatomic sites (gill, skin, digesta, and anterior kidney) and evaluated environmental and pathological factors that influence microbiome composition, including the impact of pzq treatment on microbiome stability. southern bluefin tuna gill, skin, and digesta microbiome communities are unique and potentially influenced by husbandry practices, location of pontoon growout pens, and treatment with the antiparasitic pzq. there was no significant relationship between the fish mucosal microbiome and incidence or abundance of adult blood fluke in the heart or fluke egg density in the gill. an enhanced understanding of microbiome diversity and function in high-value farmed fish species such as bluefin tuna is needed to optimize fish health and improve aquaculture yield. comparison of the bluefin tuna microbiome to other fish species, including seriola lalandi (yellowtail kingfish), a common farmed species from australia, and scomber japonicus (pacific mackerel), a wild caught scombrid relative of tuna, showed the two scombrids bluefin tuna is one of the highest value fish in the world. in , the three species of bluefin including atlantic bluefin tuna (abt), pacific bluefin tuna (pbt), and southern bluefin tuna (sbt) had a combined total value of usd $ - million for fishers with a total value of usd $ - . billion at final sale (macfadyen, ) . to meet high demands, bluefin tunas are ranched in open ocean net pens, or pontoons, which currently represents approximately % of total global bluefin production. in australia, the sbt wild-caught fishery began in (serventy, ) , peaking in at an annual catch of , tons (geen and nayar, ) ; after this peak catch restrictions were imposed on the fishery. ranching of sbt involves the collection and transfer of wild juvenile tuna into static pontoon enclosures where they are reared and fattened for up to months. over this period tuna are fed whole baitfish, which results in a near doubling of fish biomass. ranching began in port lincoln australia in and in - had an annual production value of aud$ million (françois et al., ; ellis and kiessling, ; econsearch, ) . one of the primary challenges to promoting and ensuring long term success of the industry is to maintain and improve fish health. when cultured in open ocean pontoons, fish are exposed to free-living microbes, including opportunistic pathogens and infectious parasites (nowak, ) . several parasites have been identified in ranched sbt including miamiensis avidus (scuticociliate), caligus sp. (copepod "sea lice"), cardicola spp. (blood flukes), and hexostoma thynni (gill fluke) (nowak et al., ) . digenean blood flukes, specifically cardicola spp., infect all three species of bluefin tuna during ranching which can lead to morbidities and mortalities causing significant losses for producers (balli et al., ; ellis and kiessling, ) . among the most destructive parasites for sbt are blood flukes that include two species, cardicola forsteri and c. orientalis (cribb et al., ; colquitt et al., ) . in captivity, infections peak approximately months post-relocation to pontoons with sbt having on average flukes per fish (aiken et al., ) . blood flukes produce eggs which are deposited in the gills (up to million eggs per individual) resulting in morbidity and mortality (colquitt et al., ; bullard and overstreet, ; shirakashi et al., b) . currently, praziquantel (pzq) is the only therapeutic used to treat c. forsteri and c. orientalis infections. pzq is administered orally through feeds by injecting - mg/kg body weight into sardines which are then fed to sbt (hardy-smith et al., ) . treatment with pzq ( mg/kg body weight) has a positive impact on adults sbt health by eradicating adult flukes and has shown to be effective in pbt juveniles as well (shirakashi et al., a) . lower doses ( . - . mg/kg) have also shown positive effects for adult fluke eradication with organ elimination after h (ishimaru et al., ) . in addition to therapeutic treatments, pontoon location and alterations to husbandry practices, such as ranching at greater depths, can also lower infection outcomes with a combined impact of decreasing mortalities from to < % (kirchhoff et al., ) . marine fish harbor specialized microbiomes within the gill, skin, and gastrointestinal tract that are distinct from the surrounding seawater and influenced by environmental variables (minich et al., a) . these microbial communities play important roles in mucosal barrier defenses to protect against pathogen invasion and maintain host health. manipulation and optimization of the microbiome of farmed fish through prebiotic, probiotic, or synbiotic interventions are active areas of research to promote fish health and resilience (gomez et al., ; kelly and salinas, ) . understanding how the fish mucosal microbiome is impacted by the environment and pathological status of the host, e.g., parasite infection load, is largely unknown yet may be important for the development of therapeutics and/or diagnostics (li et al., ) . secondary infections with bacteria or viruses can also be enhanced during parasitic infections but little is known about blood flukes specifically (kumon et al., ; boxaspen, ; lhorente et al., ; novak et al., ) . since blood fluke egg deposition in the gills leads to pathology and respiratory problems, evaluating microbiome changes between infected and noninfected individuals may reveal dysbiosis signatures that further influence host health. moreover, the presence of a parasite infection itself may lead to changes in the mucosal bacteria communities of the fish which could, in turn, be used to predict, diagnose, and monitor parasite infections. in atlantic salmon, skin infections by sea lice resulted in decreased richness of skin bacteria which was further driven by an enrichment of potential pathogenic microbes including vibrio spp., pseudomonas spp., and tenacibaculum spp. (llewellyn et al., ) . understanding the mechanisms and interactions between infection, treatment, host response, and the microbiome, the collective "pathobiome" (bass et al., ) , are thus important for reducing the impact of infections. in this study, ranched sbt were sampled to characterize the microbial diversity associated with mucosal body sites, including gill, skin, and gut, providing the first assessment of microbiome diversity in this ecologically and commercially important fish species. pzq treatment has previously been shown to reduce cardicola spp. parasite egg abundances in the gills (power et al., ) . to better establish the impact of antiparasitic treatment on microbiome composition, we analyzed mucosal microbiome composition as a function of parasite infection and pzq treatment. we hypothesized that prevalence of cardicola eggs in the gill would correspond to an altered microbial community. in addition, we hypothesized that among the body sites evaluated, the gastrointestinal tract would have the highest probability of being impacted by the oral treatment of pzq delivered through feeds. during the course of a harvest event, a total of sbt across six different pontoons from five spatially separate companies were sampled. assessment of blood fluke prevalence, gill egg densities, and fish biometrics (length and weight) were collected and analyzed with respect to mucosal microbiome composition. lastly, we compare the mucosal microbiome of sbt to another scombrid species to evaluate its utility as a possible model organism for marine fish microbiome studies. a meta-analysis of the sbt microbiome compared to two other commercially important fish species suggests the pacific chub mackerel, scomber japonicus, may be a suitable model to study microbiome dynamics in marine aquaculture species, including bluefin tuna. southern bluefin tuna "sbt, " thunnus maccoyii, reared in ocean pontoons (also known as ranching) in port lincoln, australia, were opportunistically sampled during a harvest event from july to . a total of fish were sampled across six pens from five total companies. between and fish were sampled from each pen. fish were measured for total length (mm) and mass (grams) and fulton's condition factor "kfactor" was calculated (fulton, ; froese, ) . fish (n = ) from four of the six pens were treated with the antiparasitic drug, anthelmintic drug praziquantel (pzq) orally (injected into baitfish) at a dose of mg/kg bodyweight - weeks after transfer to pontoons in a single treatment over consecutive days by the prescribing veterinarian (apvma permit number-per ) (power et al., ) while fish in the other two pens did not get treated with pzq (n = ). presence of adult flukes in heart and egg densities within the gill were measured per fish as previously described (aiken et al., ; power et al., ) . comparison of the sbt microbiome to other fish samples, including pacific chub mackerel (mkl) (minich et al., b) and yellowtail kingfish (ytk), was performed computationally within qiita . all mkl samples were collected from the wild while the ytk were farmed. details for fish sample collections are listed in the metaanalysis section below. southern bluefin tuna microbiome samples were collected from four body sites (gill, skin, digesta, and anterior kidney) from each of the fish for a total of samples. gill and skin communities were collected by swabbing approximately cm surface area using wooden shaft cotton swabs. for digesta and anterior kidney samples, approximately mg of fecal matter or tissue was collected with a swab and placed in a . ml tube. to preserve microbiome integrity, all microbiome samples were stored in % ethanol for - h at room temperature while on the boat and then later transferred to a − • c freezer where samples were stored until dna extraction (approximately weeks later). we chose % ethanol over rnalater because it has a superior performance in preservation of microbiome integrity, meaning there is the least bias when using % ethanol (song et al., ) . in addition % ethanol is readily available globally and has been demonstrated to successfully store samples for both bacteria (vogtmann et al., ; knight et al., ) and viruses while not interfering with the extraction (minich et al., a) . molecular methods outlined in the earth microbiome project (earthmicrobiome.org) were used to process samples. specifically, swabs of either gill, skin, or digesta stored in % ethanol were transferred to a ml bead beating tube at the beginning of dna extraction. numerous large scale studies have shown that direct extraction of swab heads are an effective way to collect and process microbiome samples (thompson et al., ; mcdonald et al., ) . dna was extracted in single tubes to avoid well-to-well contamination (minich et al., b) using the mobio powersoil kit. multiple titration replicates of -fold serial dilutions of positive controls (n = ) (escherichia coli) were included to enable detection of background contaminates and determine the limit of detection of the method using the katharoseq method (minich et al., b,c) . miniaturized ( ul) pcr reactions were used to amplify genomic dna ( nl) (minich et al., a) using s rrna v / emp primers walters et al., ) . equal volumes of amplicons ( ul) were pooled across samples and processed through the qiagen pcr cleanup kit and then sequenced on a miseq × bp run (caporaso et al., ) . sequencing analysis was performed using qiime (bolyen et al., , ) and qiita . samples were trimmed to bp and then demultiplexed and processed through the deblur pipeline to generate unique sotus (sub-operational taxonomic units) also referred to as asvs (amplified sequence variants). we use sotus terminology here. samples were rarified to reads as determined by katharoseq cutoff. all downstream analyses were performed using the rarified biom table. two sotus were removed from the dataset. the first was the sample used as a positive control (enterobacteriaceae: tacggagggtgc aagcgttaatcggaattactgggcgtaaagcgcacgcag gcggtttgttaagtcagatgtgaaatccccgggctcaac ctgggaactgcatctgatactggcaagcttgagtctcgt agaggggggtagaattccagg) and the second was a known pcr mastermix contaminant (eisenhofer et al., ) (pseudomonas veronii: tacagagggtgcaagcgttaa tcggaattactgggcgtaaagcgcgcgtaggtggtttgt taagttggatgtgaaatc cccgggctcaacctgggaact gcattcaaaactgactga ctagagtatggtagagggtgg tggaatttcctg) which we observe disproportionally in low biomass positive controls. the sotu table was annotated using both greengenes and the silva ssu . . database using a minimum identity with query sequence ( . ) and default parameters neighbors per query sequence (supplementary table s ; mcdonald et al., ; quast et al., ; yilmaz et al., ) . while both annotations are included in the table (supplementary table s ), the greengenes annotation is used for subsequent analyses and visualizations. alpha diversity was calculated using richness (total unique sotus), shannon evenness, and faith's phylogenetic diversity (shannon, ; whittaker, ; faith, ) . alpha diversity comparisons were calculated using non-parametric (kruskal and wallis, ) test with multiple comparisons done using benjamini-hochberg correction with a . fdr cutoff (benjamini and hochberg, ) . pairwise comparisons were performed using a non-parametric mann-whitney test (mann and whitney, ) . statistical comparisons were performed within prism . . beta diversity was calculated using both weighted and unweighted unifrac distances (lozupone and knight, ; lozupone et al., ) . to test which metadata categories or variables were associated with beta diversity, multivariate statistical testing was done using adonis which is a modified version of permanova (anderson, ) . specifically, the following categorical variables were assessed for their impact on the microbiome: sample type, company, and pzq treatment. following those results, body sites were independently assessed for the impacts of pzq treatment, company, parasite eggs in gill, parasite flukes in heart, tuna condition factor, tuna length, and tuna mass. differentially abundance measures were calculated within calour using the rank mean ds-fdr function to compare two groups with a . fdr (jiang et al., ; xu et al., ) . a metanalysis was performed to test the hypothesis that phylogenetically related fish species have greater similarity in microbial community composition. to enable this comparison, we used publicly available datasets in qiita . both alpha and beta diversity were compared. note, only datasets which used the earth microbiome project standardized protocols (same extraction and primer set) were used in the comparison as both dna extraction and primer choice lead to biases in analyses (brooks et al., ; fouhy et al., ) . to do this analysis, publicly available microbiome data from gill, skin, and digesta microbiomes of pacific chub mackerel (mkl), scomber japonicus, (qiita id , erp ) and yellowtail kingfish (ytk), seriola lalandi, were combined with sbt using the qiita database. mackerel samples were wild fish sampled throughout caught off the scripps institution of oceanography pier in the eastern pacific ocean, san diego. the yellowtail kingfish was sampled from pontoons in the western pacific ocean, nsw australia. mackerel are phylogenetically closer to sbt in the scombridae family (mackerels, bonitos, and tunas) (collette and nauen, ; collette et al., ) , whereas yellowtail are within the carangidae family (jacks and pompanos) (swart et al., ) . for specimens collected in this study, ytk are however similar to sbt in terms of trophic level, geographic location of sampling, and farmed rather than wild caught. southern bluefin tuna were sampled during annual harvest across days (july to july ) in port lincoln, australia. a total of six pens were sampled among five different companies (figure a) . two of the pens (n = fish) were not treated with the anthelmintic praziquantel (pzq) while the other four pens (n = fish) were treated. four anatomical sites were sampled for microbiome analysis (figure b) including gill, skin, digesta, and anterior kidney. fish biometrics including fork length (mm), mass (grams), and condition factor "k-factor" were measured for fish whereas data from three fish from july th (host_subject_id: sbt_ , sbt_ , and sbt_ ) were not recorded (figure ) . tuna ranged in length from to mm with a median length of mm (figure c) . tuna ranged in mass from to grams with a median mass of grams (figure d) . the k-factor ranged from . to . with a median of . (figure e ). blood fluke counts and parasite egg counts in the gill were recorded for all fish. blood fluke counts ranged from ( fish) to with a median and mean intensity of (figure f ). parasite egg counts in the gill ranged from ( out of ) to . , with a median of . and mean intensity of . eggs per mm filament (figure g ). a total of host-associated sbt microbiome samples were processed through the pipeline from unique fish across four body sites including gill, skin, digesta, and anterior kidney. in addition, positive controls were included to determine the limit of detection of the pipeline. after applying the katharoseq formula, the limit of detection of the pipeline, where % of the reads of a positive control map to the known control, was reads. when the % threshold is applied, as recommended by the katharoseq method, the limit of detection was reads indicating that any samples with at least reads could be included (supplementary figure s ) . to be conservative on read depth, we included samples with at least reads and thus rarified to reads. at this sequencing depth, a total of samples out of the passed qc ( table ) . the majority ( . %) of anterior kidney samples failed and thus were excluded from downstream analyses due to low successful sample size. a final table of samples with unique sotu features was annotated (supplementary table s ). the good's coverage analysis demonstrated that we were capturing the majority of microbial diversity even with just reads as the median values for gill, skin, and digesta were . , . , and . , respectively (supplementary figure s ) . digesta samples had the highest success rate ( out of ) followed by skin ( out of ) and gill ( out of ). the microbiomes of the three body sites were compared across the fish. for alpha diversity measures, richness differed across body sites (p = . , kw = . ) with gill and skin communities having a higher richness than digesta (p < . ) figure | sampling design of ranched sbt from port lincoln, australia. (a) fish were sampled from a total of six pens spanning five companies. (b) upon harvest, four body sites were sampled and stored in % etoh for the microbiome including the gill, skin, digesta (hindgut), and anterior kidney. fish biometrics across all samples collected for (c) fork length (mm: median, iqr), (d) mass (grams: median, iqr), and (e) calculated condition factor (median, iqr). blood fluke parasite counts from (f) heart (flukes per heart: median, iqr), and (g) gill (eggs per mm gill filament: median, iqr). (figure a) . shannon diversity differences across body sites was even stronger (p < . , kw = . ) with both gill and skin having higher evenness than digesta samples (p < . ) ( figure b) . however, phylogenetic diversity did not differ across body sites ( figure c) . upon comparing beta diversity, samples were most strongly influenced by body site location for both unweighted unifrac ( figure d ) (adonis: p = . , f = . , r = . ) and weighted unifrac ( figure e ) (p = . , f = . , r = . ) ( table ) . microbes sampled from the sbt mucosal sites represented different phyla, with digesta being enriched in tenericutes and spirochaetes relative to other phyla (figure f) . at the order level, the tenericutes were primarily mycoplasmatales. the spirochaetes were primarily brevinematales. both the vibrionales and pseudomonadales were also prevalent across digesta samples within the gammaproteobacteria (supplementary figure s ) . skin and gill microbes were enriched in proteobacteria, cyanobacteria, firmicutes, and bacteroidetes relative to other phyla ( figure f ). skin and gill samples were primarily enriched in vibrionales and pseudomonadales (supplementary figure s ) . microbiome variation among body sites was strongest with weighted unifrac. company (geographic location) and pzq treatment was also significant but less so than body site variation ( table ) . subsequent analyses were therefore performed on each body site independently ( table ) . the impact of pzq treatment on the microbiome was evaluated using alpha and beta diversity measures. treatment with pzq resulted in digesta samples having lower microbial richness (mann-whitney: p = . , u = ) (figure a) , shannon evenness (mann-whitney: p = . , u = ) (figure b) , and phylogenetic diversity (mann-whitney: p = . , u = ) ( figure c) . although not significant, likely due to low sample size, all measures of microbial diversity in the gill were lower when fish were treated with pzq. to determine if particular sotus were associated with pzq treatment in the various body sites, we applied the rank mean ds-fdr method for each body site comparing samples which were either treated or not treated with pzq. from the skin comparison, a sotu within the mycoplasmataceae family (tenericute phyla) was more enriched in skin samples of fish which did not get treated with pzq ( figure d ). for digesta samples, a total of sotus were also associated or enriched in fish which were pzq naïve. this included two pseudomonas sotus, two acinetobacter sotus, brevundimonas, delfita, and a sotu within the brevinemataceae family ( figure e ). all sotus classifications were verified with both greengenes and silva databases. the greengenes brevinemataceae annotated sotu was "unclassified" in the silva database, with the closest phylogenetic similarity to uncultured spirochaetes sampled from fish guts, identified from a blast search (dq . , he . ). to understand the sbt microbiome in relation to other fish species, we performed a metanalysis comparing the sbt mucosal microbiome to a geographically (australia) and trophically similar (tertiary carnivore) fish species, yellowtail kingfish (ytk), seriola lalandi (also farmed), and a phylogenetically similar yet geographically and trophically dissimilar fish pacific chub mackerel (mkl), scomber japonicus (from the wild). both mkl and sbt are within the same family, scombridae (tunas, mackerel, and bonito). the gill, skin, and digesta samples were similarly sampled across all species by the same researcher and further processed using the same molecular methods. wild s. japonicus were sampled from the eastern pacific ocean in san diego ca as part of a fish microbiome time series study (minich et al., a) . to verify that reads was sufficient to interpret and make conclusions from the data, we also compared samples rarified at , and , reads. higher sampling depth resulted in fewer samples being compared, but the trends remain the same indicating that reads is sufficient for comparisons while enabling the highest number of samples to be compared (supplementary figure s ) . body sites were independently compared across all samples for both alpha and beta diversity using richness, faith's phylogenetic diversity, unweighted unifrac and weighted unifrac (figure ) . gill microbial richness differed across species (p < . , kw = . , n = ) with richness being lowest in sbt compared to mkl (p < . ) and ytk (p < . ) (figure a) . gill phylogenetic diversity exhibited the same pattern (p < . , kw = . , n = ) with sbt having lower diversity than mkl (p < . ) and ytk (p < . ) (figure b ). skin microbial diversity was also significantly different across species for richness (p < . , kw = . , n = ) and phylogenetic diversity figure | cross fish species comparison of microbial diversity (rarified to reads). (a) richness and (b) faith's phylogenetic diversity across sbt, mkl, and ylk for three body sites: gill, skin, and digesta. beta diversity comparisons of fish species mkl and ylk to sbt. pairwise comparisons of dissimilarity from mkl and ylk each compared to sbt independent per body site using (c) unweighted unifrac distances, and (d) weighted unifrac distances (*p < . , **p < . , ***p < . , ****p < . ). (p < . , kw = . , n = ), even more so than gill microbial diversity (figures a,b) . on the skin, for both richness and phylogenetic diversity, a gradient was observed with sbt being lowest followed by mkl and then ytk. digesta richness and phylogenetic diversity was lower in sbt compared to mkl (p < . ) while mkl was also lower than ytk (p < . ) ( figure a) . for phylogenetic diversity, sbt was lower than mkl (p < . ) for all body sites and lower than ytk for both gill and skin but not digesta (p < . ) (figure b) . beta diversity was assessed using unweighted unifrac, which gives rare taxa an equal weight, and weighted unifrac, which weights taxa based on their relative abundance in the sample. for each unique body site (gill, skin, and digesta), all sbt samples were compared to mkl and ytk samples rarified at reads. for unweighted unifrac, there was no significant difference in microbial diversity in the gill (figure c ) whereas for skin, distances to sbt were lower for mkl than ytk (mann-whitney p < . , u = ). digesta distances of ytk and sbt were lower as compared to mkl (mann-whitney p < . , u = ) ( figure c) . for weighted unifrac, distances of all three body sites of mkl to sbt were lower than ytk (gill: mann-whitney p < . , u = , difference = . ; skin: mann-whitney p < . , u = , difference = . ; digesta: mann-whitney p < . , u = , difference = . ) (figure d ). parasite infections cause significant economic complications in marine aquaculture systems, particularly in high value species such as tuna (shinn et al., ) . in this study we set out to describe how the fish mucosal microbiome is associated by parasitic infection and treatment with praziquantel (pzq) across three body sites including the gill, skin, and digesta of southern bluefin tuna (sbt). results indicate that the microbiome composition is most explained by body site location, followed by geographic location (company) and lastly pzq treatment. the microbiomes at each body site were associated with company and pzq treatment, but were not associated with blood fluke infection (measured as the number of adult blood flukes in the heart, or parasitic egg density in the gills). lastly, in a meta-analysis, we show that mackerel may have a more similar microbial community to tuna. since both can be infected by the same parasite and also have a similar microbiome, we suggest the feasibility of using a mackerel as a model for sbt for understanding mucosal microbiome community dynamics. further experiments would be needed to validate the model. our findings indicate that husbandry practices, including geographic location, influence the mucosal microbiomes of ranched sbt, and that parasite infection may not have a significant impact on fish microbiomes. our overall dna sequencing success rate was rather low for a typical microbiome study (gill = . %, skin = . %, digesta = . %) and we attribute this to either poor field preservation or that the samples were too low of biomass. in the field, we used % ethanol to preserve samples, and although % ethanol was shown to best preserve the microbiome community for field collection in human stool samples, it can also inhibit particular steps of molecular assays including dna extraction and pcr, and we would instead recommend further optimization of this preservation method in the future for fish or instead use cryopreservation, e.g., dry ice (kemp et al., ; demeke and jenkins, ; song et al., ) . a second explanation is that the samples were low biomass meaning there was very few microbial cells. since we used column cleanups, it's likely that our limit of detection was between , and , cells, thus if a sample had less than this it would potentially fail sequencing. to improve this, we recommend using magnetic bead cleanup methods in the future (minich et al., b) . since % ethanol has been shown to work successfully for storage of rna viral samples (minich et al., a) , its most likely that sample dropout in this particular study was likely due to low biomass of the samples. sample type was the strongest predictor of the microbiome in the dataset. mucosal environments of fish, including the gill, skin, and gut, are inhabited by commensal microbial communities which can have both negative and positive impacts on fish health (gomez et al., ; beck and peatman, ) . recent advances in genomics methods including transcriptomics, microbiome, and proteomics have enabled the characterization and monitoring of these communities in relation to host response and environmental changes (salinas and magadán, ) . although the anterior kidney is an important organ for immune function (abelli et al., ; fänge, ; watts et al., ; geven and klaren, ) , only four of the samples had detectable microbial dna. we would expect anterior kidneys to generally lack a microbial community except in cases of systemic infection. in comparison, gill, skin, and digesta samples had a much higher success rate, indicating a rich microbial community. while various studies have focused on fish body site microbiomes independently (primarily gut followed by skin and gill), few have evaluated the cumulative microbiome across multiple body sites for individual fish (ghanbari et al., ; larsen et al., ; pratte et al., ; minich et al., a minich et al., , c . our study highlights how amongst sbt body sites, gills have a high microbial diversity which was not as pronounced in the other fish species (relative to body sites within that species). gill microbial communities in fish are understudied but have been shown to be influenced by host diet, (pratte et al., ) and environmental conditions such as suspended sediment (hess et al., ) . gill and skin communities, however, were generally more similar and stable as compared to the gut communities in sbt. this stability has also been shown in pacific chub mackerel, scomber japonicus (minich et al., a) . gill and skin communities were primarily enriched in proteobacteria, while gut communities had higher proportions of mycoplasmatales within the tenericutes phylum and brevinematales within the spirochaetes phylum. gill and skin samples were enriched in various sotus within pseudomonadales and vibrionales both of which can be a source of potential pathogens and thus important to study further for health monitoring (williams et al., ; austin, ) . mycoplasma are a common marine microbe associated with fish guts (llewellyn et al., ) . while many host associated genera within spirochaetes are intestinal pathogens including treponema, borrelia, leptospira, and brachyspira, little is known about the order brevinematales (bellgard et al., ; gupta et al., ) . fish mucosal environments including the gill, skin, and digesta can also be influenced by the rearing condition and surrounding environment including the water (minich et al., c ). when analyzing body sites independently, skin and digesta samples were significantly differentiated by the location of rearing (company). while it is probable that the five companies sampled utilized varying animal husbandry approaches (including diet) which could influence the host-associated microbial communities, it is equally likely that the pontoon location had an effect. depth of ocean floor has been shown to positively reduce parasite infection rates in sbt as the sediment and intermediate host, the polychaete longicarpus modestus, is further away from the fish and current velocities are higher (cribb et al., ; kirchhoff et al., ) . sediment is known to impact microbial communities of the water column, so it is likely this would additionally impact the fish microbiome (hess et al., ) . another possible explanation for a company effect would be that biofouling on farming structures could differ based on location in the ocean and net cleaning frequency. although it is not known if elevated biofouling leads to increased microbial diversity in fish, it is possible that exposure to different microbes could be greater. during pontoon aquaculture, many benthic organisms at the larval stage will settle on the pen infrastructure, utilizing nutrients from fish feces and excess feed. many of these organisms are common benthic invertebrates and have been shown to negatively impact aquaculture by consuming oxygen, reducing water flow in the pens, and harboring microbes which can be pathogenic (cronin et al., ; fitridge et al., ; madin and ching, ) . biofouling communities may change depending on geospatial location of the pontoons which may experience varying oceanographic conditions including currents, wind, and upwelling. our study identified pseudoalteromonas, psychrobacter, and vibrio as representing the most abundant genera across the body sites. in culture based studies, vibrio, photobacterium, pseudoaltermonas, tenacibaculum, and flavobacteria have all been isolated from the gills of sbt (valdenegro-vega et al., ) . prominent sbt skin microbial genera identified included pseudoalteromonas, vibrio, acinetobacter, psychrobacter, and mycoplasmataceae. skin microbes including vibrio, clostridium, enterobacter, klebsiella, and proteus isolated from other tunas have previously been shown to be important for causing decomposition through histamine production (yoshinaga and frank, ) . treatment of pzq had a moderate effect on microbial communities in sbt. when comparing alpha diversity, digesta samples decreased in richness, shannon, and faith's phylogenetic diversity in fish treated with pzq. when comparing beta diversity, unweighted unifrac distances in gill and digesta samples were associated with pzq treatment which may suggest that rare taxa were more influenced by pzq. for weighted unifrac, only gill and skin samples were influenced, indicating that more abundant microbial taxa were impacted by pzq treatment. children successfully cured of schistosomiasis with pzq had higher fusobacterium bacteria abundances in the gut microbiome compared to infected children who were not successfully cured (schneeberger et al., ) . the associations were not strong compared to other factors, but suggests that pzq has an impact on the human gut microbiome which could be extrapolated to fish. a caveat of this study is that only one company did not use pzq thus the effect seen could also be confounded by husbandry differences between these companies. it is also possible that microbes in the gut may metabolize pzq rendering it less effective or even toxic to the host . to the best of our knowledge, this is the first study to investigate the impact of blood fluke infection on the mucosal microbiome of a marine fish. however, no significant associations were observed between the microbiome and blood fluke prevalence or intensity in sbt. hypothetically, parasitic infections can influence the microbial communities of their hosts either directly through grazing or indirectly by promoting secondary infections post trauma. in atlantic salmon, parasitic copepod sea lice, lepeophtheirus salmonis, infections were associated with decreased bacterial richness and dysbiosis of the skin microbiome (llewellyn et al., ) . in freshwater barramundi farms, parasitic ciliate abundance was associated with changes in the gill microbiome and mortalities (bastos gomes et al., ) . despite a relatively low incidence of infection in this study, the results suggest that blood fluke infection does not impact the microbiome. initial studies on blood fluke densities in ranched sbt show that infected fish have on average flukes per fish (aiken et al., ) . the median in this study was one blood fluke per fish with a maximum of six, therefore the infection intensity was low compared to previous reports. our study demonstrated that pacific chub mackerel (mkl), scomber japonicus, has a similar gill, skin, and digesta mucosal microbiome to sbt and thus is a putative candidate model organism to study the parasitome and mucosal microbiome. previous studies have shown that platyhelminth parasites are distributed across multiple tuna hosts (aiken et al., ) . further, caridcola infections occur in other scombrid species along with other fish families (nolan et al., ) . scomber japonicus has been shown to be successfully treated for skin fluke infections with pzq (yamamoto et al., ) . for microbiome comparisons, digesta samples analyzed by unweighted unifrac and alpha measures showed yellowtail kingfish (ytk) was more similar to sbt. since ytk and sbt are both tertiary carnivores and, in this study, both are farmed, their diet is generally more rich in fish protein than mkl which are small pelagic secondary consumers (agusa et al., ; hajeb et al., ) . this association of diet and high trophic level corresponding with low diversity was first observed in mammals, and may also be conserved to some extent in fish (ley et al., a,b) . however, when relative abundance (compositional) measurements are included in the analysis, all mkl body sites were more similar to sbt, which could suggest evidence of phylosymbiosis for highly abundant taxa. for microbiome studies, a link has been demonstrated within plants and animals to suggest that phylogenetically related hosts retain more similar microbiomes (pollock et al., ; ross et al., ; lim and bordenstein, ) . while most of these studies have focused on the gut microbiome, additional body sites have recapitulated this relationship in the skin (chiarello et al., ) but not gills for fish and amphibians. our study demonstrates how gill, skin, and gut communities of the sbt were more similar to a genetically similar fish within the same family. since the mkl microbiome is similar to sbt, it provides an opportunity to explore developing mkl as a model for future sbt research. one of the challenges with marine finfish aquaculture is performing replicated experiments, including disease challenges, that are not feasible to do in a production setting (salama and rabe, ) . mackerel is an ideal species for aquaculture research as they are globally distributed, not threatened, easy to catch, inexpensive, small, and easy to culture in tanks. our findings demonstrate that southern bluefin tuna harbor a unique microbiome in each body site. the sbt microbiome is influenced by pontoon location and, to a lesser extent, treatment with the antihelminth therapeutic, praziquantel. no association between blood fluke infection and the microbiome was identified. it is possible, however, that infection intensity was not sufficient or sample size not adequate to identify a relationship. in addition, we showed genetically similar fish (scombrids) have a more similar microbial community across multiple body sites including gill, skin, and digesta, suggesting possible phylosymbiosis. based on this finding, the exploration of pacific chub mackerel as a candidate model organism for studying the microbiome and potential parasitome of sbt is proposed. tuna are highly valued, and present significant challenges for conducting controlled experiments due to their size and physiology. future work employing a smaller, highly accessible relative could enable greater gains in research and ultimately, enhanced aquaculture productivity. the datasets presented in this study can be found in online repositories (qiita id , erp ). the names of the repository/repositories and accession number(s) can be found in the article/supplementary material. ethical review and approval was not required for the animal study because all animals were harvested and euthanized as part of the standard harvest by aquaculture company personnel in accordance with standards established by the australian southern bluefin tuna association. microbiome samples were opportunistically obtained from individual specimens post-euthanization. jm, bn, nb, and ea contributed to the conception and design of the study. jm collected microbiome samples in the field, performed dna extractions and microbiome processing, analyzed the data, and wrote the first draft of the manuscript. cp performed the analyses on gill egg density and heart fluke counts. mm contributed to analysis and visualization. all authors contributed to the manuscript revision, and read and approved the submitted version. we thank the university of tasmania for hosting jm and royal melbourne institute of technology (rmit) for enabling sample processing. the australian southern bluefin tuna industry association are acknowledged for hosting the team in port lincoln, providing support for the collection of samples, including visits to commercial sbt ranching pontoons. the supplementary material for this article can be found online at: https://www.frontiersin.org/articles/ . /fmicb. . /full#supplementary-material histology of developing thymus in sea bass dicentrarchus labrax (l.) exposure assessment for trace elements from consumption of marine fish in southeast asia molecular evidence for cosmopolitan distribution of platyhelminth parasites of tunas 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interactive, microbe-centric analysis tool efficacy of oral praziquantel treatment against the skin fluke infection of cultured chub mackerel, scomber japonicus the silva and "all-species living tree project (ltp)" taxonomic frameworks histamine-producing bacteria in decomposing skipjack tuna (katsuwonus pelamis) key: cord- - tf oqa authors: wang, kai; ran, ling; yan, tao; niu, zheng; kan, zifei; zhang, yiling; yang, yang; xie, luyi; huang, shilei; yu, qiuhan; wu, di; song, zhenhui title: anti-tgev miller strain infection effect of lactobacillus plantarum supernatant based on the jak-stat signaling pathway date: - - journal: front microbiol doi: . /fmicb. . sha: doc_id: cord_uid: tf oqa transmissible gastroenteritis (tge), caused by transmissible gastroenteritis virus (tgev), is one many gastrointestinal inflections in piglets, characterized by diarrhea, and high mortality. probiotics are ubiquitous bacteria in animal intestines, which have many functions, such as promoting intestinal peristalsis and maintaining the intestinal balance. we found that the supernatant of the lp- strain of lactobacillus plantarum, isolated in our laboratory, and named lp- s had marked anti-tgev effect on ipec-j cells. lp- s could induce large amounts of interferon-β in ipec-j cells in the early stage ( h) of infection with tgev, and increased the level of phosphorylated signal transducer and activator of transcription and its nuclear translocation in the late stage ( – h) of infection. this resulted in upregulated expression of interferon-stimulated genes, and increased the transcription and protein expression of antiviral proteins, resulting in an anti-tgev effect. transmissible gastroenteritis virus (tgev) is the pathogenic agent of porcine transmissible gastroenteritis (tge), which causes vomiting, diarrhea, and high mortality in suckling piglets (masters, ) , resulting in heavy losses to the pig breeding industry (zhao et al., ) . in particular, viral diarrhea diseases are more serious because of limited treatment options. probiotics comprise microorganisms that have beneficial activities to the host, and mainly comprise clostridium butyricum, lactobacillus, bifidobacteria, actinomycetes, and yeasts. they usually occupy the human gut and reproductive system, and can improve the balance of the host microecology (fuller, ; maragkoudakis et al., ; da silva sabo et al., ; stofilova et al., ) . there is growing interest in the oral administration of appropriate probiotics to reduce the pressure in the intestines and produce an effective innate immune response (pollmann et al., ; maragkoudakis et al., ) . in recent years, probiotic animal feed supplements have been developed as viable alternatives to antibiotics because of the ban on antibiotics in feed (scharek et al., ) . the addition of probiotic feed can prevent the infection of pathogens causing intestinal diseases, directly benefiting the animal host (villena et al., ) , or can indirectly enhance the host's immune response by balancing the disordered microbiota (lee et al., ) . in addition, many basic and clinical studies have confirmed that probiotic strains have antiviral effects (lee et al., ; yuan et al., ) . studies have shown that lactobacillus plantarum can stimulate the body's innate and acquired immunity, and contributes to the production of inflammatory factors that inhibit the replication of the virus in the body. for example, l. plantarum strain yu (lpyu) not only has high interleukin (il)- -inducing activity mediated by tolllike receptor (tlr) in mouse peritoneal macrophages, but also communicates with natural killer cells (nk) in the spleen to stimulate the production of iga to enhance the body's anti-h n virus activity (kawashima et al., ) . in addition, l. plantarum l- can stimulate the production of type i interferon (ifn- ) to effectively inhibit the proliferation of h n (maeda et al., ) . tgev is an important gastrointestinal diarrhea virus; therefore, research and exploration into the antiviral mechanism of probiotics could lead to the development of oral probiotics to prevent and treat tgev infection. the body reacts rapidly to viral invasion by synthesizing and secreting type i interferon ifn- (ifn-α/β), which plays a key role in the host antiviral response. the binding of ifn- to its receptor (interferon α/β receptor, ifnar) leads to activation of the janus family kinase (jak) and subsequent signal transduction and transcriptional activator (stat) signaling cascade, resulting in the activation and upregulation of interferon-stimulating genes (isgs), ultimately activating ifn to exert its antiviral effects (zhao et al., ; chen et al., ) . a strain of l. plantarum was successfully isolated and named lp- (song han et al., ) . we wondered whether the ipec-j cells treated by lp- could induce an antiviral mechanism through the ifn-β/jaks/stat/isgs pathway after tgev infection. in the present study, we found that the supernatant of l. plantarum lp- (lp- s) could significantly inhibit tgev infection. by detecting the replication of tgev n gene in porcine intestinal epithelial cells treated with lp- s at different time points, we confirmed that lp- s had a preventive effect against tgev. then, by detecting the levels of ifn, p-stat and isgs, we further confirmed that lp- s exerts its anti-tgev role by upregulating the expression of ifn-β. porcine kidney cells (st) to amplify the virus and the experimental model pig jejunal cells (ipec-j cells) were both cultured in roswell park memorial institute (rpmi) medium (gibco, grand island, ny, united states) containing % fetal bovine serum (fbs, gibco), under • c and % co . ipec-j cells and st cells were purchased from the shanghai sur biotech co., ltd (shanghai, china). l. plantarum lp- was isolated and stored in our laboratory. its s ribosomal gene sequence has been submitted to genbank (mh ). lp- s was isolated from a culture of l. plantarum lp- after shaking for h at • c. the tgev miller strain was preserved in our laboratory. viral fluid was collected from st cells after replication for approximately h when the cells showed obvious cytopathic effects (cpes). the genbank sequences of the tgev n gene (gq- . ) coding sequence (cds) conserved region, the porcine mx cds (mx dynamin like gtpase ; ah . ), the mx cds (mx dynamin like gtpase ; ay . ), the isg cds (interferon-stimulated protein, kda; nm_ . ), the oasl cds ( - -oligoadenylate synthetase like; nm_ . ), the pkr cds (double stranded rna-dependent protein kinase; ab . ), the zap cds (zeta-chain associated protein; gu_ . ), and internal reference pig β-actin gene (actb; xm_ . ) were obtained and pairs of specific primers were designed using primer . software for quantitative realtime pcr [performed using sybr premix ex taq ii (takara, shiga, japan)] as follows: tgev-n (forward: -ttcaacccc ataaccctccaacaa- and reverse: -ggcccttcac cat gcgatagc- ), mx (forward: -atctgtaagcagg agaccatcaactt g- and reverse: -ctcgccacgtcca ctatcttgtc- ), mx (forward: -ttcactcgcatccgc acttcag- and reverse: -agctcctctgtcgcactc tgg- ), isg (forward: -ggcagcacagtcctgtt gatgg- and reverse: -tgcgtcagccagacctcat agg- ), oasl (forward: -cgttggtggtgg agacaca tacag- and reverse: -tcaggcgacaccttccagg atc- ), pkr (forward: -acaggacctgcacataact tgagg- and reverse: -tgctgtcggcagtgatgaaga ac- ), zap (forward: -gctcagtgcgaac acctgga tg- and reverse: -tgacagatgaaggcgtggag agg- ), and actb (forward: -ctcttccagc cctcct tcc- and reverse: -ggtccttg cggatgtcg- ). the designed primers were synthesized by shanghai shenggong biotechnology service co., ltd. (shanghai, china). after centrifugation of lp- with an od of . at rpm/min for min, the obtained supernatant was filtered through a . -µm filter, and then diluted with rpmi high sugar medium to six gradients at two times ratio, i.e., lp- s was diluted to obtain od values of . , . , . , . , . , and . , respectively. ipec-j cells were seeded at × /ml in -well plates and incubated overnight in % co at • c. the medium was discarded when the cells reached % confluence in the -well plate, and then µl of each gradient dilution of lp- s was added to each well. the medium in the control group was replaced with rpmi . after incubating for min, the supernatant was discarded, and the cells were washed twice with phosphate-buffered saline (pbs). culture was continued and the cytopathic effect (cpe) was observed daily. the maximum non-toxic dose of lp- s to the cells was detected using the -( , dimethylthiazol- -yl)- , -diphenyltetrazolium bromide (mtt) reagent (bbi, shanghai, china), when % of the cells in the negative control group were damaged. mtt assays for each dilution were repeated three times independently. lp- s was prepared as above and then diluted to an od of . , . , . , and . with rpmi high-sugar medium. ipec-j cells were seeded at . × /ml in -well plates and cultured in % co at • c. at % confluence, the medium was discarded, the cells were washed three times with pbs, ml of each gradient dilution of lp- s was added to each well, and the cells incubated at • c in % co for min. the medium in the control group was replaced with rpmi . for the other wells, the supernatant was discarded, tgev (multiplicity of infection (moi) = . ) in rpmi medium was added to lp- s-treated ipec-j , and incubated at • c in % co for . h. the supernatants were discarded and incubation continued in rpmi high glucose medium. after h of culture, proteins were extracted for western blotting to detect the levels of the tgev n protein after treatment with different concentrations of lp- s. ipec-j cells treated with lp- s for . h were exposed to tgev (moi = . ). the ipec-j cells treated with lp- s were sampled at h post infection (hpi), hpi, and hpi and then frozen and thawed three times to collect virus particles in the cells and supernatants. gradient dilution of ipec-j cells was performed from − and − , respectively. tgev titers of ipec-j cells treated with lp- s for different times were detected using st cells in -well plates. each dilution gradient was assayed in replicate wells. the tcid of the virus in the different groups was calculated by reed and muench methods. ipec-j cells were inoculated into -well plates at . × /ml. the ipec-j cells treated with lp- s for . h were exposed to tgev (moi = . ) for rna extraction and protein sampling. when the cells reached % confluence, rna was extracted and reverse transcribed into cdna and quantified at ng/ml. absolute fluorescence quantitative pcr was performed using fluorescence quantitative pcr. the reaction parameters were as follows: pre-denaturation at • c for min, followed by cycles of denaturation at • c for s, annealing at • c for s, and prolongation at • c for s. the reaction for each sample was repeated three times. the bio-rad cfx manager random matrix method was used to analyze the linear relationship between cycle threshold (ct) value and the copy number to calculate the copy number of the tgev-n gene. protein samples were also extracted at the same time point and detected using western blotting. the primary antibody was a mouse monoclonal antibody against tgev-n, and the secondary antibodies were horseradish peroxidase (hrp)-conjugated goat anti-mouse antibodies (proteintech,wuhan,china). the immunoreactive protein bands were visualized using a vilber fusion fx chemiluminescent imager. ipec-j cells were inoculated into -well plates at . × /ml. when they reached % confluence, three experimental groups were established: an lp- s optimal concentration treatment group infected tgev, a tgev single infection group, and the uninfected control group. cell samples at , , , and hpi were centrifuged at rpm for min, and the supernatant subjected to an enzyme linked immunosorbent assay (elisa) to detect ifn-β. transmissible gastroenteritis virus (moi = . ) was infected to ipec-j cells that had been treated with lp- s for . h, and then cell samples at , , and hpi were collected to produce protein lysates. in addition, tgev (moi = . ) was infected into ipec-j cells cultured in rpmi for . h as the tgev control group and the proteins were extracted at the same time points as the lp- s group. the protein concentration was determined using the bicinchoninic acid method and subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis. the separated proteins were transferred to a nitrocellulose membrane (bio-rad). the membranes were blocked using % skimmed milk and incubated with the following primary antibodies: anti-stat rabbit polyclonal antibodies (british biorbyt company), anti-phospho-(p)stat (tyr ) rabbit polyclonal antibodies (biorbyt), anti-zap rabbit polyclonal antibodies (abcam), anti-pkr rabbit polyclonal antibodies (abcam), anti-(p)pkr (t ) rabbit polyclonal antibodies (abcam), and anti-β-tubulin rabbit polyclonal antibodies (proteintech). secondary antibodies comprised goat anti-rabbit immunoglobulin (h + l). the immunoreactive protein bands were visualized using the vilber fusion fx imaging system (vilber), and the grayscale values of each band were analyzed by graphpad prism. ipec-j cells were seeded at a density of . × in cell slides in -well culture dishes. these cells were set as three experimental groups comprising an lp- s optimal concentration treatment group, a tgev alone infection group, and a blank (uninfected) control group, when they reached % confluence. the cells were sampled at , , and hpi; washed three times with pbs; fixed with % paraformaldehyde at • c for . h; washed three times with pbs; permeated by . % triton-x- for min; washed three times with pbs; and blocked by % bovine serum albumin for min at room temperature. primary antibodies comprising anti-p-stat protein rabbit polyclonal antibodies and anti-tgev-n mouse monoclonal antibodies were added and incubated overnight in • c in a wet box. the cells were then washed three times with pbs, proportionally added cy -goat anti-rabbit fluorescence and fluorescein isothiocyanate (fitc)-labeled goat anti-mouse fluorescent secondary antibodies were then added, the cells were incubated for h in a dark room at • c, incubated with -( -amidinophenyl)- h-indole- -carboxamidine (dapi) for min, rinsed with pbs. laser confocal microscopy was then used to observe p-stat , its nuclear translocation, and the tgev n protein. the protein levels were analyzed using the zen software. ipec-j cells were seeded at . × /ml in -well plates, grown to % confluence, and then divided into three groups: cells treated with lp- s for . h and then infected with tgev (moi = . ), tgev infection alone, and the blank control (uninfected cells). the ipec-j cells were sampled at , , and hpi for qrt-pcr. total rna was extracted using the rnaiso plus reagent (takara), and then single-stranded rna was isolated using an rna pcr (amv) ver . kit (takara). cdna was then synthesized via reverse transcription. quantitative real-time pcr was then performed using the sybr premix ex taq ii (takara) to detect the mrna levels of zap, pkr, oasl, and isg (fluorescent primers were synthesized by shanghai shenggong bioengineering technology service co., ltd.). small interfering rna (sirna) targeting stat , -ggaacagaaatacacctat- (produced by ribo, china) . transfected with the stat -specific sirna using lipofectaminetm (invitrogen, united states), according to the manufacturer's instructions, to the tgev-infected groups treated with lp- s and tgev infection alone groups, respectively, in ipec-j cells. consirna transfected as control groups. the collected samples were analyzed by western blotting using a rabbit pab recognizing stat as the primary antibody and hrp-conjugated goat anti-rabbit igg as the secondary antibody. all results were plotted and analyzed using graphpad prism software (graphpad inc., la jolla, ca, united states). the data were presented as the mean ± standard deviation (sd) of three independent experiments. data were statistically compared using the t test. a p value < . ( * p < . and * * p < . ) was considered statistically significant. the mtt assay (figure ) showed that the higher dilution ratio of lp- s, the higher the cell viability. the maximum non-toxic dose toward the cells was greater than % od ; therefore, the maximum non-toxic dose of lp- s to ipec-j cells is od . ( figure a) . the tgev inhibition rate of lp- s decreased with increasing dilution factor and was thus concentration dependent. the results showed that amount of tgev miller strain (moi = . ) was reduced by / -fold by lp- s, i.e., the od was . , and the treated ipec-j cells received the highest non-toxic dose of lp- s ( figure b) . the experimental data (figure ) showed that the tgev titers at , , and hpi after tgev infection of ipec-j cells treated with lp- s were . tcid /ml, . tcid /ml, and . tcid /ml, respectively. compared with the tgev control group, the amount of viral of lesions decreased by . , . , and . -fold at , , and hpi, respectively, in the lp- s group and no lesions appeared in the blank control group. therefore, lp- s has a significant anti-tgev effect ( * * p < . ) and is optimal for antiviral activity at hpi. we found that the tgev n gene copy number in ipec-j cells treated with lp- s was lower than that in cells infected with tgev only ( * p < . , * * p < . ). in addition, the viral n gene copy number in the tgev infection group was positively correlated with the infection duration ( figure a) . western blotting showed that the level of the tgev n protein in the lp- s group was lower than that in the tgev group at all three time points. no expression of tgev n protein was observed in the blank control group (figures b,c) . these results demonstrated that lp- s could inhibit the transcription and protein expression of tgev n. the results in figure indicate that ifn-β levels gradually increased in ipec-j cells during tgev infection. however, compared with the tgev-infected group, the ifn-β level of the tgev (moi = . )-infected group treated with lp- s increased significantly with infection time ( * * p < . ). therefore, lp- s can significantly increase the level of intracellular ifn-β during tgev infection. the results in figure show that there was almost no change in the total expression of stat in ipec-j cells under different treatments and at different time points; however, the amount of phosphorylated stat changed significantly. the amount of phosphorylated stat in the lp- s group was higher than that in the tgev group at the different time points ( * p < . and * * p < . ), while only a small amount of phosphorylated stat was detected in the blank control group at the different time points. therefore, although tgev infection could significantly increase the amount of phosphorylated stat , lp- s could further significantly increase the amount of phosphorylated stat in cells after tgev infection. the results in figure show that the amount of p-stat (red fluorescence) in the nuclei of tgev infected cells treated with lp- s at different time points was significantly higher than that in the nuclei of cells in tgev infected group. at the same time, the amount of p-stat correlated positively with the duration frontiers in microbiology | www.frontiersin.org figure | mtt cytotoxicity test and lp- s concentration screening. (a) results of lp- s cytotoxicity as detected using the mtt method on / , / , / , and / times dilutions of lp- s acting on ipec-j cells. the cell adherence state remained basically unchanged. according to the experimental data, the lp- s undiluted group, the / -fold dilution group and the / -fold dilution group showed significantly difference in cytotoxicity ( * * p < . ), and the / -fold dilution group showed no significant difference compared with that of the control group (p > . ). (b) the expression of the tgev n protein in ipec-j cells treated with lp- s at different dilutions was detected by western blotting. lane , infected tgev group after lp- s / dilution pretreatment; lane , infected tgev group after lp- s / dilution pretreatment; lane , infected tgev group after lp- s / dilution pretreatment; lane , infected tgev group after lp- s / dilution pretreatment; lane , tgev infection group; and lane , uninfected control cells (normal group). (c) grayscale analysis of the relative expression of tgev n in ipec-j cells infected with lp- s at different dilutions showing that tgev n protein levels were decreased in the / -fold dilution of lp- s treatment group after h, compared with the / -fold dilution and tgev infection group, there was a significant difference ( * * p < . ), which was significantly different from the control group ( * p < . ). of infection after lp- s-treatment of ipec-j cells infected with tgev. however, the amount of red fluorescence emitted by p-stat labeled with cy correlated negatively with the amount of green fluorescence emitted by fitc-labeled tgev n protein. we found that when tgev was directly infected into ipec-j cells, the red fluorescence of p-stat was mainly gathered around the cell wall and only a small amount appeared in the nucleus; the green fluorescence and red fluorescence of the blank control group were not obvious. meanwhile, the signal intensity of p-stat in the nuclei of the lp- s treatment group correlated positively with time ( figure ) . therefore, lp- s could significantly increase the level of p-stat and promoted its translocation into the nucleus, while simultaneously inhibiting the expression of tgev n. we found that the mrna expression levels of zap, mx , mx , pkr, oasl, and isg were significantly higher in the lp- s treated group than in the tgev infected group at various points after infection. the expression levels of zap, pkr, oasl, and isg in each experimental group increased with time. the expression levels of mx and mx peaked at h and then decreased at h (figures a-f) . the results showed that the best time to stat -sirna targeting gene silencing stat was h (figure g) , and the best interference fragment was stat -sirna ( figure h) . after gene silencing stat , we found that the expression of isgs decreased compared to the groups which no knock down stat , and the expression isgs of lp- s-treated tgev infected group was higher than that of tgev infected alone group (figures i-n) . the results showed that tgev infection of ipec-j cells treated with lp- s could stimulate the expression of isgs in cells to inhibit viral replication. as expected, the protein levels of zap, pkr, p-pkr (figures a-d) at , and hpi in the lp- s group were significantly higher than those in negative control group and tgev group (zap, * p < . and pkr, * * p < . ). there was figure e where the expression of this gene was knocked down using sirna is significantly lower than the level of unknocked down stat in figure a . after gene silencing stat , the protein levels of lp- s, lp- s pretreatment of ipec-j cells infected with tgev; tgev, cells directly infected with tgev; con, uninfected cells (normal group). there were significant differences in ifn-β levels between the tgev group and the lp- s group at different time points ( * * p < . ). e-h) at , , and hpi in the lp- s group were significantly higher than tgev group (p-stat /stat , * p < . ; zap, * p < . ; and p-pkr/pkr, * p < . ). the results showed that lp- s could there was no significant difference between the two groups (p > . ) at h; however, the difference was extremely significant at and h ( * * p < . ). lp- s, lp- s pretreatment of ipec-j cells infected with tgev; tgev, cells directly infected with tgev. showed that the level of phosphorylated stat was higher in the lp- s group than in the tgev group after h (p > . ); after h of lp- s treatment, the level of phosphorylated stat in the lp- s group was significantly higher than that in the tgev group ( * * p < . ); after h of lp- s treatment, the level of phosphorylated stat in the lp- s group was significantly higher than that in the tgev group ( * p < . ). the results showed that treatment of tgev-infected ipec-j cells with lp- s ( - h), induced increased of levels of phosphorylated intracellular stat . the nucleus was identified using zen blue software and the intensity of red fluorescence emitted by cy -labeled p-stat was analyzed. the statistical results showed that the fluorescence intensity of cy in ipec-j cells treated with lp- s was significantly higher than that in the tgev group after h ( * p < . ). in the late stage of tgev infection ( - h), the fluorescence signal intensity of p-stat in the nucleus of this group was significantly higher than that in tgev group ( * * p < . ). increase the expression of isgs and inhibit the replication of tgev in ipec-j cells, which was basically consistent with the expression trend of the pkr and zap genes. previous studies have found that many lactic acid bacteria can inhibit the infection of diarrhea-causing viruses (such as rv and pedv) in the host through a variety of methods (hou et al., ; kawakami et al., ) . some lactic acid bacteria can be recognized by toll like receptor (tlr)- or tlr- to enhance their response to stimulation by the interferon inducer poly (i: c) and induce a large amount of ifn-i (maeda et al., ; kanmani and kim, ; lee et al., ) . at the same time, they can also upregulate the transcription of il and tnfa (reyes-diaz et al., ) . in addition, some lactic acid bacteria can enhance the expression of surface molecules and cytokines in intestinal (a-f) relative mrna expression levels of mx , mx , pkr, zap, isg , and oasl, respectively. the expression levels of zap, mx , and mx in lp- s group after h were not significantly different from those in the tgev group (p > . ). the expression level of isg was significantly higher in the lp- s group than in the tgev group ( * p < . ). the expression levels of pkr and oasl were significantly higher in the lp- s group than in the tgev group ( * * p < . ). the expression levels of zap, mx , mx , pkr, oasl, and isg in the lp- s group after and h were significantly higher than (continued) figure | continued those in the tgev group ( * * p < . ). (g,h) screening of sirna targeting stat optimal treatment time and gene silencing efficiency fragment. as shown in the figure, the optimal time for sirna targeting stat is h, and the best gene silencing sirna fragment is sirna ( * p < . ). (i-n) relative mrna expression levels of mx , mx , pkr, zap, isg , and oasl, respectively, after targeting gene silencing stat . the expression levels of pkr and oasl in lp- s group after h were not significantly different from those in the tgev group (p > . ). the expression level of mx , pkr, zap, isg , and oasl was significantly higher in the lp- s group after h than in the tgev group ( * p < . and * * p < . ). the expression levels of zap, isg and oasl were significantly higher in the lp- s group after h than in the tgev group ( * p < . and * * p < . ). antigen presenting cells (apc), and enhance the molecular expression of mhc-ii and il- β (villena et al., ) . in addition, other lactic acid bacteria can also stimulate the response level of tlr- to poly (i: c) (hosoya et al., ) . they can also regulate the role of tlr- , tlr- , and tlr negative regulators in the immune response, further enhancing the production of ifn-i induced by cells, and upregulate the transcription level of related antiviral factors (such as mxa and oasl) (castillo et al., ) . other studies have shown that probiotics can inhibit tgev infection by adsorbing virus particles and stimulating cells to produce innate immunity (chai et al., ) . recent studies have shown that the main reasons why the body's interferon-beta (ifn-β) cannot fully exert its antiviral effect after tgev infection are as follows: first, the cells do not respond in time to the immune response because of the level of viral replication and the virus titer of tgev in the early stage of infection, resulting in lower levels of ifns, which is the main cause of the short burst of tgev latency (zhu et al., ) . second, ifn-β does not play a direct antiviral role. its antiviral function is produced by activating the ifn-mediated jak-stat signaling pathway to stimulate downstream interferonstimulating genes (isgs) (saha and pahan, ; li, ; proia et al., ) . and, activation of the jak-stat signaling pathway requires phosphorylated stat (tyr ) enters the nucleus to activate interferon-stimulating factors isgs (including mx , mx , pkr, oas, isg , and zap) (hovanessian, ; zhu et al., ; shi et al., ; goujon et al., ; amici et al., ; li et al., ; nigg and pavlovic, ) . zap can bind viral rna directly and prevent the accumulation of viral rna in the cytoplasm. it can also recruit rna exosomes to degrade target viral rna (li et al., ) . pkr-mediated inhibition of viral replication is activated by the formation of dsrna during the replication of single-stranded rna after viruses invade cells. the main reason is that the amino terminus of pkr can recognize the dsrna domain and the carboxyl terminus has the kinase domain. when the viral double-stranded rna is recognized, the inactive pkr protein located in the cytoplasm is phosphorylated. on the other hand, it can also regulate the cell immune response and autophagy caused by virus invasion to inhibit the virus. at the same time, pkr can activate the nuclear factor kappa b (nf-kb) signaling pathway via phosphorylation and further induce ifn production in cells (sudhakar et al., ; amici et al., ) . in the present study, we found that ipec-j cells still produced ifn-β after infection with tgev, which increased with time, (f) relative expression of p-stat /stat in lp- s treatment group was significantly higher than tgev infected alone at h ( * p < . ). (g) relative expression of zap in lp- s treatment group was significantly higher than tgev infected alone at h ( * p < . ). (h) relative expression of p-pkr/pkr in lp- s treatment group was significantly higher than tgev infected alone at h ( * p < . ). reaching a peak at h and no longer increased at h. at the corresponding time points, the level of infection, viral titer, and replication of tgev on ipec-j cells showed an increasing trend. after lp- s treatment, ipec-j cells produced a large amount of ifn-β at the early stage of tgev infection ( h), which was significantly higher than that of cells infected with tgev only. the induced level of ifn-β in lp- s-treated ipec-j cells was significantly different from that in the tgev infected group at the same time point. the induction level of ifn-β in the lp- s treated group was significantly higher than that in tgev infected group at the different time points. the level of ifn-β induction correlated positively with time, peaking at h before decreasing toward h. the early boost in ifn-β production in ipec-j cells treated with lp- s might be one of the reasons for its inhibition of tgev. in addition, although tgev delayed the expression of ifn-β in the early stage of infection, it promoted the expression of ifn-β at the peak of viral replication. the expression of ifn-β was parallel to the increase of viral rna replication level at - h, which demonstrated that tgev replication remained high in the late stage of infection when ifn-β was produced in large quantities. this might be caused by the inhibitory effect of tgev on ifn-β-mediated signaling pathways, resulting in the inability of ifn-β to regulate the transcription and expression of downstream target cytokines and exert an antiviral role. although there was no significant difference in the levels of p-stat between the lp- s group and the tgev group at hpi, the level of p-stat in the lp- s group was significantly higher than that in the tgev group at the later stages ( and h). the level of p-stat in the lp- s group correlated positively with time from - h. the results showed that ipec-j cells treated with lp- s could effectively increase stat phosphorylation in the late stage of virus infection. at the same time, the level of p-stat changed slightly from - h after infection with tgev. the results were quite different from those reported in previous studies on the infection of st cells by tgev. in addition, the level of p-stat in ipec-j cells infected with tgev from - h was significantly lower than that in st cells infected with tgev from - h, which might reflect the difference between the cell lines and viruses used. further experiments showed that the level p-stat nuclear translocation in the lp- s group was significantly higher than that in the tgev group at the same time points, and p-stat nuclear accumulation correlated positively with time. in tgevinfected cells, p-stat at the late stage of infection ( - h) accumulated in large amounts near the cell membrane and only a few nuclear translocations occurred. we speculated that the reason might be that the intracellular stat protein is activated by ifn-β after ipec-j cells are directly infected with tgev to form a homologous or heterodimer, and the virus interacts with its receptor irf . the interaction resulted in the inability of activated stat to undergo nuclear translocation through receptor-induced endocytosis, and could only dissociate around ifnar, which reduced the jak-stat signaling pathway cascade response and antagonized the antiviral effect of ifn-β. the fluorescence value of activated stat was significantly higher than that of blank control group. this indicated that tgev could not completely escape the immune mechanism of ifn-β figure | proposed mechanism of the anti-tgev effect of lp- s. lp- s increases ifn-β expression, and the binding of ifn-β to its receptor ifnar leads to activation of the janus family kinase (jak) and subsequent activation of signal transduction and transcriptional activator (stat ) signaling cascades. these signaling pathways upregulate downstream interferon-stimulated genes (isgs), including mx , mx , pkr, oas, isg , and zap), which produce the corresponding antiviral proteins, e.g., zap and pkr, ultimately activating ifn-β to exert an antiviral effect. production in ipec-j cells. at the same time, the fluorescence intensity of p-stat in the nucleus of the cells in the lp- s treatment group correlated positively with time, and the intensity of fitc-labeled tgev n protein was significantly lower than that in the tgev treatment group at the same time point. these results showed that ipec-j cells treated with lp- s could indeed activate the jak-stat signaling pathway when infected with tgev, and the intensity of jak-stat signaling pathway was significantly higher in the lp- s-treated cells than in the cells directly infected with tgev. as the signaling pathway cascade response increased, the replication level of tgev in ipec-j cells was further inhibited. finally, the transcriptional levels of isgs in the lp- s treatment group were different at different time points. mx and mx reached their peak at h, while the transcriptional levels of the two isgs decreased at h after tgev infection. the transcription levels of pkr, zap, oasl and isg were positively correlated in groups. we speculated that the decrease in mx and mx mrna transcription levels within h after lp- s treatment might be related to the cycle of infected cells. the expression of isgs of gene silenced stat decreased as a whole compared to the groups which no knock down stat , indicating that stat could affect downstream isgs. while the isgs of tgev infected group treated with lp- s showed an upward trend, which further confirmed that lp- s could activate downstream isgs. to further explore the difference in the intracellular response of lp- s treated ipec-j cells to tgev infection, we detected the changes in zap and pkr protein levels. the level of the zap protein in the lp- s and tgev groups was consistent with its transcription level at the time point. although the level of the zap protein in lp- s treated cells was significantly higher than that in the tgev infection group, the difference in the transcription level of zap was significantly lower than that in the tgev infection group. these results suggested that after lp- s treatment, the ifn-β and jak-stat signaling pathways induced in the cells are enhanced, and the expression of the zap protein is still limited, suggesting that other factors have an impact on the transcriptional regulation of zap. the protein level of pkr was consistent with its mrna transcription level. there was no significant difference in the p-pkr/pkr values between the two groups at and h; however, the p-pkr/β-tubulin protein values were in line with our expectations. the results showed that the p-pkr level in the lp- s group was significantly higher than that in the tgev group, and correlated positively with time. therefore, we believe that the increase in p-pkr is mainly determined by the expression of the pkr protein. based on the above results, lp- s plays an antiviral role by stimulating the ifn-β-mediated jak /stat pathway, resulting in upregulation interferon-stimulating genes, which induce the synthesis of antiviral proteins such as zap and pkr (figure ). the datasets generated for this study are available on request to the corresponding author. inhibition of viral protein translation by indomethacin in vesicular stomatitis virus infection: role of eif alpha kinase pkr oral administration of a probiotic lactobacillus modulates cytokine production and tlr expression improving the immune response against salmonella enterica serovar typhimurium infection in mice antiviral effects of a probiotic enterococcus faecium strain against transmissible gastroenteritis coronavirus matrix metalloproteinase facilitates hepatitis b virus replication through binding with type i interferon (ifn) receptor to repress ifn/jak/stat signaling inhibitory substances production by 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and in rat model of colitis in response to lactobacillus plantarum ls/ phosphorylation of serine in initiation factor alpha (eif alpha) promotes complex formation between eif alpha(p) and eif b and causes inhibition in the guanine nucleotide exchange activity of eif b immunobiotic lactobacillus rhamnosus strains differentially modulate antiviral immune response in porcine intestinal epithelial and antigen presenting cells egfr as a negative regulatory protein adjusts the activity and mobility of nhe in the cell membrane of ipec-j cells with tgev infection surfactin inhibits membrane fusion during invasion of epithelial cells by enveloped viruses effects of virulent and attenuated transmissible gastroenteritis virus on the ability of porcine dendritic cells to sample and present antigen endothelial cell proteomic response to rickettsia conorii infection reveals activation of the janus kinase (jak)-signal transducer and activator of transcription (stat)-interferon stimulated gene (isg) pathway and reprogramming plasma membrane integrin/cadherin signaling transmissible gastroenteritis virus does not suppress ifn-beta induction but is sensitive to ifn in ipec-j cells stat serine phosphorylation occurs independently of tyrosine phosphorylation and requires an activated jak kinase the authors gratefully acknowledge peng yuan, zhou yang, and other veterinary medicine students from the southwest university for their valuable suggestions and assistance. key: cord- -z zx gd authors: ljubin-sternak, sunčanica; meštrović, tomislav; ivković-jureković, irena; kolarić, branko; slović, anamarija; forčić, dubravko; tot, tatjana; mijač, maja; vraneš, jasmina title: the emerging role of rhinoviruses in lower respiratory tract infections in children – clinical and molecular epidemiological study from croatia, – date: - - journal: front microbiol doi: . /fmicb. . sha: doc_id: cord_uid: z zx gd rhinoviruses (rvs) are increasingly implicated not only in mild upper respiratory tract infections, but also in more severe lower respiratory tract infections; however, little is known about species diversity and viral epidemiology of rvs among the infected children. therefore, we investigated the rhinovirus (rv) infection prevalence over a -year period, compared it with prevalence patterns of other common respiratory viruses, and explored clinical and molecular epidemiology of rv infections among children hospitalized with acute respiratory infection in north-western and central parts of croatia. for respiratory virus detection, nasopharyngeal and pharyngeal flocked swabs were taken from each patient and subsequently analyzed with multiplex rt-pcr. to determine the rv species in a subset of positive children, ′utr in rv-positive samples has been sequenced. nucleotide sequences of referent rv strains were retrieved by searching the database with basic local alignment tool, and used to construct alignments and phylogenetic trees using mafft multiple sequence alignment tool and the maximum likelihood method, respectively. in our study population rv was the most frequently detected virus, diagnosed in patients ( . %), of which . % was detected as a monoinfection. median age of rv-infected children was . years, and more than half of children infected with rv ( . %) presented with lower respiratory tract infections. most rv cases were detected from september to december, and all three species co-circulated during the analyzed period ( – ). sequence analysis based on ′utr region yielded distinct strains; the most prevalent was rv-c ( . %) followed by rv-a ( . %) and rv-b ( . %). most of rv-a sequences formed a distinct phylogenetic group; only strains ri/hr - (along with a reference strain mf ) clustered with rv-c strains. strains belonging to the group c were the most diverse ( . % identity among strains), while group b was the most conserved ( . % identity among strains). despite such differences in strain groups (hitherto undescribed in croatia), clinical presentation of infected children was rather similar. our results are consistent with newer studies that investigated the etiology of acute respiratory infections, especially those focused on children with lower respiratory tract infections, where rvs should always be considered as potentially serious pathogens. rhinoviruses (rvs) are increasingly implicated not only in mild upper respiratory tract infections, but also in more severe lower respiratory tract infections; however, little is known about species diversity and viral epidemiology of rvs among the infected children. therefore, we investigated the rhinovirus (rv) infection prevalence over a -year period, compared it with prevalence patterns of other common respiratory viruses, and explored clinical and molecular epidemiology of rv infections among children hospitalized with acute respiratory infection in north-western and central parts of croatia. for respiratory virus detection, nasopharyngeal and pharyngeal flocked swabs were taken from each patient and subsequently analyzed with multiplex rt-pcr. to determine the rv species in a subset of positive children, utr in rv-positive samples has been sequenced. nucleotide sequences of referent rv strains were retrieved by searching the database with basic local alignment tool, and used to construct alignments and phylogenetic trees using mafft multiple sequence alignment tool and the maximum likelihood method, respectively. in our study population rv was the most frequently detected virus, diagnosed in patients ( . %), of which . % was detected as a monoinfection. median age of rv-infected children was . years, and more than half of children infected with rv ( . %) presented with lower respiratory tract infections. most rv cases were detected from september to december, and all three species co-circulated during the analyzed period ( ) ( ) ( ) . sequence analysis based on utr region yielded distinct strains; the most prevalent was rv-c ( . %) followed by rv-a ( . %) and rv-b ( . %). most of rv-a sequences formed introduction rhinoviruses (rvs) are small, non-enveloped viruses that belong to the family picornaviridae, genus enterovirus. to date, there are rhinovirus (rv) genotypes recognized and classified into the three species as rv-a ( types), rv-b ( types), and rv-c ( types) (royston and tapparel, ; pan et al., ) . rv-a and rv-b were discovered by isolation on monkey kidney cells in s (price, ) while rv-c genotypes, which are not cultivable using ordinary culture methods, have been identified decades after following the rise of molecular techniques (lamson et al., ; lau et al., ) . there are also differences between rv species in utilization of cell entry receptor: a majority of rv-a and rv-b attach to the intercellular adhesion molecule (icam)- (classified as the major receptor group) and the others alternatively bind low density lipoprotein receptor (ldl-r) (minor receptor group), whereas rv-c utilizes human cadherin-related family member (cdhr ) (bochkov et al., ; royston and tapparel, ) . rhinovirus genome is a . -kb single-stranded, positive-sense rna with a single open reading frame (orf) joined to a untranslated region ( utr) and a short viral priming protein (vpg) (jacobs et al., ) . orf encodes a poly-protein which is cleaved by virally encoded proteases in proteins. four proteins -vp , vp , vp , and vp -make up the viral capsid and account for the virus' antigenic diversity, while the remaining non-structural proteins are involved in viral genome replication and assembly. a rather conserved utr region that harbors internal ribosomal entry site (ires) is usually utilized for rv detection from clinical samples, while more precise genotyping is based on vp /vp or vp sequence analysis. rhinovirus have been neglected for decades, primarily because they were considered less virulent and only capable of causing mild common cold, and were not recognized, until recently, as important causative agents of lower respiratory tract infections (lrtis) and severe respiratory disease . introduction of sensitive and technically simple molecular detection assays, especially multiplex pcr, enabled affordable rv detection in line with other common respiratory viruses in clinical samples. together with coronaviruses, rvs are indeed responsible for majority of upper respiratory tract infection (urti), but also for substantial rate of lrtis in all age groups (lauinger et al., ; zhao et al., zhao et al., ,Čivljak et al., . many studies showed that rv is one of the leading causes of pneumonia, bronchiolitis and other form of severe respiratory disease (zhao et al., ) , standing side by side with respiratory syncytial virus (rsv) in children and influenza in elderly (esposito et al., ; ning et al., ; Čivljak et al., ) . there are several studies that report increased rates of asthma exacerbations and lrtis among children with rv-c when compared to those infected with rv-a and rv-b (bizzintino et al., ; lauinger et al., ) ; however, recent studies did not find any relationship between a specific rv species and the severity of clinical presentation (jacobs et al., ; van der linden et al., ; zhao et al., ) . data on rv prevalence in croatia are scarce, and molecular epidemiology was thus far not described. this study aims to determine the rv prevalence, compare it with prevalence patterns of other common respiratory viruses, as well as to explore clinical and molecular epidemiological features of rv infections among hospitalized children with acute respiratory infection. the research was performed in accordance with relevant guidelines/regulations and in line with the declaration of helsinki, as revised in . written informed consent was obtained from all participants (children's parents or their legal guardians). the study was approved by the ethics committee of the dr. andrija Štampar teaching institute of public health and conducted as part of the croatian science foundation project entitled "new and neglected respiratory viruses in vulnerable groups of patients" (no. ip- - - ). all standard biosecurity and institutional safety procedures have been adhered to. from may to april , a total of patients were included from hospitals located in north-western and central part of croatia: clinical hospital zagreb and general hospital karlovac, respectively. inclusion criteria were: age lower than years, a clinical diagnosis of acute respiratory tract infection (ari), and need for hospitalization [on ward (≥ day) or day hospital (for more than but less than h)]. exclusion criteria were presumed bacterial respiratory infection -including otitis, sinusitis and bacterial pneumonia, healthcare-associated infection, and ambulatory treated patients. patients were categorized into the four groups according to their respective age (i.e., < , - . , - . and ≥ years of age), and two groups according to the localization of infection in those with upper respiratory tract infection (urti) and lower respiratory tract infection (lrti). urti was defined by symptoms of the common cold, coryza, cough, and hoarseness with or without fever, so clinical syndromes of respiratory catarrh, rhinitis and/or pharyngitis represented urti category. lrti was defined by symptoms of tachypnea, wheezing, severe cough, breathlessness, and by specific clinical signs such as nasal flaring, jugular and intercostal retractions, cyanosis (in rare instances), as well as wheezing, crackles and inspiratory rhonchi or generally reduced breath sounds during auscultation. clinical syndromes of bronchitis, bronchiolitis and pneumonia were included in lrti category (tregoning and schwarze, ; ljubin-sternak et al., ) . to avoid unnecessary x-ray exposure, chest radiographs were taken only for some of the patients in order to exclude or confirm bacterial pneumonia. for respiratory virus detection, nasopharyngeal and pharyngeal flocked swabs from each patient were collected, combined, and placed in viral transport medium (utm tm , copan, italy). specimens were immediately transported to the molecular microbiology laboratory at the public health institute where they were stored at − • c until tested. the results of virology testing were released to the physicians periodically, approximately once per week. as a part of routine care, nasopharyngeal, pharyngeal swabs, and blood cultures were taken from hospitalized patients and submitted for bacterial diagnostics using standard cultivation methods. demographic and clinical data, antimicrobial use records, and the results of routine bacterial studies were collected by a retrospective review of patient charts. to isolate viral dna and rna from viral transport medium, µl has been extracted according to the manufacturer's protocol using ribospin tm vrd kit (geneall biotechnology, seoul, korea). multiplex rt-pcr for respiratory viruses using seeplex r rv detection kit (seegene inc., seoul, korea) was performed. briefly, multiplex pcr and cdna synthesis as onestep reaction was performed and set up in three different tubes with three sets of primers. more specifically, "a set" contained primers for simultaneous amplification of target sequences of adenovirus (adv), human coronavirus (hcov) e/nl , parainfluenza virus (piv) types - , and pcr internal control to check for the presence of substances that may interfere with amplification; "b set" contained primers for hcov oc , rv groups a/b/c, rsv type a and b, influenza (flu) type a, and pcr internal control; and "c set" contained primers for human bocavirus (hbov), flu type b, human metapneumovirus (hmpv), piv type , human enterovirus (hev), and the overall process control (human rnase p was included throughout the entire process as a control from nucleic acid extraction to amplification). amplification was performed on thermal cycler geneamp r pcr system (applied biosystems, foster city, united states). detection of pcr products was done by microchip electrophoresis on the mce r - multina device (shimadzu, kyoto, japan) -including software analysis showing results in the form of electropherograms and virtual image gels. to determine rv species, utr of the genome in rv-positive samples was sequenced. total rna was extracted from µl of rv-positive samples by the method reported by chomczynski and mackey ( ) . reverse transcription was performed at • c for min, in a reaction mix containing µl of isolated rna, × pcr buffer (ge healthcare, united kingdom), . mm of each dntp, u of rnase inhibitor (thermo fisher scientific, united states), . mm mgcl , . mm of random hexanucleotide primers and u of mulv reverse transcriptase (thermo fisher scientific, united states) in a final volume of µl. pcr reaction was performed by amplifying utr, using ntr + ( caa gya ctt ctg tyt ccc ) and ntr-( cac gga cac cca aag tag t ) primer pair, modified from wisdom et al. ( ) , corresponding to positions - and - of strain a (acc. no. x . ), respectively. final pcr mixtures contained × onetaq pcr buffer (new england biolabs, united states), mm dntp, . mm mgcl , . mm of each primer, and . u of onetaq dna polymerase (new england biolabs, united states). the amplified products were separated on a . % agarose gel, excised and purified by centrifugation through glass wool (sun et al., ) . sequencing reactions were set up with purified dna, one of the specific primers used for amplification, and a bigdye terminator v . cycle sequencing kit (thermo fisher scientific, united states) according to the manufacturer's protocol. sequencing and sequence analysis were performed on a genetic analyzer (thermo fisher scientific, united states). nucleotide sequences of referent rv strains were retrieved by searching the database with blast (basic local alignment tool) and used to construct alignments and phylogenetic trees. alignments were performed using mafft multiple sequence alignment tool available at the embl-ebi website , and edited in aliview v . (larsson, ) . phylogenetic trees were generated using the maximum likelihood method with molecular evolutionary genetics analyses (mega) software v . (tamura et al., ) , under the most appropriate model of nt substitution determined with jmodeltest v . . (darriba et al., ) . bootstrap probabilities for , iterations were calculated to evaluate confidence estimates. sequence conservation (defined as percentage of genomic positions identical in all strains; gaps were ignored during computing) and evolutionary distances (p-distances) within and between groups have been calculated using mega . . the sequences of hrsv strains obtained in this study were deposited in the genbank under acc. nos. mn -mn . data analysis was performed using stata/mp (ver. . ; statacorp llc, college station, united states). age was presented using medians with stated interquartile range (iqr). demographic and clinical parameters were compared by χ -test or fisher's exact test for categorical variables and by kruskal-wallis test for continuous variables. to assess the strength of association between dependent variable (rv positive patients) and age we used univariate logistic regression. p-value was set to < . . in total, patients were screened for respiratory viruses. the exact prevalence pattern of detected viruses can be seen in table . rv was the most frequently detected virus, diagnosed in patients ( . %); . % as monoinfection, and . % as coinfection with other respiratory viruses. this was followed by rsv ( . %), adv ( . %), pivs ( . %), flu types a and b ( . %), hcov /nl and oc ( . %), hbov ( . ), hev ( . %), and hmpv ( . %). there was a significant difference in age according to the specific virus (p < . ) (figure ) . median age in years of rv infected children ( . , iqr . ) was higher than in children infected with rsv ( . , iqr . ), pivs ( . , iqr . ), hcov ( . , iqr . ), hmpv ( . , iqr . ), and hbov ( . , iqr . ), but lower than in those infected with flu ( . , iqr . ), adv ( . , iqr . ), and hev ( . , iqr . ) (figure ) . there was no statistically significant difference in the prevalence of rv infection between age groups (p = . ). additionally, age ( year increase) was not significant predictor for rv positivity (or = . , % ci = . - . ; p = . ). according to the clinical presentation, there was a significant difference in the proportion of lrtis between the type of the respiratory viruses (p = . ) (figure ) . more than half of children infected with rv ( ; . %) presented with lrti; nonetheless, this was not significantly different from the proportion of rv positive children with urti ( ; . %) (p = . ), while children with rsv infection significantly more often presented with lrti (p < . ). out of the rv-positive samples by multiplex pcr, we were able to sequence samples ( %). sequence analysis based on bp of ' utr region of samples yielded viral types (seven strains had identical sequence) (figure ) . the most prevalent was rv-c ( / ; . %) followed by rv-a ( / ; . %) and rv-b ( / ; . %). most of rv-a sequences formed a distinct phylogenetic group; only strain ri/hr - (along with a reference strain mf ) clustered with rv-c strains (figure ) . of the three respective groups, strains belonging to the group c were the most diverse, with identity of . % ( of identical positions), while group b was the most conserved with . % identity among strains ( / ). group a comprised strains which shared an overall . % identical positions ( / ). calculated p-distances between groups showed group a and c are more closely related (p-distance . ) than to group b. similar p-value was calculated between group b and group a (p-distance . ) or group c strains (p-distance . ), respectively. no significant difference has been demonstrated in clinical symptoms based on the rv species, with the exception of increased frequency of antibiotic treatment in those infected with rv-a species (p = . ) ( table ) . most rv cases were detected from september to december, and all three species co-circulated during the analyzed period (figure ) . in this study we initially evaluated the prevalence of rv and other common respiratory viruses, as well as rv species distribution in hospitalized children with symptoms of ari over a period of years. our results are consistent with previously published studies that investigated the etiology of ari, especially those focused on children with lrtis (chen et al., ; ning et al., ) . indeed, rv is right next to rsv when addressing the most common causes of bronchiolitis in hospitalized children, as demonstrated in very low-birth-weight infants from argentina and in one multicentre prospective study from the united states, respectively (mansbach et al., ; miller et al., ) . although rv can be found both in upper and lower respiratory tract, the potential for spread and the pathophysiology of infection in those two regions differs (as evidenced by studies conducted on rsv) (kim et al., ; gonzález-parra and dobrovolny, ) . possible causes of such disparity are fundamental differences in the immune responses and virus-cell interactions between these two anatomical regions, resulting in altered disease manifestation and spread (gonzález-parra and dobrovolny, ). there is also evidence that mucus velocity is decreased in small children (akin to elderly individuals) (grubb et al., ) , making them more susceptible to lrti and creating in turn a potential niche for more detrimental effect of rv infection. when age is concerned, our study has showed that rv holds a middle ground, not affecting very young children as is the case with rsv. this is comparable with the results found in the study by cebey-lópez et al. ( ) , where rsv infection was also seen in the youngest age groups (less than year of age), rv was present in somewhat older children (mean between . and . months), while adv predominated in patients older than months. conversely, some other author groups pointed out how rv can predominate in practically all age groups (tsagarakis et al., ) . in order to determine the rv species, we decided to sequence and analyze utr, which has been established as a relatively simple and rapid technique for identifying the rv serotypes in clinical samples. moreover, it has been shown that utr rt-pcr demonstrated greater sensitivity than vp -vp pcr, as reflected by the higher positivity rate in amplification of clinical isolates, and further, no need for a nested pcr or multiple primer pairs -reducing in turn the contamination rate, cost and turnaround time (kiang et al., ) . despite using primers which target the region widely used for typing purposes, more than half of the samples produced no amplicons suitable for sequencing. such low rate of successful rv sequencing from clinical samples may be a consequence of mutations in primer regions or low viral load in original sample, but most probably arises due to rna degradation during freezing/thawing cycles. phylogenetic groups were readily distinguished and all three rv groups were detected, with group c and a predominating. the proportion of the three rv species revealed in this study (rv-a . %, rv-b . %, and rv-c, . %) is consistent with prior studies worldwide (rv-a, . - . %; rv-b, . - %; rv-c, - . %) (lauinger et al., ; rahamat-langendoen et al., ; jacobs et al., ; tsatsral et al., ; ratnamohan et al., ; van der linden et al., ; zhao et al., ) . intermixing of groups was not observed, except for a single group a sequence (ri/hr - ) which clustered with group c sequences, indicating a recombination event or co-infection as was proposed by richter et al. ( ) . furthermore, group b strains were detected sporadically during the analyzed period, which is in accordance with other studies (lauinger et al., ; launes et al., ; richter et al., ) . these results represent the first report on rv diversity in croatia. in previous reports rv-c (and in lesser extent rv-a) have been associated with more severe illness (bizzintino et al., ; lauinger et al., ; linder et al., ; chen et al., ) ; however, more recent studies failed to report the connections between species and disease severity (rahamat-langendoen et al., ; jacobs et al., ; van der linden et al., ; zhao et al., ) . in this study there were no significant differences observed in clinical symptoms among three species, except more frequent utilization of antibiotic therapy in patients with rv-a species which can be result of subjective clinical assessment of more severe disease and empirical introduction of therapy. rv circulated throughout the -year period covered by this study with peaks in autumn and winter months. previous studies reported that rv infections occur all year round, with peaks of infection usually in spring and autumn months (Čivljak et al., ) . however, recent research endeavors also report peaks in autumn and winter months (zhao et al., ) , and some of them specifically note that rv-c demonstrate peak in winter months (linder et al., ) . our study also observed no rv-c detection in spring and summer season. there are several limitations of the study. due to crosssectional study approach, a control group of asymptomatic patients could not have been included (which would facilitate assessment of rv infection severity). furthermore, we performed only utr targeted rt-pcr assay and did not confirm our results with vp /vp or vp sequences analysis. some authors report discordance between proposed phylogeny groups when sequences from the 'utr and vp /vp coding regions were analyzed, which can result in imprecise classification (ratnamohan et al., ) . more specifically, samples that clustered as rv a using utr analysis can be revealed as rv-c when vp /vp region is analyzed (ratnamohan et al., ) . also, complicated rhinovirus infections were excluded from the study. notwithstanding the aforementioned limitations, this is the first study endeavor cataloging a circulation of rv species in croatia over years. in conclusion, in our study more than half of children infected with rv presented with lrti, which (together with other newer studies in the field) underlines the need for paradigm shift where rvs will not be merely associated with urti, but also considered in cases of lrti. regardless of the diversity of rv found in this study and the purported heterogeneity of the rv strains infecting the children, the similarity of clinical presentation negates the notion that certain rv species might be more virulent, at least in our case. more clinical and epidemiological studies are warranted to further elucidate this issue, with inevitable use of animal models to study pathogenic specificities of rv infection. the datasets generated for this study can be found in the genbank-accession numbers from mn to mn . the studies involving human participants were reviewed and approved by the ethics committee of the dr. andrija Štampar teaching institute of public health and conducted as part of the croatian science foundation project entitled "new and neglected respiratory viruses in vulnerable groups of patients" (no. ip- - - ) . written informed consent to participate in this study was provided by the participants' legal guardian/ next of kin. slj-s, ii-j, and jv designed the research. slj-s, as, and df performed the experiments. mm and tt collected the data. bk, as, and df analyzed the data. slj-s, tm, mm, tt, ii-j, as, and df interpreted the data and prepared the draft of the manuscript. jv and tm critically reviewed the draft. slj-s and tm wrote the final version of the manuscript. all authors reviewed and approved the final version of the manuscript. this work has been fully supported by the croatian science foundation under the project no. ip- - - titled "new and neglected respiratory viruses in vulnerable groups of patients" (principal investigator slj-s). the funders had no role in the study design, data collection and analysis, decision to publish or preparation of the manuscript. association between human rhinovirus c and severity of asthma in children cadherin-related family member , a childhood asthma susceptibility gene product, mediates rhinovirus c binding and replication viral co-infections in pediatric patients hospitalized with lower tract acute respiratory infections epidemiologic, clinical, and virologic characteristics of human rhinovirus infection among otherwise healthy children and adults: rhinovirus among adults and children single-step method of total rna isolated by acid guanidine phenol extraction viral pathogens associated with acute respiratory illness in hospitalized adults and elderly from jmodeltest : more models, new heuristics and parallel computing impact of viral infections in children with community-acquired pneumoniae: results of a study of respiratory viruses prophylactic administration of respiratory syncytial virus immune globulin to high-risk infants and young children human rhinoviruses clinical and molecular epidemiology of human rhinovirus infections in patients with hematologic malignancy assay for noncoding region analysis of all human rhinovirus prototype strains ct findings in viral lower respiratory tract infections caused by parainfluenza virus, influenza virus and respiratory syncytial virus masstag polymerase-chain-reaction detection of respiratory pathogens, including a new rhinovirus genotype, that caused influenza-like illness in new york state during aliview: a fast and lightweight alignment viewer and editor for large datasets clinical features and complete genome characterization of a distinct human rhinovirus (rv) genetic cluster, probably representing a previously undetected rv species rv-c, associated with acute respiratory illness in children patient characteristics and severity of human rhinovirus infections in children molecular epidemiology of severe respiratory disease by human rhinoviruses and enteroviruses at a tertiary paediatric hospital in human rhinovirus c: age, season, and lower respiratory illness over the past decades etiology and clinical characteristics of single and multiple respiratory virus infections diagnosed in croatian children in two respiratory seasons prospective multicenter study of viral etiology and hospital length of stay in children with severe bronchiolitis human rhinoviruses in severe respiratory disease in very low birth weight infants the etiology of community-acquired pneumonia among children under years of age in mainland china genome sequences of rhinovirus genotype c detected in three patients with acute respiratory illness the isolation of a new virus associated with respiratory clinical disease in humans the significance of rhinovirus detection in hospitalized children: clinical, epidemiological and virological features phylogenetic analysis of human rhinoviruses collected over four successive years in sydney molecular epidemiology of rhinoviruses in cyprus over three consecutive seasons rhinoviruses and respiratory enteroviruses: not as simple as abc a quick, cost-free method of purification of dna fragments from agarose gel mega : molecular evolutionary genetics analysis version . rhinovirus-from bench to bedside respiratory viral infections in infants: causes, clinical symptoms, virology, and immunology age-related prevalence of common upper respiratory pathogens, based on the application of the filmarray respiratory panel in a tertiary hospital in greece molecular epidemiology of the human rhinovirus infection in mongolia during - a molecular epidemiological perspective of rhinovirus types circulating in amsterdam from screening respiratory samples for detection of human rhinoviruses (rvs) and enteroviruses: comprehensive vp -vp typing reveals high incidence and genetic diversity of rv species c genotypic diversity and epidemiology of human rhinovirus among children with severe acute respiratory tract infection in shanghai the authors wish to thank matea kvaternik celjak for her technical assistance. key: cord- -kwzo puo authors: si, lulu; meng, yu; tian, fang; li, weihua; zou, peng; wang, qian; xu, wei; wang, yuzhu; xia, minjie; hu, jingying; jiang, shibo; lu, lu title: a peptide-based virus inactivator protects male mice against zika virus-induced damage of testicular tissue date: - - journal: front microbiol doi: . /fmicb. . sha: doc_id: cord_uid: kwzo puo zika virus (zikv) was a re-emerging arbovirus associated with guillain–barré syndrome in adult and congenital zika syndrome in fetus and infant. although zikv was mainly transmitted by mosquito bites, many sexual transmission cases have been reported since the outbreak in . zikv can persist in testis and semen for a long time, causing testicular tissue damage and reducing sperm quality. however, no drug has been approved for prevention or treatment of zikv infection, especially infection in male testicular tissue. previously reported peptide z could inactivate zikv, inhibiting zikv infection in vitro and in vivo. importantly, z could inhibit vertical transmission of zikv in pregnant mice, reducing zikv infection in fetus. here we showed that intraperitoneally administered z could also be distributed to testis and epididymis, resulting in the reduction of zikv rna copies in testicular tissue and protection of testis and epididymis against zikv-induced pathological damage and poor sperm quality in type i interferon receptor-deficient a mice. thus, z , a zikv inactivator, could serve as an antiviral agent for treatment of zikv infection and attenuation of zikv-induced testicular tissue damage. zika virus was a re-emerging arbovirus (gao, ; pettersson and bohlin, ) , like dengue virus (denv) and japanese encephalitis virus (jev), belonging to flavivirus genus in the flaviviridae family. since the outbreak in brazil in , zikv has rapidly spread to countries and territories (baud et al., a; gorshkov et al., ) , attracting global attention. about % of those infected with zikv presented with asymptomatic, or only mild illness (petersen e. et al., ; pierson and diamond, ) . however, zikv infection has been associated with more severe complications: guillain-barré syndrome in adult (brasil et al., ; peixoto et al., ) and congenital zika syndrome in fetus and infant (baud et al., b; gurung et al., ) . zikv is mainly transmitted through mosquito bites (petersen l.r. et al., ) , while it can also be transmitted via in utero from mother to fetus (besnard et al., ) , blood transfusion (tai et al., ) and intercourse (duggal et al., ; mead et al., ; sakkas et al., ) . it is reported that zikv was transmitted through sexual contact, perhaps up to days after the onset of symptoms (turmel et al., ) , and infective virions were still isolated from semen days after infection (arsuaga et al., ; garcia-bujalance et al., ) . the viral load in semen was , times that in the blood at weeks postinfection (mansuy et al., ) , and viral rna was still detected up to days after illness onset (barzon et al., ) . it is also reported that zikv infection caused patients to have a decreasing total sperm count in the acute phase of infection (joguet et al., ) and abnormal spermogram results year after infection (avelino-silva et al., ) , suggesting zikv was harmful to human spermatozoa production. testis explants from uninfected donors were also proven to be susceptible to zikv infection (matusali et al., ) . as determined from an in vitro human testicular organoid culture system, zikv-infected testicular organoids may lead to multiple kinds of cell death (strange et al., ) . although little was known about zikv infection in human testis and epididymis, except for semen, many murine models were used to study damage to testicular tissue. govero et al. ( ) performed a study in wild-type c bl/ mice in the presence of the anti-ifnar antibody and revealed that zikv preferentially infected spermatogonia and sertoli cells in the testis. this led to cell death and destruction of the seminiferous tubules in association with testis damage and poor sperm quality (govero et al., ) . ma et al. ( ) also established a mouse model using ifnα/β receptor-deficient mice (ifnar −/− knockout mice), and demonstrated that zikv infection induced inflammation in the testis and epididymis, leading to severe damage to testes at days post-infection. taken together, these findings suggested that zikv could persist in testicular tissue for a long time, causing severe damage to testis and epididymis and reducing sperm quality. currently, no approved drug is available to inhibit zikv infection (da silva et al., ) , especially infection in testicular tissue. ebselen (ebs), an antioxidant in clinical trials, was reported to alleviate testicular pathology in zikv-infected mice by reducing the level of oxidative stress and proinflammatory cytokines. however, it only had a weak effect on zikv directly, and its safety for pregnant women was unknown (simanjuntak et al., ) . this calls for the development of safe and effective drugs to prevent zikv-induced testicular damage. the testis is a male reproductive organ, mainly producing spermatozoa and androgen. specifically, spermatogenesis is a complex cellular event taking place in the seminiferous epithelium of seminiferous tubules and protected by sertoli cells that form the blood-testes barrier (btb) by tight junction protein (su et al., ) . the btb provides a specialized microenvironment for spermatogenesis by preventing harmful agents from entering the seminiferous tubule, but this was found to pose a major obstacle to the delivery of therapeutic drugs to the seminiferous epithelium (cheng and mruk, ) . therefore, any drug able to prevent zikvinduced damage in testicular tissue should be able to cross the btb into seminiferous tubules, or reach the testicular tissue, to inhibit zikv from entering into seminiferous tubules. the most promising anti-zikv drugs so far include small-molecule compounds (deng et al., a; chan et al., ; li et al., ) , antibodies (zhang et al., ; wang et al., wang et al., , , and peptides (yu et al., ; jackman et al., ) . compared with small-molecule compounds, peptides were safer, especially for pregnant women. as a macromolecular substance, passing through the btb was challenging for antibodies, and it is reported that the concentration of specific igg entering into the rete testis was . % of that in blood serum (knee et al., ) . therefore, the safer and cheaper peptide drugs, which consisted of dozens of amino acids, began to gain gradual acceptance. we previously demonstrated that an amphipathic peptide z , derived from the stem region of zikv e protein (figure a) , inhibited zikv infection in vivo and in vitro, suggesting its promise as an anti-zikv candidate drug. what's more, z had a protective effect in pregnant mice and their fetuses, suggesting it was able to cross the placental barrier (yu et al., ) . however, whether z could enter the seminiferous tubules and protect testicular tissue against zikv infection remained unknown. in this work, we showed that intraperitoneally injected z could be distributed in the testicular tissue. it then inhibited zikv infection, resulting in significant reduction of viral loads and protection of testis, epididymis, and sperm from zikvinduced pathological damage. these results suggest that z has the potential to be further developed as an anti-zikv agent for treatment and prevention of zikv-induced damage in testicular tissue. (sirohi et al., ) ]; di, dii, diii, stem and transmembrane domain are shown in red, yellow, blue, orange, and wheat, respectively; the fusion peptide is shown in cyan, and z ( - ) is shown in green. tm cells were infected with zikv strain sz (b), mr (c) and flr (d), treated with z at different concentration, and then viral copies in the supernatant at h were measured by qrt-pcr. data were presented as means ± sd. (e) z -treated zikv of different strain lost infectivity on tm cells irreversibly. after incubation with z at • c for h, zikv particles were separated from the unbound z by peg to measure their infectivity on tm cells. each sample was tested in triplicate and the experiments were repeated at least once. the data from two independent experiments were presented as mean ± sd. mixture f- (dmem/f , thermo fisher scientific, waltham, ma, united states) supplemented with % horse serum and . % fbs at • c and % co . zika virus (zikv) strain sz / (genbank no. ku ) was kindly provided by dr. cheng-feng qin (deng et al., b) and preserved in our laboratory. zikv strains mr (#vr ) and flr (#vr ) were obtained from atcc. zikv was propagated in vero-e cells. briefly, vero-e cells were infected with the virus at multiplicity of infection (moi) of . . the supernatants were harvested at days post-infection, centrifuged at , rpm for min to remove cellular debris, and stored at − • c as stock. peptides z (mavlgdtawdfgsvggalnslgkgihqifg aaf), z -cy and scrambled peptide (ldiiaglsagfq ggatfvdahgmvkasflggnw) were synthesized at kangbei bio, co., ltd. (ningbo, china) with % purity. peptides were solubilized in dimethyl sulfoxide (dmso) at mm and stored at − • c. virus titer was detected by plaque forming assay as shown below. bhk- cells were seeded onto a -well plate with × cells per well and cultured overnight at • c and % co . serially fold diluted virus were added to each well and incubated for h at • c. then the supernatant was replaced with ml dmem containing % low melting agarose and % fbs. after agarose solidification, the cells were cultured at • c and % co for days. then cells were fixed with % formalin and stained with % crystal violet overnight. finally, the plaque forming units were counted, and virus titer was calculated. six male a mice ( - weeks old) were randomly divided into two groups. mice in each group (n = ) were injected intraperitoneally with z -cy ( µg in µl pbs) or pbs (vehicle in µl pbs) as control. after anesthetization with pentobarbital sodium, mice were imaged by the ivis lumina k series iii in vivo imaging system from perkinelmer (waltham, ma, united states) for h. to determine the distribution of z -cy in the testicular tissue, mice were sacrificed using pentobarbital sodium, and all testes and epididymides were removed for imaging. the radiant efficiency (ps − cm − sr − ) (µw − cm − ) in mouse body and testis and epididymis was calculated by living image . . software. to determine the protection of z against zikv-induced testis damage, male a mice ( - weeks old) were randomly divided into three groups (n = ): z -treated group, vehicletreated group, and mock-infected group. mice in the z -or vehicle-treated group were intraperitoneally injected (i.p.) with pfu zikv with z ( mg/kg) or vehicle on day , followed by i.p. administration of z ( mg/kg) or vehicle once daily for six consecutive days, respectively. mice in the mock-infected group only received pbs as normal control. the body weight was monitored daily for days, and blood was collected at , , , , and days post-infection (d.p.i.) for detection of zikv copies in sera. at d.p.i., mice were euthanized by co inhalation, and the testes and epididymides were removed. the weight and size of testes were measured as previously described (de la vega et al., ) . after imaging, the left testes and epididymides were immersed in bouin's for hematoxylin-eosin (h&e) staining. the right testes and epididymides were soaked in rnaiso plus reagent at − • c for further detection of zikv copies. for safety analysis of z in testicular tissue, male balb/c mice ( - weeks old) were randomly divided into two groups. five mice in each group were i.v. administered with z at mg/kg of body weight or pbs control for days. the body weight of mice was monitored every other day for days. blood was collected at h, as well as , , and days post-injection and sera were separated from the blood samples for use in elisa to detect the concentration of testosterone and inhibin b as well as the titer of z -specific antibody. at days, mice were sacrificed, and the testes and epididymides were removed for histological examination. mature sperm in the cauda epididymis of the three groups of male a mice were collected and placed in ml pbs (preincubated at • c) immediately after euthanasia. the sperm suspension was analyzed for total sperm count and motility by computer-assisted sperm analysis (casa), as previously described (goodson et al., ) , using hamilton thorne ivos ii (beverly, ma, united states). then, after smear, desiccation and fixation, remaining sperm were stained by the papanicolaou staining method for manual morphological analysis. sperm morphology was observed in each mouse. zikv-infected mice were euthanized at days post-infection. testes and epididymides were homogenized with beads in ml rnaiso plus reagent (takara, japan) using tissuelyser- (jingxin, shanghai, china) after weighing. homogenized tissue were centrifuged for min at , rpm at • c, then total rna in tissues was extracted according to the operating manual and stored at − • c for the next step. sperm collected in pbs were placed in rnaiso to extract total rna under the same procedure. viral rna in sera samples on specific days was extracted using the easypure r viral dna/rna kit (transgen, china) and stored at − • c for the next step. zikv rna was examined by one-step real-time quantitative reverse transcription pcr (qrt-pcr) using the mastercycler r ep realplex real-time pcr system (eppendorf, germany). zikv rna copies were calculated based on the standard curve which was determined by plasmid containing specific sequence. the following primers were used: zikv-f: -ttggtcatgatactgctgattgc- ; zikv-r: -ccttccacaaagtccctattgc- ; zikv-probe: -fam-cggcatacagcatcaggtgcataggag-bhq - . the concentration of testosterone and inhibin b in the sera of balb/c mice was detected by mouse testosterone (ml , mlbio, shanghai, china) and inhibin b elisa kit (ml , mlbio, shanghai, china), respectively. according to the manual, µl standard or testing samples were added to a -well plate, which was coated with purified mouse testosterone or inhibin b antibody combined with hrp labeling. hrp-conjugate reagent was added to each well, except blank well (no sample; hrpconjugate reagent added as background). the plate was closed with closure plate membrane and incubated at • c for min. after washing, chromogen solution was added and incubated for min at • c. stop solution was added to each well, and absorbance was read at nm. peptide z was dissolved in dmso and diluted to different concentration by dmem/f . then µl of different zikv strains were incubated with z for h at • c. the mixture was added to × cells seeded into a -well plate and incubated at • c for h. after the culture supernatant was replaced by dmem/f- with % horse serum, cells were cultured for h at • c. then the culture supernatant was collected to detect zikv rna copies by qrt-pcr, as described above. the ability of z to inactivate different zikv strains was determined as follows. briefly, µl z or z -scr, at graded concentration were added to µl zikv ( × pfu/ml), followed by incubation at • c for h. then, peg- and nacl were added to the treated virus at final concentration of % and . m, respectively. after incubation on ice for h, the mixture was centrifuged at , rpm for h. the supernatants were removed, and the pellet was resuspended in µl dmem with % fbs. the infectivity of the zikv particles in the pellet was determined by cck- on bhk- cells or qrt-pcr on tm cells. the testis and epididymidis of z -or vehicle-treated zikvinfected mice and mock-infected mice were all collected post mortem. tissues were fixed in bouin's overnight, dehydrated, embedded in paraffin and sectioned. then the sections ( µm thick) were stained by h&e. subsequently, observation was made via panoramic scanner ( d histech pannoramic midi, hungary). student's unpaired two-tailed t-test was used to monitor the distribution of z in male a mouse body and testicular tissue and to analyze the difference of viral rna level in sera or tissues between z -and vehicle-treated a mice. one-way anova was used to examine the effect of z on the weight, length and width of testes, as well as sperm count, sperm motility and progressive sperm motility among the three groups. p-value was calculated by graphpad prism software, v. . , and significant difference was achieved with p-value less than . . * p < . ; * * p < . ; * * * p < . ; * * * * p< . . to determine the protective effect of z on zikv infection of testicular tissue, we tested if z could inhibit infection by different zikv strains of asian and african lineages in mouse sertoli tm cells, which are nurse-like cells that support spermatogenesis (wei et al., ) and important target cells for zikv testicular infection. zikv sz (asian lineage), flr (asian lineage), or mr (african lineage) was pretreated with z at different concentration before addition of tm cells and incubation at • c for h. after replacement of culture medium and further incubation for h, the viral copies in the supernatant were examined by qrt-pcr. as shown in figures b-d, z treatment resulted in a decrease of zikv copies in a dosedependent manner. considering that z -mediated inhibition of zikv infection is possibly attributed to its viral inactivation activity (yu et al., ) , different strains of zikv were incubated with z at different concentration for h at • c, followed by separating virions from the unbound free peptide by peg and detecting the infectivity of z -treated zikv in tm cells ( figure e ) and bhk- cells (supplementary figure s ) . we found that z -treated zikv strains lost their infectivity in a dose-dependent manner with % effective concentration (ec ) of . ± . µm (for sz ), . ± . µm (for mr ) and . ± . µm (for flr), respectively, suggesting that z inhibits infection of zikv strains of both asian and african lineages in tm and bhk- cells via inactivation of virions. to determine whether z entered testicular tissue of male mice, we employed cy -conjugated z peptide (z -cy ) to detect the distribution of z in the organs of male mice. as shown in figure a , the bodies of the z -cy -treated mice showed a strong fluorescence signal with average radiant efficiency of about . × (ps − cm − sr − ) (µw − cm − ), which is significantly higher than that in pbs-treated mice (p = . , student's two-tailed t-test; figure b) . then, the testes and epididymides were collected for examination of the fluorescence signal in the testicular tissue. as expected, both testes and epididymides of mice treated with z -cy showed a strong fluorescence signal, while those in the pbs-treated mice displayed no significant fluorescence signal (figure c) . the average radiant efficiency in testes and epididymides of z -cy -treated mice was significantly figure | distribution of z in the testicular tissue of male a mice. (a) imaging of male a mice treated with z -cy or pbs by the ivis lumina k series iii from perkinelmer. male a mice were injected intraperitoneally with µg z -cy (n = ) or pbs (n = ) as control, followed by imaging analysis. (b) the statistical analysis of fluorescence signal intensity in mouse body. data were presented as means ± sd. (c) imaging of the testes and epididymides from male a mice. (d) statistical analysis of fluorescence signal intensity in testis. each sample was tested in triplicate and the data were presented as mean ± sd. (e) statistical analysis of fluorescence signal intensity in epididymis. data were presented as means ± sd. * p < . ; * * * * p < . , student's two-tailed t-test. higher than that of pbs-treated mice (p < . , student's twotailed t-test; figures d,e) . these results suggest that z peptide can be distributed in the testis and epididymis of male mice. to determine the protective effect of z against zikv infection of male mice, pfu zikv were intraperitoneally injected into male a mice (type i interferon receptor-deficient). the infected mice were i.p. administered with z at mg/kg of body weight or vehicle, respectively, daily for days (figure a) . mice in the mock-infected group received pbs as normal control. results showed that the z -treated mice had neither weight loss ( figure b ) nor obvious clinical symptoms, consistent with mice in the mock-infected group (data not shown). however, in the vehicle-treated group, mouse body weight began to decline from the fifth day post-infection (d.p.i.) (figure b) , and some symptoms, like hunched posture and ruffled fur, appeared. we then examined viral copies in sera of z -or vehicle-treated mice at different time points by qrt-pcr. a high level of viral load was detected in sera of vehicle-treated mice, e.g., about copies/ml at d.p.i. (figure c) . however, viral load in sera of z -treated mice (figure c ) was as low as copies/ml at all time points tested, significantly lower than that of vehicle-treated mice. these results suggest that z can exert protection against zikv infection of male mice. to further evaluate the protective effect of z against zikvinduced damage of testicular tissue in male mice, all mice were sacrificed at d.p.i. and their testes were collected for analysis of weight and size. we found that testis weight in vehicle-treated mice was around mg, which was significantly lower than that in z -treated mice (∼ mg) (p < . , one-way anova, figure a ). the length and width of testes in vehicle-treated mice were both significantly decreased compared with those of testes in z -treated mice the change of mouse body weight was monitored daily for days. (c) the change of zikv rna level in mouse sera were detected by qrt-pcr at days , , , , and after zikv infection. male a mice were intraperitoneally injected with pfu zikv with z ( mg/kg) or vehicle on day , followed by daily injection of z ( mg/kg) or vehicle for six consecutive days. each sample was tested in triplicate and the data were presented as mean ± sem, * * * p < . ; * * * * p < . , student's two-tailed t-test. (p = . and p < . , one-way anova, figures b,c) . no significant difference in weight (p > . , one-way anova, figure a ), as well as length (p > . , one-way anova, figure b ), and width (p > . , one-way anova, figure c ) of testes were noted between z -treated zikvinfected mice and mock-infected mice. the representative image of testes from the three groups of mice were shown in figure d . subsequently, we examined the testes and epididymides for histopathological changes. the results of h&e staining of testes in vehicle-treated mice revealed that the normal architecture of the seminiferous tubule was seriously destroyed and replaced with an infiltrate of mixed inflammatory cells and necrotic debris, accompanied by degeneration of the spermatogenic lineage ( figure e, upper panel) . the connective tissue areas surrounding the seminiferous tubule had also been infiltrated by a large number of inflammatory cells (figure e, upper panel) . however, the architecture of the seminiferous tubule in the testes of both z -treated and mock-infected mice was intact and clear. spermatogenic cells at different stages were organized tightly and identified clearly (figure e, upper panel) . histological analysis of epididymis showed that epididymides from mice in the vehicle-treated group were also damaged. sperm in the lumen of caput epididymides decreased precipitously, only to be replaced by secretions and numerous necrotic epithelial cells (figure e, middle panel) . the lumens of cauda epididymis contained degenerating spermatozoa and a small number of normal spermatozoa, accompanied by scattered necrotic epithelial cells and inflammatory cells (figure e , lower panel). however, histological analysis of the caput epididymis and cauda epididymis showed no apparent microscopic differences between z -treated and mock-infected mice (figure e middle, lower panel). the architecture of the caput epididymis and cauda epididymis in these two groups was normal with no obvious morphological damage, suggesting that z protected testicular tissue against zikv-induced pathological damage. we used casa to evaluate the protective effect of z on the count and motility of mouse sperm. as shown in figure , the figure | z effectively attenuated damage to testes and epididymides of zikv-infected male a mice. (a) the weight, (b) length, and (c) width of testes from z -or vehicle-treated zikv-infected and mock-infected male a mice at day . each symbol represents one testis; all horizontal bars indicate mean, and error bars reflect sem. (d) the representative image of testes from z -or vehicle-treated zikv-infected and mock-infected male a mice at day . scale bar, mm. (e) histopathological analyses of testes and epididymides collected from z -or vehicle-treated zikv-infected male a mice and mock-infected mice used as a control. scale bar: µm. upper panel, testes; middle panel, caput epididymides; lower panel, cauda epididymides. * * p < . ; * * * * p < . ; one-way analysis of variance with tukey's multiple comparison post hoc tests. sperm count of z -treated mice was significantly higher than that of the vehicle-treated group (p = . , one-way anova; figure a ). meanwhile, the percentages of total ( figure b ) and progressively ( figure c ) motile sperm in z -treated mice were dramatically higher than those in the vehicle-treated group (p = . and p = . , one-way anova), but similar to that in the mock-infected mouse group (p > . , oneway anova). papanicolaou staining of morphological spermatic features revealed more noticeable teratogenesis of sperm in vehicle-treated mice compared to the other groups ( figure d) . to investigate whether the protective effect of z on testicular tissue results from the reduction of local viral load, we examined the viral copies in different testicular tissues. results showed a high level of viral rna ( - equivalents per g) detected in the testis and epididymis of vehicle-treated mice at d.p.i., much higher than that ( - equivalents per g) in z -treated mice (figures a,b) . notably, zikv rna was also detected (up to equivalents per ml) in the mature sperm collected from cauda epididymis in vehicle-treated mice, which was significantly higher than that in z -treated mice (p = . , student's twotailed t-test; figure c ). finally, to examine the safety of z for male mice, male balb/c mice were injected intravenously with z at mg/kg of body weight (n = ) or pbs (n = ). results showed that body weight change of mice was nearly consistent in the two groups ( figure a) , indicating that z peptide did not cause significant harm to the male mice. since the levels of testosterone and inhibin b reflect testicular function and sperm count, the concentration of these hormones in mouse sera at the indicated time points was measured. we found no significant difference between the z -and pbs-treated groups at all time points tested (figures b,c) , suggesting z may not affect the function of testis or sperm. we then compared the potential histopathological changes between the two groups. as shown in figure d , h&e analysis of testis and epididymis revealed no obvious pathological abnormality in mice treated with z compared with the pbs group. besides, the titer of z -specific antibody in sera of mice at and days post-injection was detected. as shown in figures e,f , no significant level of z -specific antibody was detected in the sera of mice that were intravenously injected with high doses of z peptide, consistent with the finding from our previous report for studying anti-mers-cov peptides (xia et al., ) . this result suggests that z peptide consisting of amino acids is unable to elicit a significant z -specific antibody response after it is intravenously administered in the absence of figure | z inhibited zikv replication in testes, epididymides and sperm. zikv rna copies in (a) testes, (b) epididymides, and (c) sperm of z -or vehicle-treated zikv-infected male a mice at day were detected by qrt-pcr. each symbol represents data from individual mice; all horizontal bars indicate mean, and error bars reflect sem. experiment was repeated at least twice. * * * p < . or * * * * p < . respectively, student's two-tailed t-test. figure | safety analysis of z for male balb/c mice. balb/c mice were injected with z ( mg/kg/day, i.v.) for days (n = ), and another group of mice (n = ) received pbs as a control. (a) body weight change of balb/c mice at different time points. data were presented as means ± sem. concentration of (b) testosterone and (c) inhibin b in sera before and after z injection. data were presented as means ± sem of triplicate experiments. (d) histological analysis of the testis and epididymis collected from z -or pbs-treated male balb/c mice. scale bar: µm. (e) z -specific antibody response in mice days after i.v. administration of z or pbs. (f) z -apecific antibody response in mice days after i.v. administration of z or pbs. each sample was tested in triplicate and the data were presented as mean ± sd. adjuvant. therefore, z is safe for male mice, especially for their testicular tissue. currently, many studies have reported the deleterious effects of zikv on male testicular tissue, causing severe damage of testis and epididymis, even leading to infertility uraki et al., ) . two dna vaccines were reported to reduce zikv persistence in the testicular tissue and zikv-associated pathological lesion (griffin et al., ) , or partially prevent infertility of male mice (de la vega et al., ) . however, no effective and safe antiviral agent has ever been reported to prevent or treat zikv infection in testicular tissue. our previous study has demonstrated that z peptide is highly effective in inhibiting zikv infection in vivo and in vitro (yu et al., ) . noticeably, it can penetrate the placental barrier to inhibit vertical transmission of zikv in pregnant mice. however, whether z could cross the btb and protect testicular tissue against zikv infection remained unknown. several studies have reported that sertoli cells play an important role in the entry of zikv into the seminiferous tubules and support long-term replication of zikv in the testicular tissue (siemann et al., ; kumar et al., ) . we found that z peptide possesses potent antiviral activity against zikv infection in bhk- and vero cells (yu et al., ) . in this study, we found that z was also highly effective in inhibiting infection of divergent zikv strains with asian and african lineages in tm cells, the mouse sertoli cell line. particularly, z treatment via intraperitoneal injection resulted in dramatically decreased zikv rna level in the testis of a mice, suggesting that z can protect testis against zikv infection in sertoli cells. meanwhile, we employed z -cy to examine whether z could enter seminiferous tubule, and we found that intraperitoneally injected z could be distributed in the testicular tissue of male a mice, consistent with the observation in mice intravenously administered with z (yu et al., ) . however, because of the intricate structure of capillary vessel and seminiferous tubule in mouse testis, we could not obtain sufficient evidence to prove that z crossed btb into seminiferous tubule. h&e analysis showed no obvious pathological damage in the testicular tissue of z -treated mice, but it did reveal severe pathological damage in the testis and epididymis of vehicle-treated mice, consistent with the findings of other studies (govero et al., ; ma et al., ) . when combined with evidence that viral load in mature sperm of z -treated a mice was significantly decreased, we speculate that z may, indeed, cross the btb and enter seminiferous tubule to inhibit zikv infection in the sperm. zika virus infection in the testicular tissue not only damages male testicular tissue, resulting in pathological lesion of testes and epididymides, but also produces zikv-infected semen, causing infertility. in addition, zikv in semen of an infected male can be sexually transmitted to his pregnant partner (russell et al., ; nelson et al., ) , who can further pass the virus to her fetus, causing congenital zika syndrome in the newborn (yarrington et al., ) . sexual transmission may also contribute to the spread of zikv in regions where the aedes mosquito is not endemic (rowland et al., ) . here we found that z treatment could significantly reduce viral load in sperm of zikv-infected a mice and improve the number and motility of sperm, implying that application of z can limit the damage to testicular tissue and sperm caused by zikv infection and reduce the risk of sexual transmission of zikv. conclusion z administered via intraperitoneal or intravenous injection could be distributed in mouse testicular tissue, protect the tissue against zikv infection and zikv-induced pathological damage and poor sperm quality, suggesting that z peptide has the potential to be further developed as an anti-zikv therapeutic for treatment of zikv infection and attenuation of zikv-induced damage in the testicular tissue. all datasets generated for this study are included in the manuscript/supplementary files. the animal study was reviewed and approved by shanghai public health clinical center animal welfare and ethics committee institutional laboratory animal care and use committee at fudan university. probable sexual transmission of zika virus from a vasectomised man potential effect of zika virus infection on human male fertility? 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Å resolution cryo-em structure of zika virus human testicular organoid system as a novel tool to study zika virus pathogenesis drug transporters, the blood-testis barrier, and spermatogenesis transfusion-transmitted zika virus infection in pregnant mice leads to broad tissue tropism with severe placental damage and fetal demise late sexual transmission of zika virus related to persistence in the semen zika virus causes testicular atrophy a human bi-specific antibody against zika virus with high therapeutic potential structural basis for neutralization and protection by a zika virus-specific human antibody b -h promoted proliferation of mouse spermatogonial stem cells via the pi k signaling pathway a pancoronavirus fusion inhibitor targeting the hr domain of human coronavirus spike congenital zika syndrome arising from sexual transmission of zika virus, a case report a peptide-based viral inactivator inhibits zika virus infection in pregnant mice and fetuses neutralization mechanism of a highly potent antibody against zika virus we are very grateful to the staff at the animal experiment department of shanghai public health clinical center for their contribution to this study. the supplementary material for this article can be found online at: https://www.frontiersin.org/articles/ . /fmicb. . /full#supplementary-material key: cord- -f fctcru authors: wang, changlin; shan, lingling; qu, shuxin; xue, mei; wang, keliang; fu, fang; wang, lu; wang, ziqi; feng, li; xu, wanhai; liu, pinghuang title: the coronavirus pedv evades type iii interferon response through the mir- c- p/socs axis date: - - journal: front microbiol doi: . /fmicb. . sha: doc_id: cord_uid: f fctcru porcine epidemic diarrhea virus (pedv) is an economically important pathogen that has evolved several mechanisms to evade type i ifn responses. type iii interferon (ifn-λ), an innate cytokine that primarily targets the mucosal epithelia, is critical in fighting mucosal infection in the host and has been reported to potently inhibit pedv infection in vitro. however, how pedv escapes ifn-λ antiviral response remains unclear. in this study, we found that pedv infection induced significant ifn-λ expression in type i ifn-defective vero e cells, but virus-induced endogenous ifn-λ did not reduce pedv titers. moreover, we demonstrated that pedv escaped ifn-λ responses by substantially upregulating the suppressor of cytokine signaling protein (socs ) expression, which impaired the induction of ifn-stimulated genes (isgs) and dampened the ifn-λ antiviral response and facilitated pedv replication in vero e cells. we further showed that pedv infection increased socs expression by decreasing host mir- c- p expression. mir- c- p suppressed socs expression through targeting the ′ untranslated region (utr) of socs . the inhibition of ifn-λ elicited isgs expression by socs was specifically rescued by overexpression of mir- c- p. collectively, our findings identify a new strategy by pedv to escape ifn-λ-mediated antiviral immune responses by engaging the socs /mir- c axis, thus improving our understanding of its pathogenesis. porcine epidemic diarrhea virus (pedv), a member of the alphacoronavirus family, is an enteropathogenic coronavirus with economic importance (madson et al., ; wang et al., ; zhang and yoo, ) . pedv infection in newborn piglets is characterized by vomiting, anorexia, watery diarrhea, and dehydration (song and park, ) . the virus primarily infects small intestinal epithelial cells in vivo and causes high morbidity and mortality in piglets (li et al., ) . interferons (ifns) are the key components of innate immunity in response to viral infection . among three types of ifns (types i, ii, and iii), type iii ifn-lambda (ifn-λ) primarily acts on mucosal surfaces, including epithelial surfaces of the liver, respiratory, and gastrointestinal systems, and plays vital roles in controlling viral infection within mucosal surfaces (mordstein et al., ; pott et al., ; lazear et al., ) . we and other groups previously demonstrated that porcine ifn-λdisplays powerful antiviral activity against pedv infection in both vero e cells and porcine intestinal epithelia (li et al., (li et al., , . pedv has evolved multiple strategies to escape ifn responses, including the degradation of stat and the suppression of type i ifn production . although type i and type iii ifns have a large overlap in the spectrum of induced antiviral isg responses, recent studies demonstrated that type iii ifn is a critical non-redundant antiviral mediator of type i ifns in the gi tract and elicits a unique transcriptional profile that does not completely overlap with that induced by ifn-α (wells and coyne, ) . it is necessary to clarify how pedv evades type iii ifn following infection. unlike ample studies reporting that pedv escapes type i ifns, limited studies demonstrate that pedv escapes ifn-λ response. pedv suppresses irf -mediated type iii ifn responses by reducing the number of peroxisomes and counteracting type iii ifn response by pedv nsp endoribonuclease deng et al., ) . deng et al. showed that type i and type iii ifns exhibit different modulation in response to pedv infection and that the discrepancy of type i and type iii ifn responses is independent of pedv endoribonuclease activity (deng et al., ) , suggesting that there are distinct strategies to modify host type i and type iii ifn responses during pedv infection. because cells generally produce both type i and type iii ifns in response to viral infection, it is challenging to elucidate how viruses escape ifn-λ response separately to type i response. in this study, we used vero cells, a cell line with a defective function, to produce endogenous type i ifns. vero cells are widely used as an in vitro model to study the interactions between viruses and hosts including pedv. we and others reported that vero cells respond well to both porcine type i and type iii ifns shen et al., ; li et al., ) . ifn-λ is rapidly produced after infection and following engagement with its receptor induces ifn-stimulated gene (isg) expression to mediate antiviral activity (kotenko et al., ; dellgren et al., ; lazear et al., ) . binding of ifnλ to its receptor, which consists of two subunits, ifn-λr and il- r , leads to activation of jak and tyk , which mediates the phosphorylation of stat and stat proteins (sheppard et al., ; palma-ocampo et al., ) . the suppressor of cytokine signaling protein (socs ), a negative regulator of janus family kinase (jak) signal transducer, simultaneously binds the receptors and jaks and prevents stats from accessing the receptor kinase complex (de weerd and nguyen, ; palma-ocampo et al., ) . previous reports demonstrated that socs is an inducible negative regulator of ifn-λ-induced gene expression in vivo (blumer et al., ) . socs was also associated with denv- escape from ifn-λ response during infection (palma-ocampo et al., ) . however, the role of socs during pedv infection remains unclear. micrornas (mirnas), as important post-transcriptional modulators of gene expression, participate in modulating the host innate and adaptive immune responses in response to pathogen invasion (baltimore et al., ; gottwein and cullen, ; o'neill et al., ) . increasing evidence has shown that mirnas of viral and cellular origin can help viruses evade host immune responses by targeting critical components in the host immune system (cullen, ; sullivan et al., ; kincaid and sullivan, ) . for example, mir- c is a potent negative regulator of type i ifn signaling by targeting jak , resulting in the enhancement of prrsv infection . the mir- family is a well-studied host mirna that plays an important role in viral infection by modulating ifn signaling (zhu et al., ; liu et al., ; ma et al., ) . our previous study revealed that tgev escapes type i ifn response by engaging the ire -mir- a- p/socs / axis (ma et al., ) . the potential role of mirnas in coronavirus escape from ifn-λ response remains elusive. in this study, we showed that pedv escaped ifn-λ responses by upregulating socs expression in type i ifn-defective vero e cells. in addition, we demonstrated that pedv infection increased socs expression by decreasing the expression of host mir- c- p, which modulates socs expression by specifically targeting the ′ utr of socs . our findings identify a new strategy by pedv to escape ifn-λ-mediated host innate immune defenses. african green monkey kidney cells (vero e cells) were stocked by our laboratory and grown in dmem (gibco, gaithersburg, md, usa) supplemented with % fbs (gibco) and antibiotics ( u/ml of penicillin and µg/ml of streptomycin) at • c in a humidified atmosphere of % co . pedv-cv (genbank accession no. kt ) stocked in our laboratory was propagated in vero e cells as previously described (hofmann and wyler, ; sun et al., ) . to evaluate the anti-pedv activity of porcine ifn-λ (prosit sole biotechnology, co., ltd., beijing, china), vero e cells were pretreated with designated concentrations of ifn-λ for h and then infected with pedv (moi of . ). all of the mirna mimics, mirna inhibitors, and short hairpin rnas (shrnas) were synthesized by gene pharma (shanghai, china). the mirnas and shrnas sequences are listed in table . lipofectamine (invitrogen, carlsbad, ca, usa) or lipofectamine rnaimax (invitrogen) was used to transfect cells with plasmid dna or synthetic oligonucleotides according to the manufacturer's instructions. the cells were infected with pedv as previously described after transfection for h. the cells were harvested for quantitative real-time pcr (rt-qpcr) or treated with np- lysis buffer for western blotting after infection for h. sequences total rna isolation, reverse transcription, and qpcr total cellular rna was isolated using an rneasy mini kit (qiagen sciences, hilden, germany) according to the manufacturer's instructions. total rna was extracted and reverse transcribed as previously described (ma et al., ) . for mirna reverse transcription, cdna was prepared with a mirna first strand cdna synthesis kit (sangon biotech, shanghai, china). qpcr was conducted in triplicate with power sybr green pcr master mix reagents (takara) on a lightcycler ii system (thermo fisher scientific, waltham, ma, usa) as previously described (ma et al., ) . the mirna expression levels were normalized to the internal control of u . the sequences of rt-qpcr primers for pedv, ifn-λ, socs , ifit , isg , mxa, gapdh, mir- c, and uni-mir transcription are listed in table . the results are presented as the means ± sem from three separate trials. targetscan release . (http://www.targetscan.org) was used to predict the targets of mir- c- p. socs ′ utrs as a prospective target was cloned and constructed as previously described (ma et al., ) . to construct the monkey socs expression vector, the full-length cds region of monkey socs was amplified from vero e cellular mrna pcr and cloned into pcaggs-ha vector (clontech, mountain view, ca, usa) using ecor i and kpn i restriction sites. the ′ utr of socs (genbank: ) was amplified and inserted into the pmirglo luciferase reporter vector. the socs ′ utr mutant vector was produced by mutating five seed nucleotides using a site-directed mutagenic kit (stratagene, la jolla, ca, usa) according to the manufacturer's instructions. the constructed plasmids were verified by sequencing. the luciferase activities were tested using a dual-luciferase reporter assay system (promega, madison, wi, usa) based on the manufacturer's instructions. wild-or mutant-type socs ′ utr luciferase reporter vectors were co-transfected with mir- c- p mimics (mir- c), mimic nc (nc), mir- c- p inhibitor (mir- c-i), or inhibitor nc (nc-i) into vero e cells for h. then prl-tk was co-transfected with either mir- c, nc, mir- c-i, or nc-i for h. the cells were collected and the luciferase activity was evaluated with a dual-luciferase reporter assay system (promega). the prl-tk vector expressing the renilla luciferase gene was used as a normalization control. vero e cells were fixed with % paraformaldehyde for min at • c and permeabilized with . % triton x- for min, then blocked with blocking buffer (pbs with % fbs) for h at • c. the cells were incubated with an anti-ha monoclonal antibody (sigma-aldrich, munich, germany, : ) at • c for h, followed by labeling with an alexa fluor goat anti-mouse igg antibody (thermo fisher scientific, : ) at • c for h. ′ , -diamidino- -phenylindole (dapi, : ) was used to stain the cellular nuclei. the stained cells were visualized using an amg evos f fluorescence microscope. vero e cells were lysed with np- lysis buffer (beyotime, china) supplemented with . mm of phenylmethylsulfonyl fluoride (pmsf) (roche, indianapolis, in, usa). target proteins were separated on sds-page gels then transferred onto nitrocellulose membranes (ge healthcare, chicago, il, usa). after blocking with tbs-t containing % non-fat milk at room temperature (rt), the membranes were incubated with primary antibody at • c for h. antibodies included: β-actin (sigma-aldrich, : ) and socs (sigma-aldrich, : ). the membranes were incubated with secondary antibody goat antimouse-hrp or goat anti-rabbit-hrp, diluted at : for h at room temperature, and visualized using an ecl system (thermo fisher). the results were analyzed using imagej software. all of the data are described as the means ± the standard error of the mean (sem). graphpad prism (graphpad software, inc.) was used to analyze the data using student's t-test. each experiment was repeated three times. p-values < . were considered significant: * p < . ; * * p < . ; * * * p < . ; * * * * p < . , and ns, not significant. kinetic curve of pedv replication in vero e cells. vero e cells were inoculated with pedv at an moi of . , and the level of pedv infection compared to mock controls at , , and h was quantified by rt-qpcr and tcid . the results were obtained from three independent experiments. mean ± sem, *p < . ; **p < . ; ***p < . ; ****p < . , and ns, not significant. previous research showed that the pretreatment of porcine ifn-λ inhibits pedv infection in vero e cells and ipec-j (li et al., ) . it is well-established that pedv replicates efficiently in vero e cells. to determine whether pedv elicits an endogenous ifn-λ response in vero e cells following infection, we initially infected vero e cells with pedv at moi = . and monitored the ifn-λ expression. compared with a mock uninfected control, pedv did not increase the expression of ifnλ transcripts as observed until hpi, and then gradually induced ifn-λ expression, indicating that pedv infection elicits type iii ifn expression at the late stage of infection instead of the early stage of infection in the vero e cells (figure a) , which was consistent with the results in porcine enteroids (li et al., ) . and pedv propagated efficiently in vero e cells by quantifying viral genomes and titers (figures b,c) . the virus titer increased up to / . ml at hpi ( figure c) . interestingly, despite the increased expression of endogenous ifn-λ at the late stage of infection, the pedv virus titer did not decrease. this indicates that there are mechanisms explored by pedv to antagonize the endogenous ifn-λ isg response at the late-stage infection. pedv infection increased the expression of socs in vero e cells socs , a typical member of the socs family of proteins, is a well-known negative feedback inhibitor of jak/stat signaling pathway induced by cytokines (ma et al., ) . to explore the underlying mechanisms exploited by pedv to escape ifnλ-induced antiviral responses, we initially assessed whether pedv infection induces socs expression in vero e cells. the mrna levels of socs significantly increased following pedv infection and displayed a time-dependent response (figure a) . the induction of socs by pedv infection was further verified by socs western blotting ( figure b ). counteracted the anti-pedv activity of ifn-λ socs is a potent inhibitor of the type i and type ii ifn signaling pathway (skjesol et al., ) . we next investigated whether socs suppresses ifn-λ-mediated antiviral activity. first, we silenced endogenous socs expression by specific shrnas. socs shrnas or a non-targeting shrna (nc) were transfected into vero e cells. the efficiency of socs knockdown was confirmed by western blotting (figure a) . shsocs # and # led to a and % decrease in socs expression, respectively, compared with nc ( figure a) . silencing of endogenous socs by shsocs # or # significantly reduced pedv replication in vero e cells without the addition of exogenous ifn-λ ( figure b) . the decreased levels of pedv infection were in line with the knockdown efficiency of socs shrnas, indicating the specific effect of socs shrnas. as previously reported, exogenous ifn-λ significantly inhibited pedv infection, whereas silencing of endogenous socs by shsocs # or # further enhanced the pedv inhibition by ifn-λ in vero e cells compared with untreated ifn-λ mock control ( figure b) . the knockdown of endogenous socs by shsocs # resulted in a more than . -fold decrease in pedv titer ( figure b ) and degraded to . -fold of pedv titers with ifn-λ treatment in vero e cells (figure b) , indicating that socs knockdown increases the antiviral effects of ifn-λ. inconsistent with the viral results, socs knockdown increased the mrna levels of isg , mxa, and ifit ( figure c) . we subsequently investigated the role of socs overexpression on the anti-pedv effects of ifn-λ. the transient overexpression of socs in vero e cells was verified by ha ifa (figure d ). as expected, socs overexpression substantially elevated pedv figure | mimics or inhibitor for h, cells were pretreated with ifn-λ or dmem for h and then infected with pedv (moi = . ) and harvested at hpi for viral rna quantification and tcid . (f) the socs expression levels in vero e cells were measured by rt-qpcr at hpi at different mois. p values represent the difference from the mock-infected control for time kinetics, the socs , and mir- c- p levels. error bars, mean ± sem. (n = independent experiments). *p < . , **p < . , ***p < . , and ns, not significant. infection ( figure e ) and blunted the expression of isg , mxa, and ifit ( figure f) . thus, these data indicate that socs counteracts the anti-pedv activity of ifn-λ. modulating mir- c- p the porcine mir- family (five members: mir a-e) has been demonstrated to modulate host type i ifn response during virus infection (zhu et al., ; liu et al., ; ma et al., ) . the targetscan (http://www.targetscan. org) prediction program indicated that socs was targeted by mir- c- p through a site in the ′ utr conserved in the socs of seven representative mammals ( figure a ). to investigate whether mir- c- p is involved in modulating ifnλ signaling by directly targeting socs and downregulating endogenous socs expression, we conducted a computational analysis using targetscan release . (http://www.targetscan. org). the result showed that mir- c could directly target the site on the ′ utrs of socs ( figure a) . we cloned the predicted target sites in porcine socs ′ utr, and constructed the firefly luciferase reporter vector of porcine socs ′ utr ( figure a) . overexpression of mir- c- p, the luciferase reporter containing the socs wild-type target sequence, decreased to ∼ % relative to nc mimics, whereas the blockage of mir- c by mir- c inhibitor increased socs ′ utr luciferase activity. however, the mutation of the socs target ′ utr site of mir- c- p disrupted the effects of mir- c- p on modifying the luciferase activity in vero e cells relative to the ncs ( figure b) . these results confirmed that mir- c- p directly targets the ′ utr of socs . consistent with the luciferase results, mir- c- p overexpression reduced socs expression measured by western blotting (figure c) . conversely, blockage of endogenous mir- c- p increased the expression of socs in vero e cells compared with the nc inhibitor. to further validate the modulation of socs expression by mir- c- p during pedv infection, we examined the expression of socs in pedvinfected vero e cells with overexpression or inhibition of mir- c- p, and found that the expression pattern of socs in pedv infected vero e cells was similar to that in pedv-uninfected e cells ( figure c) . taken together, these data demonstrated that mir- c- p downregulates the expression of socs by directly targeting socs ′ utr. to verify whether pedv escape the ifn-λ antiviral signaling through mir- c- p mediated modification of socs expression, we then explored the effect of mir- c- p on pedv infection and ifn-λ antiviral signaling. transient mir- c expression reduced pedv titers and promoted ifn-λ anti-pedv activity compared with the mock control ncs, whereas mir- c- p inhibitor significantly increased pedv infection and undermined the anti-pedv activity of ifn-λ (figures d,e) . furthermore, the socs expression increased starting at hpi and substantially increased at hpi, which was inversely correlated with the kinetic expression profiles of mir- c- p ( figure f ). in agreement with the kinetics pattern of mir- c- p and socs in vero e cells, pedv infection reduced the levels of mir- c- p and increased socs expression in ipec-j starting at h post-infection (data not shown). collectively, pedv infection upregulates socs expression by modulating host mir- c- p abundance at the late stage of infection. to further verify whether pedv escape ifn-λ response through the mir- c- p/socs axis, we co-transfected mir- c- p with socs and measured the replication of pedv with or without ifn-λ treatment. socs overexpression promoted pedv replication with or without ifn-λ treatment. mir- c- p largely abolished the role of socs in promoting pedv replication, and the effect was more pronounced in the presence of ifn-λ stimulation ( figure a) . consistent with this, socs inhibited ifn downstream isgs expression such as ifit and isg expression (figures b,c) , whereas overexpression of mir- c- p abrogated the isg inhibition of socs , which was more pronounced in the presence of ifn-λ priming (figures b,c) . in summary, these data indicate that pedv escapes the response of ifn-λ through the mir- a- p/socs axis. ifn-λ is an antiviral innate cytokine induced by virus infection that plays vital roles in controlling mucosal infection (blumer et al., ) . we and other groups previously showed that ifnλ substantially inhibits pedv (li et al., ; zhang et al., ) . however, whether pedv has evolved a mechanism to counteract endogenous ifn-λ just as pedv does the type i ifn response remains unclear. in this study, we found that pedv propagated well despite the significant production of endogenous ifn-λ induced at the late stage of infection in vero e cells, indicating that pedv escaped the ifn-λ response at the late stage of infection not through suppressing ifn-λ production. we further defined the mechanism that pedv counteracted ifnλ-elicited antiviral isg responses by exploiting the mir- c- p/socs axis. pedv has evolved multiple strategies to escape type i ifn response. whether pedv exploits similar mechanisms to counteract type iii ifn remains elusive. one previous study demonstrated that pedv escaped type iii ifn by suppressing irf -mediated ifn-λ production through pedv viral nsp protein . in that study, pedv actually upregulates ifn-λ expression at h post-infection and then decreased to minimal levels of ifn-λ expression until hpi figure | mir- c- p inhibited the infection of pedv by regulating the expression of socs . (a) socs overexpression increased pedv infection and undermined the anti-pedv activity of ifn-λ. vero e cells were transfected as described with pcaggs-ha, pcaggs-socs , and mir- c- p for h, followed by incubation with porcine ifn-λ ( ng/ml) or dmem for h. the cells then were infected with pedv at an moi of . ; pedv infection was determined at hpi. (b,c) mir- c- p abolished the impairment of the overexpression of socs to ifn-λ signaling under ifn-λ-stimulated or pedv-infected conditions. e cells were treated as described in the legend for panel a. the cells were collected for rt-qpcr analysis of ifit and isg expression relative to that of gapdh. error bars, mean ± sem (n = independent experiments). *p < . , **p < . , and ns, not significant. . in agreement with this, we did not observe increased ifn-λ expression at hpi ( figure a) . they did not show the ifn-λ expression at the late stage of pedv infection. in the current study, we observed that pedv elicited substantially increased ifn-λ expression in vero e cells only after hpi (figure a) , which is consistent with the results observed in porcine enteroids following pedv infection (li et al., ) , indicating that pedv has evolved mechanisms to escape ifn-λ antiviral response instead of ifn-λ production at the late stage of infection. it is possible that pedv exploits varying strategies at different infection stages. this is also observed in other rna viruses such as influenza virus (chung et al., ) . to prevent over-activation of the ifn signaling pathways, the host evolves a few negative regulators of ifn signaling, and socs is one of the canonical inhibitors of ifn signaling (shao et al., ) . socs has been reported to be exploited by multiple viruses to abrogate ifn antiviral signaling (shao et al., ; wei et al., ; ma et al., ) . we showed that pedv significantly induced the expression of socs at the late stage of infection (figure ) . as expected, increased socs impaired the antiviral isgs expression and impaired the anti-pedv activity of ifn-λ (figure ) . this is in agreement with the results of tgev, another swine alphacoronavirus (ma et al., ) . therefore, unlike previously published studies with the modification of ifn production mediated by viral proteins such as nsp , our study found that pedv largely evades innate immunity of ifn-λ by modulating the antiviral signal of ifn-λ rather than manipulating the production of ifn-λ at the late stage of infection. mirna plays a vital role in regulating gene expression through post-transcription modification. increasing evidence demonstrates that viruses escape ifn antiviral activity for optimal infection by modifying the cellular abundance of mirna targeting vital components of the ifn response (zhu et al., ; liu et al., ; ma et al., ) . jev infection downregulates the expression of mirna mir- , which directly targets the suppressor of cytokine signaling protein (socs ) and manipulates the jak-stat signaling cascade (sharma et al., ) . the mir- family has been reported to target socs family members and manipulate the jak/stat signaling pathway (zou et al., ; ma et al., ; yuan et al., ) . in this study, we showed that pedv infection suppressed mir- c- p expression, which was conversely related to socs expression during pedv infection (figure ) . just as other members of mir- , mir- c- p specifically targeted the ′ utr of socs and inhibited socs expression (kobayashi et al., ; ma et al., ; yuan et al., ) (figure ) . however, the mechanism of pedv decreasing mir- a- p remains unclear and deserves further study. in summary, we determined that pedv escaped ifn-λ response at the late stage of infection by downregulating mir- c- p, thus increasing socs expression. therefore, unlike previously published studies with defined mechanisms such as nsp , we demonstrated that pedv escapes ifn-λ response through another pathway of the mir- c- p/socs axis. our results highlight the important role of mir- c- p in the regulation of interferon pathways during pedv infection, improve the current knowledge of pedv infection, and expand the role of micro-rna in viral infection. the datasets generated for this study are available on request to the corresponding author. pl, cw, ls, and wx designed the research studies and analyzed and interpreted the data. cw, ls, sq, mx, kw, ff, lw, zw, and lf conducted the experiments and collected the data. pl, cw, and ls drafted the manuscript. all of the authors contributed revisions. micrornas: new regulators of immune cell development and function socs is an inducible negative regulator of interferon lambda (ifnlambda)-induced gene expression in vivo construction of a transcriptomedriven network at the early stage of infection with influenza a h n in human lung alveolar epithelial cells viruses and micrornas the interferons and their receptors-distribution and regulation human interferon-lambda is a potent member of the type iii interferon family coronavirus endoribonuclease activity in porcine epidemic diarrhea virus suppresses type i and type iii interferon responses viral and cellular micrornas as determinants of viral pathogenesis and immunity porcine epidemic diarrhea virus infection inhibits interferon signaling by targeted degradation of stat propagation of the virus of porcine epidemic diarrhea in cell culture virus-encoded 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hypoxia-reoxygenation-induced tubular epithelial cell injury via hif alpha stabilization by targeting socs the authors declare that the research was conducted in the key: cord- -y u e authors: sun, wuping; gao, hong; luo, yuhui; zheng, hushan; liao, xiang; xiong, donglin; xiao, lizu title: management of immunity alteration-induced chronic pain during the coronavirus disease- (covid- ) pandemic date: - - journal: front microbiol doi: . /fmicb. . sha: doc_id: cord_uid: y u e nan it has been reported that sars-cov- infections in elderly and weak individuals cause more severe syndromes, such as acute respiratory distress syndrome (ards) and acute lung injury (ali), which are associated with lung malfunction and death (matuschak and lechner, ) . patients with chronic obstructive pulmonary disease (copd) and smoking history have also been reported to experience worse progression and outcomes when diagnosed with covid- . in cancer patients has a % lethality, and worse prognosis has been observed among older individuals (stroppa et al., ) . human immunodeficiency virus (hiv)-positive patients may be more vulnerable to covid- due to their immunecompromised status. a recent clinical observation reported that covid- results in a death rate of approximately % among patients living with hiv (harter et al., ) . besides, some patients treated with opioids could be more susceptible to sars-cov- infections because treatments with morphine and fentanyl have been reported to be the most immunosuppressive (mellon and bayer, ; shavit et al., ) . opioids act on the hypothalamic-pituitary-adrenal (hpa) axis and the autonomic nervous system, further suppressing the immune system (mellon and bayer, ; shavit et al., ; plein and rittner, ) . these studies suggest that hypo-immune individuals might have higher risks and worse outcomes associated with covid- . on the other hand, the possibility of sars-cov- infection in hyper-immune individuals is also worldwide discussed because of their immunological background and therapies. it has been reported that hyper-immunity individuals have received treatment with immunosuppressive or modulatory agents; these approaches may increase the possibility of sars-cov- infection (cai et al., ) . therefore, individuals with altered immunity (hypo-immune & hyper-immune) may require additional attention to prevent the infection of sars-cov- during the covid- outbreak. it is known that the induction of a cytokine storm is the basic cause of pathogenic inflammation in covid- (jose and manuel, ; mehta et al., ) . cytokine storm is an acute hyperinflammatory response responsible for critical illness in many conditions, including viral infections, cancer, sepsis, and multi-organ failure (bhaskar et al., ) . the elevation of cytokines in the blood is crucial to induce cytokine storm and immunosuppression in the transition of severity in covid- patients (bhaskar et al., ) . laboratory results have shown that dysregulation in the immune system has occurred in covid- patients. sars-cov- infection increases the plasmatic secretion of interleukin β, interferon-γ, interferonγ-induced proteins, monocyte chemoattractant protein- , il- , and il- (vinciguerra et al., ) . moreover, it has been reported that sars-cov- infection induces the up-regulation of a series of interferon-stimulated genes, indicative of immune and interferon responses to the virus (blanco-melo et al., ) . these results demonstrated that sars-cov- infection-induced immune alteration in covid- patients. the sars-cov- infection causes systemic inflammation and dysregulation of immunity, resulting in various delayed neurological complications. both central and peripheral nervous systems have been reported to be involved in the immunemediated manifestations in covid- patients (abdelnour et al., ; mao et al., ) . it is known that the virus could enter the brain carried by infected immune cells (bergmann et al., ) . several regions in the brain, including vasculature, meninges, and choroid plexus, could be the entry sites for virus-infected immune cells (engelhardt et al., ) . besides, sars-cov- -induced cytokines, il- , il- β, tnf, and il- could facilitate the entry of the virus into the brain through disrupting the blood-brain barriers (bbb) (erickson and banks, ) . on the other hand, the hypothalamus in the brain also contributes to the dysregulation of immunity. il- , il- β, and tnf-α have been reported to be robust activators of the hpa axis (dantzer, ) . hpa axis plays a central role in regulating systemic immunity and is majorly activated by bbb dysfunction and neurovascular inflammation (dantzer, ) . these studies suggested that sars-cov- infection-induced immune alteration could further result in the concurrence of chronic pain since it affects the nervous system. chronic pain is a complex and distressing problem, which significantly impacts the life quality of each individual. chronic pain is not merely an accompanying symptom and can be associated with various underlying causes, including immunity alterations, and viral infections. patients with chronic pain must be given effective and continuous treatments to manage their long-term pain at an acceptable level. to date, the prevalence of covid- appears to continue increasing exponentially worldwide. constant exposure risk to covid- is currently expected to become the new normal. in this situation, the management of immunity alteration-induced chronic pain may require additional attention. here we summarize several types of chronic pain, which are closely related to the alteration of immunity in individuals, and give some recommendations from a clinical view. hz is an acute, cutaneous viral infection caused by the reactivation of the vzv (saguil et al., ) . the incidence of hz has been estimated to be . % in china, with a lifetime risk of % in each individual (yang et al., ) . the risk factors associated with the reactivation of vzv have not yet been clarified; however, malignancies, immune deficiencies, solid organ, and bone marrow transplantations, autoimmune diseases, psychological conditions, emotional stress, and immunosuppressive therapies have been identified as possible major risk factors (wei et al., ) . in hypo-immune conditions, especially those associated with decreased cellmediated immune status, the risk of vzv reactivation is fold higher than in healthy subjects (singh et al., ) . it has been reported that sars-cov- infected individuals have also developed severe acute herpetic neuralgia despite the early initiation of antiviral therapy (saati et al., ; shors, ) . herpes zoster (hz) might be an indicator of latent sars-cov- infection because the clinical presentation of hz even in patients having mild or no upper respiratory symptoms should be considered as an alarming sign for sars-cov- infection (elsaie et al., ) , which highlights the possibility of covid- -induced hz. postherpetic neuralgia (phn) is the most common complication in approximately one-fifth of hz patients, especially among elderly individuals (gershon et al., ) . phn is defined as skin-distributed pain that persists for at least months after acute hz (salvetti et al., ) . the treatment for phn focuses on symptom control, including the use of topical lidocaine or capsaicin and oral gabapentin, pregabalin, or tricyclic antidepressants (hempstead et al., ; kopel and brower, ) , as well as the improvement of immunity and antiviral treatments (huning et al., ; hunter et al., ) . the analgesic effects of traditional treatments have not been reported to have good efficacy among phn patients with older age, serious skin lesions, or long disease courses. neuromodulation, such as spinal cord stimulation, may represent a new approach for pain relief among these patients . acquired immune deficiency syndrome (aids) is associated with various infection symptoms, and peripheral neuropathic pain is the most common and severe neurological manifestation that has been reported in hiv-positive, immunocompromised individuals (amaniti et al., ) . statistical analysis has revealed that up to one-third of hiv-infected individuals suffer from neuropathic pain, which presents as distal, symmetrical, axonal, and peripheral sensory neuropathic pain, accompanied by a burning sensation and paraesthesia, which primarily affects the legs and hands (gabbai et al., ) . the possible pathogenesis of hiv infection-induced neuropathic pain includes tumor necrosis factor-α (tnf-α) (zheng et al., ) , ccaat/enhancer binding protein β (cebpβ) phosphorylation (yi et al., ) , and mitochondrial oxidative stress (kanda et al., ) . the current clinical treatment for hiv-induced neuropathic pain includes nonopioid pain relievers, opioid analgesics, adjuvant medications, and psychosocial therapies (krashin et al., ) . reactive arthritis (ra), an autoimmune disease, is the most common chronic inflammatory disease. ra is characterized by the progressive, symmetric inflammation of affected joints and tendon (tenosynovitis), resulting in both cartilage destruction and bone erosion (lin et al., ) . the clinical manifestations of ra vary greatly among individuals and can become more severe without medical intervention. the prevalence of ra has been reported to range from to per , individuals, and the risk factors include age, gender, genetics, smoking, obesity, exposure to ultraviolet (uv) light, drugs, changes in the microbiome of the gut, mouth, and lungs, periodontal disease (periodontitis), and infections (deane et al., ) . besides, sars-cov- infection also causes ra (ono et al., ) . joint inflammation in ra is mediated by t-cells, b-cells, macrophages, fibroblasts, and inflammatory cytokines (lin et al., ) . currently available therapeutic drugs include the administration of non-steroidal anti-inflammatory drugs (nsaids), immunosuppressive glucocorticoids (hajialilo et al., ) , and disease-modifying anti-rheumatic drugs (dmards) (fries, ) . ankylosing spondylitis (as) is both an autoimmune rheumatological arthritis and a chronic inflammatory disease. as is reported to be highly correlated with the presence of human leukocyte antigen (hla)-b (hill et al., ) . moreover, genome-wide association studies (gwass) have identified numerous single-nucleotide polymorphisms (snps) related to as susceptibilities, such as those in il- r, il- a, runx , and bcl b . as is caused by chronic inflammatory disorders, manifested as structural damage to the spinal and sacroiliac joints, which subsequently develops into ankylosis, due to new osteogenesis (sieper and poddubnyy, ) . the clinical symptoms of as include chronic back pain, morning stiffness, fatigue, and the loss of spinal mobility (sieper and poddubnyy, ) . the clinical treatment of as typically involves suppressing immunity and anti-inflammatory medications (yang et al., ) . nsaids are the first-line drugs used clinically and are considered the most effective therapeutic approach, according to current management recommendations (ward et al., ) . chronic pain involves complex brain circuits, which include sensory, emotional, cognitive, and interoceptive processing (simons et al., ) . an association exists between psychosocial factors and the severity of chronic pain. psychosocial factors have been reported to affect the development of chronic pain, as well as pain treatment outcomes. the severity of chronic pain can be evaluated by pain-related distress. covid- has caused an international public health emergency and poses a tremendous challenge to psychological resilience. these adverse psychological impacts and psychiatric symptoms include depression, anxiety, panic, somatic symptoms, selfblame, guilt, post-traumatic stress disorder (ptsd), delirium, psychosis, and even suicide (steenblock et al., ) . a recent survey has revealed that . , . , and . % of participants reported sleep difficulties, paranoia regarding the acquisition of covid- infection, and distress related to social media. a perceived mental healthcare need was reported for more than % of participants (roy et al., ) . these phycological challenges could affect the management of chronic pain. abnormal psychological status results in more severe chronic pain and increased difficulty experiencing clinical relief. moreover, the reasons for adverse psychological outcomes among patients ranged from inadequate access to personal protective equipment, shocking news media, feeling not supported, helpless and hopeless, experiencing insomnia, undergoing complicated medical procedures and environments, and the relatively high infection rate among medical staff and patients (spoorthy et al., ) . therefore, phycological counseling became critical for the management of chronic pain during the covid- outbreak. cognitive-behavioral therapy is recommended as a psychological intervention for patients with chronic pain. strengthening the patient's confidence could be necessary to relieve psychological stress in chronic pain patients during the covid- outbreak. patients in an emergency or eager for treatment in the hospital have to receive sars-cov- nuclear acid assay and ct image check before entering into the hospital. for those suspected covid- patients, if medical treatment is critically necessary, medical staff has to make adequate preparation to protect themselves and patients. besides, disposable materials, including masks and gloves, should not be used repeatedly, and particular attention should be paid to cleaning and sterilization. all procedures must follow local instructions and guidelines. the pandemic of covid- has made great challenges to the social, medical system, particularly in light of the redistribution of medical staff, beds, equipment, and resources against sars-cov- infection. chronic pain is suffering, significantly impacts the quality of life. chronic pain patients have received limited treatment and discounted services 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in china: results from a cross-sectional study clinical features and complications of scleritis in chinese patients phosphorylated ccaat/enhancer binding protein beta contributes to rat hiv-related neuropathic pain: in vitro and in vivo studies the impact of copd and smoking history on the severity of covid- : a systemic review and meta-analysis tnfalpha is involved in neuropathic pain induced by nucleoside reverse transcriptase inhibitor in rats a pneumonia outbreak associated with a new coronavirus of probable bat origin this work was supported by grants from the national natural science foundation of china (no. ), and shenzhen municipal science, technology, and innovation commission (no. jcyj ). the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © sun, gao, luo, zheng, liao, xiong and xiao. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- -hg qslyx authors: camelo-castillo, anny; henares, desirée; brotons, pedro; galiana, antonio; rodríguez, juan carlos; mira, alex; muñoz-almagro, carmen title: nasopharyngeal microbiota in children with invasive pneumococcal disease: identification of bacteria with potential disease-promoting and protective effects date: - - journal: front microbiol doi: . /fmicb. . sha: doc_id: cord_uid: hg qslyx background and aims: the risk of suffering from some infectious diseases can be related to specific microbiota profiles. specifically, the nasopharyngeal microbiota could play a role as a risk or protective factor in the development of invasive disease caused by s. pneumoniae. methodology: we analyzed the nasopharyngeal microbiota of children with invasive pneumococcal disease (ipd) and that of healthy controls matched by age, sex, and seasonality from catalonia, spain. epidemiological, microbiological and clinical variables were considered to compare microbiota profiles, analyzed by sequencing the v –v region of the s rrna gene. results: twenty-eight children with ipd (median age months) and controls ( . months) were included in the study. ipd children presented a significantly higher bacterial diversity and richness (p < . ). principal coordinate analysis revealed three different microbiota profiles: microbiota a, dominated by the genus dolosigranulum ( . %); microbiota b, mostly represented by streptococcus ( . %) and staphylococcus ( . %) and a high diversity of anaerobic genera including veillonella, prevotella and porphyromonas; and microbiota c, mainly containing haemophilus ( . %) and moraxella ( . %). the only explanatory factor for the three microbiotas was the classification of children into disease or healthy controls (p = . ). a significant negative correlation was found between dolosigranulum vs. streptococcus (p = . ), suggesting a potential antagonistic effect against pneumococcal pathogens. conclusions: the higher bacterial diversity and richness in children with ipd could suggest an impaired immune response. this lack of immune competence could be aggravated by breastfeeding < months and by the presence of keystone pathogens such as porphyromonas, a bacterium which has been shown to be able to manipulate the immune response, and that could favor the overgrowth of many proteolytic anaerobic organisms giving rise to a dramatic dysbiosis. from an applied viewpoint, we found suggestive microbiota profiles associated to ipd or asymptomatic colonization that could be used as disease biomarkers or to pave the way for characterizing health-associated inhabitants of the respiratory tract. the identification of beneficial bacteria could be useful to prevent pneumococcal infections by integrating those microorganisms in a probiotic formula. the present study suggests not only respiratory tract samples, but also breast milk, as a potential source of those beneficial bacteria. the human microbiota communities and their genes are an intriguing ecosystem that play an essential role for human body functions including food digestion, nutrition, regulation of human metabolism, and regulation of immune defense against infections, among others (zhu et al., ) . the majority of microorganisms of the human ecosystem act as symbionts that co-evolve and co-adapt with their human host according to their diet, life-style, human genetic characteristics, immune modulation, or environment parameters (dethlefsen et al., ) . particularly in samples from the respiratory tract, the variability in bacterial composition between individuals has been described to be so high that a core microbiome could not be defined at the species level (bogaert et al., ) . despite this interindividual variability, the bacterial composition of each person has been proposed to fall within discrete categories where some bacterial taxa dominate the community. the presence of these structured microbial consortia was first described in the gut, where three groups or "enterotypes" were found, dominated by bacteroides, prevotella, or ruminococcus (arumugam et al., ) . however, the notion of enterotypes is currently under debate, and it is still not clear whether enterotypes can be generalized to the general population, or whether microbiota follows in fact a gradient in taxonomic composition (jeffery et al., ) . in children between and months of age, for instance, the hypopharynx was found to contain four or five community types, and these "pneumotypes" followed specific trajectories during the child's development (mortensen et al., ) . in adults, bronchoalveolar lavage samples showed two distinct pneumotypes, one of which was enriched with oral microorganisms (particularly prevotella and veillonella) and appeared to be associated with higher inflammatory markers (segal et al., ) . thus, studying the bacterial community structures in the respiratory tract could be important to understand susceptibility to chronic obstructive pulmonary disease (copd) or to pulmonary infections like pneumococcal pneumonia or, more extensively, invasive pneumococcal disease (ipd). it has been proposed that the degree of stability of the microbiota depends partly on the first colonization by keystone species in the first days of life (relman, ) . natural feed of humans in their early days is breastfeeding, so human milk is expected to be a major supplier of keystone species for human microbiota development, including the respiratory tract, given the high microbial diversity in colostrum, transition, and mature milk (hunt et al., ; cabrera-rubio et al., ) . in addition, breast milk provides macronutrients, micronutrients and bioactive molecules that protect against infections and inflammation and are key factors for human growth and development (ballard and morrow, ) . another source of resilience to infection is provided by the antagonistic interactions between the local microbiota and the invasive species. for instance, the nasal passage inhabitant corynebacterium accolens appears to be over-represented in children without streptococcus pneumoniae nasal colonization and has been shown to release antipneumococcal free fatty acids from human skin surface triacylglycerols (bomar et al., ) . other commensal bacteria produce antimicrobial peptides that have been shown to inhibit oral pathogens (lópez-lópez et al., ) and prevent pharynx infections in children (di pierro et al., ) . thus, understanding microbiota composition and structure in respiratory samples will be instrumental for establishing disease risk and for the development of probiotics that could be used to colonize the respiratory tract and contribute to the prevention of infections (tagg and dierksen, ; medina et al., ) . our hypothesis is that the nasopharyngeal microbiota plays a role as a risk or protective factor in the development of invasive disease caused by s. pneumoniae. to test this hypothesis, the objective of the present study was to characterize the nasopharyngeal microbiota profiles in two groups of children: (i) children with invasive pneumococcal disease (ipd), considered as a case group whose nasopharyngeal microbiota was suffering an important disturbance; and (ii) a matched control group of healthy children representative of a healthy nasopharyngeal niche. a series of epidemiological, microbiological, and clinical variables related with major risk of developing ipd were considered to compare microbiota profiles in the two groups. for robust characterization of bacterial composition, a long-fragment s sequencing approach was used, in which a bp region of the gene was amplified and sequenced in order to improve taxonomic assignment. this was an observational prospective study including all children and adolescents (< years) with ipd admitted to hospitals in catalonia, spain: hospital sant joan de deu, hospital de nens, hospital de mataro, hospital de vic and hospital de calella (hc), during the period january to march . one healthy control that was treated at sant joan de deu hospital for minor surgery (i.e., phimosis or minor dermatologic surgery) was selected for each case. cases and controls were matched by age, sex, and seasonality, given previous evidence of seasonal influence on the microbiota composition of respiratory samples (bogaert et al., ) . children whose parents or guardians did not sign the informed consent were excluded of the study. the study was performed following the guidelines of the ethics committee of hospital sant joan de deu which approved the study. all information collected has been treated confidentially and in accordance with applicable laws on personal data. ipd was defined as the presence of clinical findings of infection (which were used for classification of the disease) together with isolation of streptococcus pneumoniae and/or dna detection of autolysin (lyta) gene and an additional capsular gene of s. pneumoniae by real-time pcr in plasma, cerebrospinal fluid or pleural fluid. clinical diagnoses were mutually exclusive. relevant clinical, epidemiological, immunological, and microbiological variables were collected for each subject including delivery mode, duration of breastfeeding (mixed or exclusive), fulfillment of breastfeeding world health recommendations (minimum duration of months) exposure to crowding, pneumococcal vaccination status, parents' smoking habits, and previous occurrence of respiratory infections. among microbiological variables we assessed carriers of valent pneumococcal conjugate vaccine (pcv ) serotypes, invasiveness potential of nasopharyngeal s. pneumoniae serotypes and co-colonization with respiratory viruses. serotypes were classified according to the studies of brueggemann et al. ( ) , sleeman et al. ( ), and del amo et al. ( ) . serotypes , , , , f, , a, v, f, , c, and a were considered to have a high-attack rate or highly-invasive serotypes whereas the remainder were considered as low-attack rate or non-highly-invasive or opportunistic serotypes. in addition, we recorded data of clinical diagnosis, presence of underlying disease, previous antibiotic intake, white blood cell count, c-reactive protein level, occurrence of complications, length of hospitalization, provision of intensive care, and course of disease for the cases. nasopharyngeal aspirate samples collected in sterile plastic tubes from cases and controls and eluted with . ml of phosphate buffered saline (pbs) were extracted and stored at − • c until further genomic/microbial analysis. whenever samples were needed for the sole purposes of the study they were taken by experienced nurses of the hospital sant joan de deu clinical trials unit. the definition of invasive pneumococcal disease (ipd) is the presence of clinical findings of infection together with isolation of streptococcus pneumoniae by culture and/or dna detection of s. pneumoniae by real-time pcr in plasma, cerebrospinal fluid or any other sterile fluid. the microbiological confirmation of the patients is based on one technique, the other or both together. all pneumococcal isolates were identified by standard microbiological methods, including the optochin sensitivity test and an antigenic test targeting the capsular polysaccharide (slidex pneumo-kit, biomérieux, marcy-l'etolie, france). dna detection of pneumococcal lyta gene was performed by a duplex real-time pcr as previously reported del amo et al., ) . this duplex real-time pcr also included as target the rnase p human single-copy gene to detect and measure the number of human cells (brotons et al., ) , using primers and probes recommended by cdc (meningitis, ) . absolute quantification of rnasep using pcr can be used to estimate the number of human cells present in the clinical samples. the measure of human dna load was then used to normalize by potential differences in sample amount and avoid potential bias between samples. in addition, dna was measured prior to pcr and concentrations adjusted to minimize amplification bias between samples. capsular typing was carried out by a molecular technique based on automated fluorescent fragment analysis which allows differentiation of serotypes . quellung reaction performed at the national center for microbiology (majadahonda, madrid) was used to complete serotyping in invasive strains isolated by culture. all nasopharyngeal samples were tested by anyplextm ii rv detection v . dna extraction of nasopharyngeal aspirates was performed by nuclisens r easymag r according to manufacturer instructions (biomérieux, marcy-l'etolie, france). this is an automated system for total nucleic acid extraction based in magnetic silica particles. after extracting the dna, its quality and quantity was measured with nanodrop (thermo fisher scientific, massachusetts, usa) and only samples with a / absorbance ratio between . and were processed for next generation sequencing (ngs). ngs libraries were created with ng of total dna for each sample, to which a unique multiplex identifier (mid) was assigned. pcr amplification of the s rrna gene was performed using high fidelity extensor long range pcr enzyme (thermo fisher scientific, massachusetts, usa), with the degenerate universal bacterial primers of s rrna gene f ( ′ -cagagtttgatcmtggctcag- ′ ) and r ( ′ -ggccvgggtatctaatcc- ′ ) (simón-soro et al., ) and the following cycling conditions to minimize amplification biases (simón-soro et al., ) : min at • c, followed by cycles of s at • c, s at • c, s at • c, and a final step of min at • c. these primers amplified the variable regions to (v -v ) of s rrna gene with an expected size of pb. the pcr products were first purified using a minielute pcr purification kit (qiagen, venlo, the netherlands) and then using agencourt ampure beads (beckman coulter, munich, germany). finally, the amplicons were pyrosequenced using a gs flx titanium chemistry (roche, basel, switzerland) with lib-l type microspheres, pooling samples per / of a pyrosequencing plate. negative controls (pbs) were extracted by the same method of the studied samples (easymag) and s rrna gene and human rnase p gene were analyzed by pcr, producing no amplification. dna concentrations for negative controls were also below the quality control thresholds required at macrogen inc. (republic of south korea). in addition, a negative control (water) was included in each sequencing run to discard contamination during library preparation, producing no results. raw sequences were analyzed using "quantitative insights into microbial ecology" (qiime) v . . software (caporaso et al., ) and were separated using the -bp "barcodes" assigned to each sample. chimeric sequences were eliminated by chimeraslayer program (haas et al., ). an end-trimming quality filtering was performed by removing -bp windows with quality values < using prinseq. only reads > bp were included for taxonomic classification. sequences with differences in the primer binding region or with more than four ambiguities in homopolymeric regions were excluded from the analysis. the resulting high quality sequences of the s rrna gene were classified using the rdp database (ribosomal database project) (cole et al., ) with a bootstrap cutoff of %. samples were classified in operational taxonomic units (otus) at % sequence identity as the standard species-level boundary (yarza et al., ) . only the otus representing over . % of the total sequences of each sample were considered for further statistical analysis, as low-frequency reads, including singletons, are more likely to represent sequencing errors, contaminants, or transient organisms without a biological role at the niche under study (de la cuesta-zuluaga and escobar, ). bacterial taxonomic composition was determined for each sample and means for each group (cases and controls) were calculated. data analysis was performed using qiime . . software. rarefaction curves (mean ± s.e.) were calculated by including , randomly selected reads per sample. alpha diversity indexes were calculated from rarefied samples ( , sequences per sample), using the shannon index (shannon, ) for diversity and the chao index (chao, ) for richness. beta diversity was also calculated with the rarefied , reads per sample using weighted and unweighted unifrac (lozupone et al., ) distance matrices, and the principal coordinate analysis (pcoa) generated d and d plots for all mapping fields. in addition, we clustered samples using upgma (unweighted pair group method with arithmetic mean, also known as average linkage) (michener and sokal, ) . two-way comparisons in bacterial composition using the unifrac metric (lozupone et al. ) were used to measure whether the microbial communities in the microbiota types were significantly different. a tree with , reads from each sample was obtained, and microbiota types were considered significantly different if the unifrac distance value for the tree was larger than expected if the sequences were randomly distributed. a thousand permutations were performed to obtain a p-value, using bonferroni corrections for multiple comparisons. in addition, we also performed constrained correspondence analysis (cca), which is a statistic tool which emphasizes variation, and tests whether the factor provided (the microbiota type) can explain part of the total variability. this analysis was performed by the r software ade package (dray and dufour, ) using the chi-squared distances-based function cca. adonis tests were done with the r library "vegan" (oksanen et al., ) to determine corrected p-values. microbial community comparisons and statistical analysis were performed using the statistical software r v . . with the packages for community ecology (vegan), euclidean methods in environmental sciences (ade ), the bee swarm plot (beeswarm) and gplots packages (http://r-project.org), r (the r foundation for statistical computing). we used lefse (linear discriminant analysis effect size) with default parameters (segata et al., ) , available in the galaxy web server toolkit to determine significant differences (alpha value = . ) in the proportions of bacterial genera between cases and controls groups. lefse uses the non-parametric factorial kruskal-wallis (kw) sum-rank test to detect features with significant differential abundance with respect to the class of interest. normality of the data was analyzed with the shapiro-wilk test. continuous data with normal distributions were described using mean and standard deviation (sd). in case of non-normal distribution, median and interquartile range (iqr) were used. significance (p-values) of continuous and normally distributed data was determined using student's t-test. when data did not follow a normal distribution, wilcoxon signed-rank test was used. significance of categorical data was established by using chi-square test. when chi-square expected frequency was equal or less than five, fisher's exact test was applied. when variables followed a normal distribution, the anova test was used to determine if there were statistically significant differences in mean values between the microbiota groups. if distribution was not normal, we used the non-parametric kruskal-wallis test. multiple testing correction was performed when needed. the study was performed following the guidelines of the ethics committee of hospital sant joan de deu which approved the study (project approval code pic- - ). all information collected has been treated confidentially and in accordance with applicable laws on personal data. written consent was obtained from parent or legal guardians of children included in the study. during the study period a total of patients were diagnosed of ipd in hospital sant joan de deu (n = ), hospital de nens (n = ), hospital de mataro (n = ), hospital de vic (n = ), and hospital de calella (n = ). fourty-two of them ( . %) accepted to participate in the study under informed consent. twenty-eight ( . %) controls were available for matching with cases so the study finally comprised subjects. no significant differences of total human dna load was found between samples of cases and controls (log . vs. . gene copies/reaction, p = . ), indicating that both types of samples did not differ in the amount of material. the median age of the cases was . months (iqr . - . months) and ( . %) were male. the clinical diagnoses of these patients were pneumonia (including complicated pneumonia with empyema and necrotizing pneumonia), bacteremia, meningitis, and fulminant sepsis. invasive samples collected for diagnosing ipd included blood (n = , . %), pleural fluid (n = , . %), blood and pleural fluid (n = , . %), and cerebrospinal fluid (n = , . %). detection of s. pneumoniae in invasive samples was performed by pcr (n = , . %), by culture (n = , . %), and both by pcr and culture (n = , . %). median white blood cell count of , /l and median c-reactive protein levels of . mg/l indicated inflammatory activity. an underlying disease was reported in cases with chronic pulmonary disease, neuroblastoma, and splenic dysfunction, respectively. median length of hospitalization was days (iqr - days). five cases ( . %) received intensive care, ( . %) suffered sequelae, and one child died. pneumococcal nasopharyngeal carriage was observed in ( . %) cases and ( . %) controls. a total of pcv serotypes were identified among carriers in the case group, specifically serotype (n = ), serotype (n = ), serotype (n = ), serotype a (n = ), and serotype f (n = ). in contrast, only pcv serotypes, classified as serotype , were found in control carriers. nasopharyngeal samples were taken from cases after a mean of h of fever (iqr - h) and all of them except two (one patient with necrotizing pneumonia and another one with fatal fulminant sepsis and viral respiratory coinfection), were exposed to beta-lactamic antibiotic treatment, during a mean period of days. no patients were exposed to macrolide treatment or other class of antibiotics. table shows epidemiological and microbiological variables for cases and controls. proportions of coinfection with respiratory virus and pneumococcal nasopharyngeal carriage, were higher in cases compared to controls, but the differences between groups did not reach statistical significance. there was a significantly higher proportion of pcv pneumococcal serotype carriers in cases compared to controls ( . vs. . %, p = . ) as well as significantly higher number of high-attack rate serotypes ( . vs. . %, p = . ). a trend for significance was observed for breastfeeding through months (the minimum time recommended by world health organization) in cases ( . vs. . %, p = . ). a total of . s rrna sequence reads were obtained. after sequence length and quality filtering, we obtained . reads > bp ( - bp), with an average of . reads of the s rrna gene per sample (range between , and , reads). rarefaction curves (using otus at % sequence identity) showed that mean bacterial richness had higher values in children with ipd compared with healthy patients (figure ) . individual rarefaction curves with , reads show that most bacteria present were detected by this level of coverage and show a shift toward higher otu richness in children with ipd (supplementary figure ) . species richness (chao index) was significantly higher in cases (mean: , % confidence interval: . - . ) compared to healthy controls (mean: . , % ci: . - . , p < . ). the shannon diversity index was also significantly higher in cases (mean: . , % ci: . - . ) vs. healthy controls (mean: . , % ci: . - . , p < . ). the taxonomic assignment of the s rrna reads revealed that the most common phyla in ipd patients were firmicutes ( . % of the total number of reads), proteobacteria ( . %), bacteroidetes ( . %), actinobacteria ( . %), and a lower proportion of fusobacteria. within the control group, the most common phyla were proteobacteria ( . % of the total number of reads), firmicutes ( . %), bacteroidetes ( . %), and actinobacteria ( . %). figure a shows differences between the two groups in the mean proportions of each bacterial genera. in the cases group the most prevalent bacteria were streptococcus, haemophilus, moraxella, dolosigranulum, veillonella, and staphylococcus with . , . , . , . , . , and . % of the total, respectively. whilst in the control group their proportions were . , . , . , . , . , and . %, respectively. the higher bacterial diversity detected in children with ipd corresponded to the high prevalence of lactate fermenting veillonella, and mainly to genera found at low frequencies. the taxonomic assignment of those genera present at < % of the total, which accounted for over % of the reads in ipd patients, is shown in figure b and reveals the presence of many oral species like corynebacterium, neisseria, actinomyces, or rothia, among others. lefse ranking analysis shows that moraxella and staphylococcus were found to be significantly more abundant in healthy individuals, according to this high-dimensional class comparison test. on the other hand, the genera streptococcus, megasphaera, veillonella, atopobium, oribacterium, prevotella, granulicatella, porphyromonas, actinomyces, rothia, lachnoanaerobaculum, capnocytophaga, alloprevotella, and acinetobacter were significantly more abundant in patients data indicate percentages of s rrna reads from a given genus with a hit against each species. frequencies of each genera are shown in figure . to minimize taxonomic assignment errors, only hits with > % nucleotide identity and > bp alignment were selected. only species with frequencies > . % are shown. with ipd ( figure c) . most of these bacteria are common oral inhabitants . supplementary figure shows a heatmap profile showing abundances of bacterial genera in individual cases and controls indicating the age of each child. the use of long-amplicon ( bp) sequencing allowed us to taxonomically assign the s sequences at the species level with a higher degree of accuracy (shin et al., ) . in order to minimize errors in taxonomic assignment, only sequence alignments > % sequence identity over > bp were considered, which would encompass the s rrna hypervariable regions v -v /v . our results are shown in table . according to this assignment, the genus staphylococcus would be dominated by s. aureus in the controls (> % of the reads in this genus) whereas this species would be totally absent in ipd patients, that would be dominated by s. epidermidis and s. haemolyticus. virtually, all haemophilus species in controls would correspond to h. influenzae, whereas in the cases, % of the reads in this genus would correspond to uncultured or unknown species. it is surprising to note that over % of the reads matching streptococcus would correspond to s. pneumoniae in the controls while in ipd patients, only % of the streptococcal reads would correspond to s. pneumoniae and over % of them gave top matches to s. mitis, s. oralis, or uncultured species. when all samples were analyzed by two-dimensional principal coordinates analysis ( d pcoa), % of the variability in the data could be explained by the first two components. samples appeared to cluster in three different groups, corresponding to three different types of respiratory tract microbiota, or nasopharyngeal-types (figure ) . the microbiota a was mainly composed of reads assigned to the genera dolosigranulum ( . %), moraxella ( . %), and haemophilus ( . %) (figure a) . the microbiota b was represented mostly by the genera streptococcus ( . %), staphylococcus ( . %), veillonella ( . %), together with a high diversity of anaerobic genera as prevotella and porphyromonas ( figure b) . finally, the microbiota c was composed mainly of the genera haemophilus ( . %), moraxella ( . %), and streptococcus figure | principal coordinates analysis (pcoa) of all nasopharyngeal samples according to bacterial composition. data include ipd patients (red-dots) and healthy controls (blue dots). pcoas were performed with weighted unifrac analysis with clustering at the species taxonomic level ( % sequence identity) with , reads per sample. the taxonomic composition (proportion of bacterial genera) of the microbiota types ("nasopharyngeal-types") is shown to the right. data in (a-c) were obtained from , randomly selected sequences per sample. ( . %) ( figure c) . thus, nasopharyngeal-type a was dominated by dolosigranulum, nasopharyngeal-type c by haemophilus, and nasopharyngeal-type b by streptococcus, with a high presence of oral microorganisms. the three microbiota types were found to be significantly different from each other (unifrac distance, corrected p < . in all cases). in addition, cca analysis showed that microbiota type significantly explained the variability in bacterial composition among samples (adonis p-value: . ). in agreement with the existence of specific bacterial communities dominated by a given genus, negative correlations were found (figure ) between the three dominant genera (p-values for significant hyperbolic regressions were, respectively, . , . , and . for comparisons between dolosigranulum and streptococcus, dolosigranulum and haemophilus, and streptococus and haemophilus). in order to understand the features influencing nasopharyngeal-types, all available epidemiological, microbiological, immunological and clinical variables were compared by bivariate analysis with the three microbiota types. no significant differences were found in any of the variables considered that could explain the grouping in the three different (table ) . however, the classification of patients into case and control groups was significantly associated with the nasopharyngeal-types (p = . , chi-square test). among cases, nasopharyngeal-type b was the most frequently detected pattern ( . %), followed by nasopharyngeal-type c ( . %), while nasopharyngeal-type a was detected only in . % of children with ipd. conversely, nasopharyngeal-type b was only detected in controls ( . %). overall, children with nasopharyngeal-type a showed to have markedly lower inflammatory activity measured by the c-reactive protein level when compared to those with nasopharyngeal-type c (p = . ) or nasopharyngeal-type b (p = . ). in addition, no case with nasopharyngeal-type a was diagnosed with complicated pneumonia, meningitis, and sepsis, in contrast to the relatively high occurrence of these serious ipd manifestations among nasopharyngeal-type b and nasopharyngeal-type c cases ( . , . , and . %, respectively, p = . ). similarly, none of the cases with nasopharyngealtype a required intensive care but a noticeable proportion of cases with nasopharyngeal-type b and c did ( . , . , and . %, respectively). of note, % of the ten healthy controls with a microbiota a were fed with maternal milk whereas breastfeeding was less frequent in controls with microbiota b ( . %) and c-types ( . %). in spite of these differences, the association between breastfeeding and type of microbiota was not found to be statistically significant. on the other hand, there were no significant correlations between any specific bacterial genera over-represented in ipd patients and inflammatory parameters. the current work describes for the first time the nasopharyngeal microbiota in a case-control study of children with ipd and healthy children. our data show that bacterial richness and diversity were significantly higher in ipd patients. in such cases, a clear dysbiosis was observed with a high frequency of veillonella and other oral microorganisms which appeared to be relatively absent in controls. this over-representation of anaerobic and proteolytic oral species in children with ipd was also found by segal et al. ( ) in adult bronchoalveolar lavage samples in association with increased inflammation. in our samples, the microbiota related with ipd was also associated with higher levels of the c-reactive protein inflammatory biomarker. de steenhuijsen piters et al. ( ) similarly found higher nasopharyngeal microbiota diversity in elderly pneumonia patients compared to elderly healthy controls, whilst this difference was not found in adult patients. in ecological terms, these results are surprising since higher bacterial diversity is usually related with health (turnbaugh et al., ) , while lower bacterial diversity is associated with disease. it is noteworthy that patients with primary immunodeficiencies presented a higher microbiota diversity compared with healthy controls. this fact might correlate with an increase of immune system permissiveness for microbe colonization (oh et al., ) . similarly, a local increase in bacterial diversity has been detected in polyps and tumor biopsies from patients with colorectal cancer (mira-pascual et al., ) , a disease which has been associated with immune suppression at the affected tissues. the best studied case of immune-driven dysbiosis is probably gum disease, where the presence of the "keystone pathogen" porphyromonas gingivalis has been shown to induce a profound alteration of the immune system (hajishengallis, ) facilitating the settlement of many species that produce inflammation. however, the increase in microbial diversity associated with dysbiosis in gum disease and cancer could not only be due to immune alteration but also to a nutritionally richer environment . in the respiratory tract of children with ipd, future investigations should elucidate whether the observed dysbiosis is a consequence of immune changes, increased nutrient availability or both. in periodontal disease, destructive inflammation generates abundant gum tissue breakdown products that serve as nutrients for proteolytic and saccharolytic bacteria to obtain essential amino-acids and iron, including degraded collagen, and heme-containing compounds . it is important to underline that porphyromonas was one of the over-represented genera in ipd patients ( figure c) . however, all reads belonging to this genus appeared to correspond to uncultured or unknown species ( table ) . we hope the present study stimulates research into the characterization of these potentially pathogenic species, including their potential involvement as keystone pathogens . in addition, ipd samples presented significantly higher levels of veillonella (figure c) , which is a bacterium that uses lactate as a carbon source, as a consequence of which it is usually found physically and functionally associated to lactate producers like streptococcus (dige et al., ; gaspar et al., ) . in our data, however, the correlation between streptococcus and veillonella levels in cases was not significant (r = . , p > . , supplementary figure ) and therefore it is unknown whether streptococcal serotypes associated with ipd produce more lactate that those found in healthy children. future work should determine whether the seemingly higher acidic environment in children with ipd is a consequence of lactate production by microorganisms, by lactate-dehydrogenase over-expression in the human tissue (tan et al., ) or both. our data identified three bacterial clusters or nasopharyngealtypes, dominated by dolosigranulum, streptococcus, or haemophilus. similarly, other studies have described the presence of bacterial nasopharyngeal clusters. in a danish cohort ( - months of age) and a dutch cohort (from birth to months of age), up to and nasopharyngeal-types were described (mortensen et al., ) , respectively. although there are methodological, geographical and age-related differences between these studies, all available data from respiratory tract samples point toward the presence of precise microbial communities with a given dominant bacteria on each nasopharyngeal-type, although larger samples sizes are required to validate this. in the current manuscript, the only statistically significant explanatory factor for these three microbiota profiles was the classification of patients into case or control groups. streptococcus-dominated community (microbiota b) was clearly associated to ipd patients. given that dolosigranulum abundance in the nasopharynx has been reported to be inversely associated with episodes of wheezing and mild respiratory tract infections (biesbroek et al., ) we speculate that the dolosigranulumdominated community (microbiota a) could be more resistant to pneumococcal infection occurrence and severity. our hypothesis was supported by the lower number of cases with a microbiota a-type ( . % of the total) and by the fact that such cases experienced less severe manifestations of ipd and did not require intensive care. in this respect, it is suggestive that dolosigranulum and haemophilus are found in the present study to have a negative correlation with streptococcus, the dominant genera in ipd cases (figure ) . moreover, the nasopharyngeal presence of dolosigranulum has been associated with breastfeeding. it has been shown that lactation has a profound impact in the microbial community composition of the infant upper respiratory tract, increasing the prevalence and levels of dolosigranulum and reducing the levels of staphylococcus, prevotella, or veillonella (biesbroek et al., ) . in agreement with this, we found a considerably higher proportion of microbiota a healthy controls that were breastfed and a trend for statistical significance in the relation between exclusive breastfeeding up to months of age and case-control classification (p = . ) ( table ) . despite the lack of statistical power to observe significance in these associations, the results obtained suggest that breastfeeding could have an important role in protection against pneumococcal infection. as far as for haemophilus-dominated community (microbiota c), both ipd cases and healthy controls appear to fall within this microbiota profile. this genus, together with moraxella, has been associated with higher rates of parentreported upper respiratory infections and wheezing in the first years of life (hyde et al., ; bogaert et al., ) . in fact, the group of children with haemophilus and moraxella-dominated microbiota showed a higher percentage of patients presenting a respiratory infection in the previous days before the sample collection ( . %), with a trend for significance (p = . ). however, future experimental work with a larger sample size should further explore the significance of these microbiota profiles and test whether the presence of a given nasopharyngeal microbiota makes individuals more sensitive or resistant to pneumococcal infection. our study describes the use of a long-amplicon sequencing approach of the s rrna for species-level identification. computer simulations show that taxonomic assignment accuracy, especially at the species level, decreases dramatically in short reads, like those of current illumina or ion torrent technologies (claesson et al., ) , while the use of longamplicon (≥ bp) has been postulated as a good tool for species-level taxonomical assignments (shin et al., ) and has been widely used as a diagnostic tool of bacterial infections in clinical samples (guembe et al., ; martínez et al., ) . however, we have found surprising results using s rrna for species level identification since s. pneumoniae would be more frequent in controls than in cases. these results are discordant with the results generated from a real-time pcr targeting lyta gene, the target recommended by cdc for pneumococcal detection. lyta pcr indicated the presence of s. pneumoniae in . % of cases vs. . % in controls ( table ) . according to our results, taxonomic assignment by using s rrna could be limited in some genera and accurate identification at the species level should be done with more conserved genes. it is important to keep in mind that streptococci are particularly similar in the s rrna gene sequence, and therefore species-level assignment in this genus must be treated with caution (ing et al., ) . even more, despite lyta is the recommended gene utilized for pneumococcal diagnosis according to cdc (national center for immunization and respiratory diseases, ), this target has been reported to misidentify the pathogen (morales et al., ; simões et al., ) , may be showing the occurrence of genetic exchange among streptococcal species and opening the possibility that other etiological agents could be involved. thus, future work should increase our understanding of these microbial consortia by accurate species-level identification. in conclusion, we found a higher bacterial diversity and richness in children with ipd which could suggest an impaired immune response. this lack of immune competence could be aggravated by limited breastfeeding lower than recommended by who and by the presence of keystone pathogens which need to be characterized and that could favor the overgrowth of many proteolytic anaerobic organisms from the oral cavity, giving rise to a dramatic dysbiosis. from an applied point of view, we found suggestive microbiota profiles associated to ipd (streptococcus-dominated microbiota profile) or asymptomatic colonization (dolosigranulum-dominated microbiota profile) that could be used, respectively, as disease biomarkers or to identify health-associated inhabitants of the respiratory tract. the characterization of beneficial bacteria could be useful to prevent pneumococcal infections by integrating those microorganisms in a probiotic formula. the present study suggests not only respiratory tract samples, but also breast milk, as a potential source of those beneficial bacteria. sequence files and metadata for all samples used in this study are stored in the mg-rast server to be publicly available by accessing the project invasive pneumococcal disease (ipd) in children is associated with a highly diverse nasopharyngeal microbiota: a case-control study, id mgp . all remaining data are contained within the paper and/or supporting information files. am and cm-a contributed to the conception, design, and analysis of data. dh and jr contributed to the case and control recruitment and microbiological characterization of samples. ac-c and ag contributed to the bioinformatics analysis. pb contributed to the coordination, statistical analysis, and data/sample management. ac-c, pb, am, and cm-a were the major contributors in writing the manuscript. all authors read and approved the final manuscript. this work was supported in part by fondo europeo de desarrollo regional (feder) enterotypes of the human gut microbiome human milk composition the impact of breastfeeding on nasopharyngeal microbial communities in infants the role of the local microbial ecosystem in respiratory health and disease variability and diversity of nasopharyngeal microbiota in children: a metagenomic analysis corynebacterium accolens releases antipneumococcal free fatty acids from human nostril and skin surface triacylglycerols nasopharyngeal bacterial load as a marker for rapid and easy 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of sars-cov- by direct amplicon-based sequencing through comparison of minion and illumina iseq (tm) system date: - - journal: front microbiol doi: . /fmicb. . sha: doc_id: cord_uid: r m o dp global human health is increasingly challenged by emerging viral threats, especially those observed over the last years with coronavirus-related human diseases, such as the severe acute respiratory syndrome (sars) and the middle east respiratory syndrome (mers). recently, in late december , a novel betacoronavirus, sars-cov- , originating from the chinese city of wuhan, emerged and was then identified as the causative agent of a new severe form of pneumonia, covid- . real-time genome sequencing in such viral outbreaks is a key issue to confirm identification and characterization of the involved pathogen and to help establish public health measures. here, we implemented an amplicon-based sequencing approach combined with easily deployable next-generation sequencers, the small and hand-held minion sequencer and the latest most compact illumina sequencer, the iseq (tm) system. our results highlighted the great potential of the amplicon-based approach to obtain consensus genomes of sars-cov- from clinical samples in just a few hours. both these mobile next-generation sequencers are proven to be efficient to obtain viral sequences and easy to implement, with a minimal laboratory environment requirement, providing useful opportunities in the field and in remote areas. global human health is increasingly challenged by emerging viral threats, especially those observed over the last years with the severe acute respiratory syndrome (sars) and the middle east respiratory syndrome (mers) caused by coronaviruses. recently, in late december , a novel betacoronavirus, sars-cov- , originating from wuhan, hubei province, china, emerged and was identified as the causative agent of a new form of severe pneumonia, named coronavirus disease (covid- ) zhou et al., ) . the outbreak spread further to europe and america mid-march to become a global public health emergency, with the vast majority of countries and territories reporting confirmed cases. by june , , the number of confirmed cases in the world has increased to over , , (dong et al., ) . sars-cov- , an enveloped positive-sense (+) single-stranded (ss) rna virus, is a new member of the coronaviridae family, betacoronavirus genus, and the seventh coronavirus known to infect humans. genetically distinct from sars-cov and mers-cov, sars-cov- was included in the monophyletic clade , within subgenus sarbecovirus, due to its closest sequence similarity with chinese bat sars-like coronaviruses (lu et al., ; wu et al., ; ye et al., ) . despite its rna proofreading capacity during replication like all members of the coronaviridae family, sars-cov- could nevertheless acquire some nucleotide mutations along the genome, allowing for tracking its spread and thus clade definition. the implementation of short turnaround time and userfriendly sequencing tools in affected regions to track the spread, analyze variants, and identify clusters of transmission of emerging viruses, like sars-cov- , is becoming essential. although whole genome sequencing of viral pathogens has become a routine procedure in epidemiological monitoring and surveillance, these molecular investigations essential for public health support are still challenging in remote areas with poor technical resources. thus, the development of easily deployable viral genome toolkits can be proposed to rapidly obtain sars-cov- genomic sequence data in the field without the requirement to send biological samples to suitably equipped and reference laboratories. these molecular investigations also depend on efficient and direct sequencing of viral material from clinical samples, i.e., often with low viral genetic loads and a high concentration of contaminant host nucleic acid background. to circumvent this difficulty, different techniques of enrichment can be achieved, either directly through laborious and timeconsuming culture isolation or, even better, the use of specific primers targeting the whole genome of the virus (houldcroft et al., ) . moreover, specific amplification-based approaches are proven successful since they can generate sufficient quantities of the genetic material needed for next-generation sequencing (quick et al., ) . recently, mobile next-generation sequencers have provided new opportunities in infectious diseases diagnostics and surveillance, such as the rapid sequencing of viral genomes during outbreaks. the pocket size nanopore technology sequencer minion (oxford nanopore technologies, ont) is portable and field-deployable and enables real-time outbreak surveillance of threatening pathogens, such as ebola, lassa, and zika viruses (faria et al., ; hoenen et al., ; kafetzopoulou et al., ) . similarly, the recent illumina iseq tm system is the most compact, accessible, and affordable next-generation illumina sequencer. this easy-to-use system is ideal for small whole-genome sequencing, e.g., viruses, and specific genomic targeted approaches (colman et al., ) . in this study, we aimed at implementing an ampliconbased sequencing approach to obtain sars-cov- consensus genomes directly from clinical specimens, adaptable into the field conditions, with the two easily manageable nextgeneration sequencers, the nanopore minion and the illumina iseq tm system. we also evaluated the performance of these two sequencers, in terms of ease of use, hands-on time, simplification of the sequencing process, and capacity for realtime genome sequencing in settings lacking laboratory resources for outbreak response. the two samples used in this study were sputum (cibu- and cibu- ) vero e cells (mycoplasma-free) seeded in -well plates ( × cells/well) were cultured in dmem (dulbecco's modified eagle medium; thermo fisher scientific, united states) containing % ps (penicillin , u/ml; streptomycin , µg/ml) and supplemented with % fbs (fetal bovine serum). samples were diluted : in dmem % ps without fbs and supplemented with µg/ml tpck-trypsin (sigma-aldrich, united states), added to the cell monolayers and incubated for h at • c in the presence of % co . the inoculum was then removed and replaced with fresh dmem % ps containing µg/ml tpcktrypsin. after incubation for days at • c in the presence of % co , cells were observed for the presence of a cytopathic effect (cpe) under the microscope, and culture supernatants were harvested, aliquoted, and stored at − • c. total rna from clinical samples was extracted using the nucleomag kit on the kingfisher automate (macherey nagel, germany) and from isolates using the nucleospin dx virus (macherey nagel, germany), following the manufacturer's instructions. sputum specimens and respective isolates rna extracts were tested with the sars-cov- real-time rdrp gene duplex reverse transcription (rt)-pcr developed by the french national reference center for respiratory viruses and the real-time e gene rt-pcr from the charité protocol (see who coronavirus disease covid- technical guidance: laboratory testing for -ncov in humans, available from https://www.who. int/docs/default-source/coronaviruse/whoinhouseassays.pdf). samples were considered sars-cov- positive if at least two out of three sars-cov- gene targets were detected by the rt-pcr assays. for each sample, the protoscript ii first strand cdna synthesis kit (new england biolabs, neb, united states) was used to obtain single-strand cdna from rna extracts using random hexamers. rt was performed at • c for min and • c for min. pools of specific primer sets were used to generate amplicons from the cdna using the q hot start high-fidelity dna polymerase (neb, united states) (supplementary table) . these multiplex primer sets (divided into two separate pools) were designed using the primal scheme software on the sars-cov- reference sequence (genbank accession number nc_ ) to sequentially amplify , bp using the following pcr conditions: • c for s, cycles of • c for s, • c for min, and ended by • c for min. clean-up of pcr products was performed with ampure xp magnetic beads (beckman coulter, united states). the quantity of amplicons was measured with the qubit . fluorometer using the dsdna hs assay kit (thermo fisher scientific, united states), and the quality was assessed by electrophoresis in e-gel r ex % agarose (invitrogen r ) and visualized with e-gel powersnap electrophoresis system (invitrogen r ). the amplicons obtained from the two clinical samples and their corresponding isolates were normalized to equal concentrations, multiplexed (x ), and sequenced using the minion and illumina iseq tm system (figure ). library preparation for the minion sequencing was performed using the ligation sequencing kit sqk-lsk and natives barcoding kits exp-nbd /exp-nbd (ont) according to the manufacturer's instructions and modifications as per quick et al. ( ) . briefly, pcr amplicons pools were end-repaired and da-tailed using an ultraii end prep reaction module (neb, united states) followed by ligation of native barcodes using the nebnext ultraii ligation module (neb, united states). all the pooled barcoded libraries were purified using ampure xp beads (beckman coulter) followed by adapter ligation with the nebnext ultraii ligation module. library clean-up was performed using ampure xp beads and short fragment buffer (sfb) and then eluted in µl of ont's elution buffer. the library was loaded onto an r . flow cell (flo-min ) and sequenced on a minion mk b device within h (figure ). ont minknow software (version . . ) was used to collect raw data and perform basecalling (guppy v . ). the rampart tool (read assignment, mapping, and phylogenetic analysis in real time) developed by the artic network has been used to visualize genome coverage in real time and reference matching for each barcode. from the fastq files, only reads with a minimum q score of were selected for the analysis. data were demultiplexed using the guppy_barcoder with the option require_barcodes_both_ends to figure | sars-cov- next-generation sequencing workflow, from the sample to sequence analysis. created with biorender.com. frontiers in microbiology | www.frontiersin.org ensure barcodes are present at each end of the fragment. the reads were filtered on the expected length of amplicons. reads between and , base pairs were kept; thus, potential chimeric reads were removed. selected reads were mapped against sars-cov- reference (nc_ ) using minimap (v . ) (li, ) . samtools (v . ) were used to sort the aligned bam files, to obtain coverage data and a consensus sequence. alignment statistics were also calculated with samtools. data were manually inspected using tablet (v . ) (milne et al., ) . the appropriate volume of each pool of amplicons was adjusted to have a total quantity of ng of amplicon input ( ng of each) per sample. the sequencing-ready libraries were prepared using the nextera dna flex prep kit (illumina, united states). the libraries were qualified on an agilent technologies bioanalyzer using a high-sensitivity dna chip following the manufacturer's instructions and quantified with the qubit . fluorometer using the dsdna hs assay kit (thermo fisher scientific, united states). the resulting libraries were sequenced using the iseq tm system (illumina, united states) and in two multiplexed runs were performed generating × bp read length data during a h run time (figure ). to remove low quality reads, trim off low-quality and contaminant residues, and filter out duplicated reads, fqcleaner v. . . was used, with phred quality score of . filtered reads were mapped against sars-cov- reference (nc_ ) using burrows-wheeler aligner mem algorithm (bwa-mem) (v . . ). samtools (v . ) was used to sort bam files and to generate alignment statistics, coverage data, and a consensus sequence. data were manually inspected using tablet (v . ) (milne et al., ) . the two sputum samples cibu- and cibu- , were tested with the specific sars-cov- real-time rt-pcr protocol. they were both found positive with the three sars-cov- gene targets. the threshold cycle (ct) values for cibu- and cibu- were respectively - and - for the rdrp gene duplex and and for the e gene. the two clinical specimens were also inoculated on vero e cells, and after incubation for days, a clear cytopathic effect was observed. culture supernatants of the first passage were harvested and tested for the presence of the virus with the specific sars-cov- real-time rt-pcr protocol. the ct values of the two corresponding isolates cibu- c and cibu- c were respectively - and - for the rdrp gene duplex and and for the e gene, confirming the isolation of sars-cov- . as soon as the first sars-cov- reference genome was released in genbank (nc_ ), we designed two sets of primers to generate a tiling path along the genome using the primal scheme tool. the full-length genome was amplified directly from the rna extracts from the clinical samples and the corresponding isolates, to obtain pcr products overlapping by bp and covering the entire , bp viral genomic sequence. thirtysix amplicons, split into two pools, were successfully generated for each specimen with a size expected range between and , bp (with an average length of bp) and controlled by gel electrophoresis. the amplicons obtained from the two clinical samples cibu- and cibu- were multiplexed and sequenced using the minion platform with r . . flow cell during h. the minion run produced , and , raw reads with a good quality score, respectively, for the two clinical samples, with an average of , bp read length. thus, the mapping against wuhan sars-cov- reference (nc_ ), by using minimap , retrieved , ( . %) and , ( . %) reads, respectively, for the two clinical samples ( table ) . the run yielded a median read depth of x and , x for the two clinical samples, respectively (table and figure ) , with the average base error rate ranging between . and . %. a near fulllength consensus sequence of , and , bp was generated for cibu- and cibu- , respectively. in the same way, a h minion run, applied to the two corresponding isolates cibu- c and cibu- c , produced , and , high-quality raw reads, respectively, with an average of , bp read length ( table ) . the run yielded a median read depth of , x and , x for the two isolates, respectively (table and figure ) . the mapping against wuhan sars-cov- reference (nc_ ) using minimap used . % of raw reads in both cases and allowed to generate the genome consensus sequence of , and , bp, for the two isolates, respectively. regardless of using a read depth cutoff of x or x, we obtained around and % of sars-cov- genome coverage, for the clinical samples, respectively. the genome coverage variation between both clinical samples reflected the initial viral load difference, which was even more marked at x (table ) , as is the difference between the median read depths. however, due to the high sequencing error rate around %, we observed that a reliable consensus sequence should be analyzed at a minimum depth coverage at x, or even x for some nucleotides. conversely, the genome coverage for the isolates was quite similar, with at x . % and . %, respectively ( table ) . for the clinical sample with the lower viral load cibu- , the genome coverage was significantly improved by the isolation step, less so for the second clinical sample with a very low ct value (figure ) . concerning the bioinformatics analysis, the rampart tool (read assignment, mapping and phylogenetic analysis in real time) developed by the artic network was helpful and time-saving to visualize genome coverage in real time and allowed to stop the run when sufficient data were generated. globally, using our amplicon-based approach, combined with the minion platform, we were able to obtain the near fulllength genome of the studied viral specimens in around h, from samples to sequences data. (figure ) . these observed variations in read depth are the direct consequence of the variable amplification efficiency of the primer pairs used in the multiplex pcr pool, impacted by the viral load of the samples as well as the secondary structures of the viral genome which affect primer binding. these results underlined that high-quality sequences could be obtained with greater samples multiplexing. using an amplicon-based approach, combined with the iseq tm system platform, we were able to obtain the near full genome of the studied sars-cov- in around h. viral consensus genomic sequences were rapidly and easily obtained for the two sars-cov- clinical specimens and their respective isolates, by using the two different sequencing platforms, minion and iseq tm system. we noted that the sequences obtained for the same specimen are identical, regardless of the sequencer used. sequence analysis of the sars-cov- consensus genomes showed that they belong to the proposed phylogenetic clade a , which is consistent with the phylogenetic analysis of the other human french sars-cov- sequences (gámbaro et al., ) . compared to the wuhan reference sequence (nc_ ), the viral genomic sequences cibu- and cibu- (gisaid accession numbers epi_isl_ and epi_isl_ ) display seven and six nucleotide mutations, respectively. both share five nucleotide mutations (c t, c t, c t, a g, and g t), among which three have led to amino acid mutations p l in orf ab, d g in s, and q h in orf a. it is noteworthy that the mutation d g in the spike glycoprotein is specific of clade a , encompassing most of the french sars-cov- sequences. the cibu- additionally contains two more mutations in orf ab, c t (t i) and c t, whereas the cibu- includes an additional one, c t. comparison of the genomes obtained for the clinical specimens and their respective isolates show that the sequences are strictly identical, suggesting that culture of the sars-cov- on vero e cells did not induce any molecular change, at least during the first passage. with many challenges still to be overcome in real-time genomics in rapid-response diagnostics, we evaluated the performances of these both mobile benchtop instruments. while the library preparation time is similar (approximately h) between the two approaches and require the same benchtop laboratory equipment (figure ) , the minion is more efficient and versatile in terms of run time, with the procurement of robust results after - h, compared to an incompressible run time of h for the iseq tm system. it is important to take into account the thawing time of the illumina cartridge reagents which takes at least h in comparison to the immediately available minion loading reagents which are stored at room temperature. minion needs a powerful laptop computer and a stable broadband internet connection, whereas illumina system is stand-alone at least in the first bioinformatics steps, as basecalling and demultiplexing. concerning the sequencers themselves, the two systems are equivalent in terms of ease of use and maintenance and are therefore both suitable for experiments in resourcelimited settings. the read length was fixed to nt by the illumina chemistry, and nanopore reads were longer and entirely covered the length of the generated amplicons with an average of , nt, after trimming with porechop (table ) . while ont improved sequencing accuracy, a still relatively high error rate of the minion raw reads (around %) was noticed in comparison with illumina ones (around . %), requiring a higher read depth to obtain a confident consensus sequence. overall, for each sample, we obtained a better coverage of the genome with the illumina approach, and this as early as x of read depth. by comparing minion and iseq tm system produced consensus genomes, we observed that the necessary coverage threshold is higher for minion than for iseq tm system. novel mammalian coronaviruses are regularly identified and involved in epidemics and severe diseases, such as sars-cov, which emerged in southern china in (drosten et al., ) , and mers-cov, first described in saudi arabia in and still circulating (song et al., ) . at the source of viral outbreaks, readily adaptable methods with rapid turn-around times are necessary for pathogen identification, surveillance programs, and public health strategies. whole genome sequencing with next-generation sequencers is now widely used in specialized laboratories, which have required equipment and long experience for this implementation. however, it is also essential that this approach is accessible at the pen-side of infected humans to investigate outbreaks in remote areas. in this regard, we therefore evaluated two space-saving and easily portable next-generation sequencers, minion and iseq tm system, through the current pandemic of covid- . although the initial investment gives an advantage to the minion, the cost of reagents on the other hand is similar, and both approaches require the identical minimum of laboratory equipment to generate the libraries. by using the primal scheme software on the first released sars-cov- reference sequence, we immediately developed an amplicon-based approach to sequence around , bp amplicons on our small portable sequencers. it has been reported that the ampliseq panel proposed by artic network, based on amplicons, may lead to coverage bias due to dimer formation between primers (itokawa et al., ) . our choice to produce longer amplicons, with lower number of primers, could be more efficient. nevertheless, for all the specimens, the extremities of the genome could not be obtained. as often in the genome sequencing of singlestrand rna viruses, the ' and ' non-coding regions are quite difficult to directly acquire, which would rather require an approach such as rapid amplification of cdna ends pcr (race-pcr). we successfully proved that such targeted approach performs well directly with clinical samples. the counterpart of this workflow requires prior knowledge of the pathogen but, on the other hand, could be promptly adapted to all viruses of interest, including those with very large genomes. we found that viral genomic sequencing, even with low-throughput sequencers, could be performed with high confidence and be achieved directly from clinical specimens with significantly shortened run time. although higher samples multiplexing is suitable, these relatively low throughput sequencers can also run economically with a small number of samples, which is a great advantage when the information is needed as quickly as possible. both sequencers generated the correct consensus genomes, with a slightly lower coverage for minion and a higher error rate than the iseq tm system. however, we were able to obtain the genome of the studied specimens in around h with the minion, illustrating the quasi real-time sequencing capacity to effectively support health authorities. the main defect of the iseq tm system finally lies in its run time. nanopore is regularly considered to have the advantage over other technologies of high portability and fast turnaround time (gardy and loman, ) . it should be noted that today illumina offers a sequencer, which tends toward the same interesting features, while maintaining the high quality of the reads, which is at the origin of illumina's success. the minion retains the advantage of greater versatility, with the possibility of analyzing data in true real time, particularly with tools such as rampart, and thus being able to stop the run after a few hours as soon as the amount of data is sufficient. concerning the further generation of the alignment of amplicon reads against the reference and the variant calling, both devices usually offer tools in the cloud, such as local run manager (dna amplicon analysis module) for iseq tm system, and epi me for minion. in outbreak situations, these two sequencers therefore make rapid sequencing accessible to a very large number of laboratories, requiring a minimum of molecular biology skills. nevertheless, the improvement of the dedicated bioinformatics user-friendly resources should still be developed to take into account the low network and informatics resources in the field. in conclusion, our investigation led to the development of an amplicon-based sequencing approach and the characterization of the viral genome as sars-cov- within a few hours, adaptable in field conditions, with two easily manageable next-generation sequencers, the nanopore minion and the illumina iseq tm system. they provide faster and more cost-effective small-scale runs, which avoid outsourcing for laboratories in remote areas, and allow control of the whole sequencing process. the minion and iseq tm system bring the power of next-generation sequencing to virtually any laboratory, with amplicon-based whole-genome sequencing protocols rapidly transposable to all molecular investigations of epidemic prone pathogens. the datasets generated for this study can be found in online repositories. the names of the repository/repositories and accession number(s) can be found in the article/supplementary material. ethical review and approval was not required for the study on human participants in accordance with the local legislation and institutional requirements. written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements. vh performed all the data analysis. ak and cb conducted the experiments. vh, ak, cb, jv, and vc conceived the study. vh, ak, cb, and vc analyzed the results and prepared the manuscript. sm, cb, and j-cm provided scientific guidance. all authors reviewed the manuscript. this work was made possible thanks to financial support obtained through the «urgence nouveau coronavirus» fundraising campaign of institut pasteur. this project also received funding from the french institut carnot pasteur "microbes et santé" (anr carn - ) and institut carnot "france futur Élevage" (anr carn - ), in the framework of the field project. whole-genome and targeted sequencing of drug-resistant mycobacterium tuberculosis on the iseq and miseq: a performance, ease-of-use, and cost evaluation an interactive web-based dashboard to track covid- in real time identification of a novel coronavirus in patients with severe acute respiratory syndrome mobile real-time surveillance of zika virus in brazil introductions and early spread of sars-cov- in france towards a genomics-informed, real-time, global pathogen surveillance system nanopore sequencing as a rapidly deployable ebola outbreak tool clinical and biological insights from viral genome sequencing a proposal of alternative primers for the artic network's multiplex pcr to improve coverage of sars-cov- genome sequencing. biorxiv metagenomic sequencing at the epicenter of the nigeria lassa fever outbreak minimap : pairwise alignment for nucleotide sequences genomic characterisation and epidemiology of novel coronavirus: implications for virus origins and receptor binding using tablet for visual exploration of second-generation sequencing data multiplex pcr method for minion and illumina sequencing of zika and other virus genomes directly from clinical samples from sars to mers, thrusting coronaviruses into the spotlight a new coronavirus associated with human respiratory disease in china zoonotic origins of human coronaviruses a pneumonia outbreak associated with a new coronavirus of probable bat origin we acknowledged all the members of the laboratory for urgent response to biological threats (institut pasteur) who are involved in the covid- diagnosis and india leclercq for her support in viral isolation. the supplementary material for this article can be found online at:https://www.frontiersin.org/articles/ . /fmicb. . /full#supplementary-material key: cord- - fvlhuo authors: guo, kangkang; xu, lei; wu, mengmeng; hou, yufeng; jiang, yanfen; lv, jiangman; xu, panpan; fan, zhixin; zhang, ruiqi; xing, fushan; zhang, yanming title: a host factor gpnmb restricts porcine circovirus type (pcv ) replication and interacts with pcv orf protein date: - - journal: front microbiol doi: . /fmicb. . sha: doc_id: cord_uid: fvlhuo porcine circovirus type (pcv ) is the infectious agent of postweaning multisystemic wasting syndrome (pmws). the recently discovered open reading frame (orf ) in pcv genome encodes a non-structural protein. previous study revealed that orf protein inhibits cell proliferation and may interact with host transmembrane glycoprotein nmb (gpnmb). however, whether the gpnmb affects pcv replication and the underlying molecular mechanisms are still unknown. in this study, the transcriptome maps of pcv -infected and orf -transfected porcine alveolar macrophages d / (pam) cells were profiled. the gpnmb gene was down-regulated in pcv -infected and orf -transfected pams. by using glutathione s-transferase (gst) pull-down, co-immunoprecipitation (co-ip) and confocal microscopy approaches, we convincingly showed that pcv orf protein interacts with gpnmb. furthermore, by utilizing lentivirus mediated overexpression or knockdown approach, we showed that the cellular gpnmb significantly inhibits pcv replication and orf expression. moreover, gpnmb overexpressing leads to an increased cyclin a expression and a reduced s phase, whereas gpnmb knockdown causes a decreased cyclin a expression and a prolonged s phase. in conclusion, we identified a novel host factor gpnmb that interacts with pcv orf protein and restricts pcv replication. porcine circovirus type (pcv ) is the leading cause of postweaning multisystemic wasting syndrome (pmws) which brings tremendous economic losses to swine industry (darwich et al., ; opriessnig et al., ) . pcv is a non-enveloped, single-stranded, closed-circular dna virus, belonging to the genus circovirus in the family circoviridae with or nucleotides (meehan et al., ) . eleven pcv open reading frames (orfs) have been predicted with six have been well characterized (ellis et al., ; li et al., ) . the orf (nucleotides - ) gene encodes the rep protein to initiate replication (mankertz et al., ) . the orf -encoded cap protein is the only structural protein and is an immune-associated protein (nawagitgul et al., ) . the orf protein has been identified in as an inducer of apoptosis (liu et al., ) . the orf protein is not essential for viral replication but involved in host cell apoptosis inhibition (he et al., ) . the orf was characterized by our group and has been shown localizes to the endoplasmic reticulum (er) and induces er stress (lv et al., ) . notably, it is reported that pcv orf does not affect host cell apoptosis but inhibits host cell proliferation via the prolongation of s phase (lv et al., ) . the yeast two-hybrid assay has showed five host proteins interact with orf , including transmembrane glycoprotein nmb (gpnmb), cytochrome p a (cyp a ), - - protein beta/alpha (ywhab), zinc finger protein isoform x (znf ) and serine/arginine-rich splicing factor (srsf ; yu et al., ; lv et al., ) . gpnmb is a type i transmembrane protein containing an n-terminal signal peptide, an integrin-binding (rgd) motif and a polycystic kidney disease (pkd) domain in extracellular domain (ecd), a single pass transmembrane domain and a amino acid (aa) cytoplasmic tail (selim, ; singh et al., ) . previous studies have showed the gpnmb is involved in various physiological and pathological processes, including immune system activation, cell proliferation, angiogenesis, tissue-repair, especially the invasion and metastasis of malignant tumors (rose et al., ; oyewumi et al., ) . emerging studies have generated a more complex picture regarding the expression of gpnmb in various cancer progression, including lung cancer, ovarian cancer, stomach cancer and breast cancer (singh et al., ; zhou et al., ; maric et al., ) . viral replication is strictly relied on host cellular physiological processes. accumulating evidence demonstrated that the subversion of host cell cycle is a common mechanism employed by virus to facilitate its replication (swanton and jones, ; he et al., ; laichalk and thorley-lawson, ; grey et al., ; balistreri et al., ) . as one indispensable physiological process, cell cycle contains a series of consecutive biochemical switches allowing the dna replication of cell genome at the s-phase, subsequently generating daughter cells (g and g phase) via the equal division during mitosis (m-phase) and quiescent cells are referred as being in g -phase (harper and brooks, ) . the binding of cyclins and cyclin-dependent kinases (cdks) is required for the entry into the cell cycle phases (morgan, ) . it has been reported that the activation of p pathway induced by pcv infection causes the s phase accumulation, which provides favorable conditions for efficient viral replication (xu et al., ) . although the gpnmb has been reported interact with pcv orf by yeast two-hybrid assay (lv et al., ) , whether the gpnmb affects pcv replication and the underlying molecular mechanisms are still unknown. in this study, we convincedly demonstrated that pcv orf protein interacts with cellular gpnmb, which was also identified as a novel cellular factor that inhibits pcv replication. in addition, we also revealed that gpnmb positively regulates cyclin a expression and triggers a higher proportion of cells to enter s-phase. taken together, our study identified a novel host factor gpnmb that interacts with pcv orf and restricts pcv replication and the molecular mechanisms was further deciphered. porcine alveolar macrophages d / (pams) (atcc: crl- ) were grown in rpmi medium (solarbio, china) with % fetal bovine serum (fbs) (gibco, united kingdom). human embryonic kidney (hek t) and porcine kidney (pk- ) cells were cultured in dulbecco's modified eagle's medium (dmem) (solarbio, china) with % fbs. pcv yangling strain (wtpcv ) was isolated from one pig with naturally occurring pmws and propagated in pams or pk- cells (tang et al., ) . pegfp-orf and pegfp-c plasmid was stored in laboratory of veterinary public health and food safety, northwest a&f university, yangling, china (lv et al., ) . pcv orf gene was amplified and inserted into pegfp-c with flag tags both at its n and c terminus to generate flag-orf . orf was inserted into pgex- p- to generate gst-orf . gpnmb gene was amplified from pams cdna and cloned into pcdh-cmv-mcs-ef with flag tags both at its n and c terminus to generate flag-gpnmb. gpnmb was inserted into pdsred-n to generate pdsred-gpnmb. three pairs of shrnas targeting to gpnmb gene and a random sequence negative control naming shn were predicted and designed. the fragments were cloned into pcdh-u -mcs-ef -greenpuro after annealing to generate gpnmb-sh , gpnmb-sh , gpnmb-sh and shn lentivectors. all primers used were listed in supplementary table s . all plasmids were verified by sequencing. pams cultured in six well plates were incubated with pcv at a multiplicity of infection (moi) of . for h, then replaced with fresh rpmi medium with % fbs. all eukaryotic expression plasmids were transfected into cells cultured in six plates using the turbofect transfection reagent (thermo fisher scientific, #r ) according to the manufacturer's instructions. to investigate the cellular response after pcv infection, pam cells were infected with pcv yangling strain. pam cells were infected at a moi of and then replaced with fresh rpmi medium containing % fbs. meanwhile, to elucidate the role of orf played in pcv -induced cellular response, pams cells were transfected with pegfp-orf plasmid. total rna was isolated from pams (control), pegfp-orf transfected and pcv infected pams using trizol (takara, china) following the manufacturer's instruction. the rna samples were submitted to gene denovo co. (guangzhou, china) for high-throughput sequence (each group was triplicate) and the analysis of the data was also conducted by this company. the rna samples were assessed for integrity, quality and quantity. µg mrna was purified from total rna, adsorbed to oligo (dt) magnetic beads and converted to cdna used for pcr following size selection by agarose gel electrophoresis (age). the obtained rna was fragmented and subjected to reverse transcription by the random n primer. next, double-stranded dna was synthesized from the cdna. then fill-in and phosphorylate the synthesized doublestranded dna at the ' end. the ' end forms a sticky end and then ligate with an adapter that have a bubbling t shape at the ' end. the ligation product is pcr amplified by specific primers. the pcr product is heat-denatured into a single strand, and a single-stranded dna is cyclized with a bridge primer to obtain a single-stranded circular dna library. the illumina sequencing was conducted with illumina cluster station and illumina hiseq system. q and q were used to assess the accuracy of base sequences. the software soapnuke was used to evaluate the quality of sequencing reads (cock et al., ) . the raw sequencing data were filtered as follows: (i) remove the reads with adapter sequences. (ii) remove the reads with ambiguous bases (n) > %. (iii) remove reads with low quality (defined as reads with > % of the bases with quality scores < ). to insure clean and high-quality reads, all raw samples for sequencing were purified by illumina pipeline, which were mapped to the reference sequences in the unigene database of sus scrofa (li et al., ) . the reads per kilobase million (rpkm) values, eliminating the interference of gene length and quantity, were used to estimate each gene expression level (mortazavi et al., ) . the false discovery rate (fdr)adjusted p-value and log -ratio were employed to screen the degs (benjamini et al., ) . the criteria of a two-fold fdr (fdr) < . and an absolute value of log -ratio > were chosen to determine the significance of up and down-regulated genes. in gene expression profiling analysis, degs were mapped to the terms of go database, to obtain the annotation of go functional classification and enrichment analysis measured in p-value (xu et al., ) . kyoto encyclopedia of genes and genomes (kegg) were chosen to perform pathway associated with metabolic or signal transduction enrichment analysis of degs (kanehisa et al., ) . total cell rna was isolated using trizol (invitrogen, united states). the cdna with oligo (dt) primers were synthesized using the primescript rt reagent kit (takara, china) with gdna eraser, according to the manufacturer's instructions. real-time quantitative rt-pcr was conducted on the iq multicolor real-time pcr detection system (bio-rad, united states), with sybr premix ex taq ii (cwbio, china) reagents, following the manufacturer's instructions. sequences of primer pairs used are presented in supplementary table s . the porcine β-actin gene served as a reference gene. each sample was carried out in technical duplicates. melting curve analysis and quantitative analysis of the data were performed on iq data analysis software. the relative level of pcv , gpnmb, orf and cyclin a were tested using specific primers in supplementary table s . the ct method was used to analyze relative expression levels of target genes. exogenous expression and endogenous verification were performed for co-immunoprecipitation (co-ip). for endogenous verification, pams co-transfected with µg of pegfp-orf and µg of flag-gpnmb plasmids were harvested at h with western blot and ip lysis buffer (beyotime, china) containing phenylmethanesulfonyl fluoride (pmsf, solarbio, china). followed by centrifugation for min at • c, a quarter of the supernatant was subjected to input assays. the rest were incubated with anti-flag m affinity gel (sigma-aldrich, united states) overnight at • c, which had been centrifuged and rinsed with tbs. followed by washed with tbs and boiled in × sds sample buffer, the proteins samples were subjected to western blot with mouse anti-gfp monoclonal antibody (mab) (zsgb bio, china) or mouse anti-flag polyclonal antibody (pab) (cwbio, china). for endogenous verification, pams transfected with µg of flag-orf plasmid were harvested, and protein samples were detected with rabbit anti-gpnmb pab and mouse anti-flag pab (santa cruz biotechnology, united states). the other steps were like those in exogenous verification. for gst pull-down assays, gst or gst-orf protein was expressed in escherichia coli rosetta (de ) cells and flag-gpnmb protein was expressed in hek t cells. the pierce gst protein interaction pull-down kit (thermo fisher scientific, united states) was used according to the manufacturer's instructions. the proteins produced in e. coli were treated with pull-down lysis buffer and then conjugated to glutathione beads glutathione agarose resin for h at • c. the beads then were washed with : wash solution (tbs: pull-down lysis buffer) for five times and incubated with flag-gpnmb harvested from hek t cells overnight at • c. after washing five times, target protein was detected by western blot. cell lysates were prepared in radioimmunoprecipitation (ripa) buffer with protease inhibitor pmsf and halt tm phosphatase inhibitor cocktail (roche, switzerland). protein concentrations were confirmed by bca protein assay reagent (cwbio, china). equivalent amounts of protein samples were separated by % sds-page, and the target proteins were transferred to pvdf membranes (millipore, united states). membranes were blocked in tbst, a blocking buffer containing % skim milk for h at room temperature, followed by incubation with primary antibodies, including rabbit anti-gpnmb polyclonal antibody, rabbit anti-cyclin a polyclonal antibody ( : , santa cruz biotechnology, united states), and rabbit anti-β-actin polyclonal antibody ( : , cwbio, china) at • c for overnight. after washes with tbst for five times, membranes were incubated with horseradish peroxidase hrp-conjugated goat anti-rabbit (or mouse) igg ( : , cwbio, china) for h at room temperature. after washes with tbst for five times, immunoreactive bands were detected using chemiluminescent reagent ecl (solarbio, china) by genegnome xrq chemidoc system (syngene, cambridge, united kingdom). the cellular protein β-actin was measured as an internal control. pams cultured in glass bottom dishes ( mm) (solarbio, china) were co-transfected with pegfp-orf and pdsred-gpnmb recombinant plasmids with turbofect transfection reagent (thermo fisher scientific, #r ). a set of control cells were also subjected to the same experimental conditions. the transfected cells were further cultured for h were rinsed with pre-cooled pbs, then fixed with % paraformaldehyde for min at room temperature and washed with pbs buffer for five times, and then stained with ' , -diamidino- -phenylindole (dapi) (solarbio, china) for min. laser confocal scanning the medium was refreshed at - h post-infection and kept incubation for an additional h. stable cell lines for gpnmb overexpression or knockdown were seleted by using puromycin at a concentration of µg/ml (thermo fisher scientific, united states). pcdh-cmv-mcs-ef -greenpuro (cmv) and random sequence vector (shn) were treated as controls. pams stable exhibiting gpnmb overexpression or knockdown were fixed with % ethanol • c overnight and were then incubated with propidium iodide (pi) in the dark for . h. a set of control cells was also subjected to the same experimental conditions. the nuclear dna content was tested by a coulter epics xl flow cytometer (beckman, united states). data analysis was performed using the cxp software (beckman) based on the fsc-ssc (forward light scatter-side scatter) dot plot. statistical analyses were performed on microsoft excel and graphpad software. results are presented as mean ± the standard deviations (sd). student's t-test was used for the statistical comparisons analysis. a p-value < . was considered significant. rna samples from the pcv -infected and orf -transfected pams were collected at h post-infection or transfection. each condition was assayed in triplicate and pam cell without any treatment was used as mock-control. rna concentration of mock, orf and pcv were , , and ng/µl, respectively. the integrity number (rin) values, yielded from an agilent bioanalyzer (agilent technologies) were . , . , and . , respectively, which was an indication of high quality rna for cdna library construction and sequencing. the major characteristics of the three cdna libraries were summarized in table . pcv infection and orf transfection stimulated a wide range modification of the host transcriptional profile. as shown in figure a , total degs (fdr < . , log ratio > ) were identified by pairwise comparison between different groups (mock vs. orf and mock vs. pcv ). analysis of go distributions in orf and mock samples showed different patterns of biological processes, cellular components and molecular functions (figures b,c) . we also noticed a significant difference in pathways in pairwise comparison in pcv and orf , including the cell cycle, protein degradation and absorption, various heart-related clinical diseases (supplementary figure s ) . notably, the cell cycle pathways were changed in orf transfected cells (supplementary figure s ) . to analysis the common cellular response between orf -overexpressed and pcv infected cells, the pathway enrichment analysis was performed. as shown in supplementary tables s , s , up-regulated and down-regulated genes were both detected in orf -transfected and pcv -infected samples. these genes mainly associated with translation, metabolism, cell cycle, mitogen-activated protein kinases (mapk) signaling pathways. interestingly, orf transfection and pcv infection down-regulated gpnmb expression level in pams, suggesting its potential role in interacting with pcv orf . to further investigate the effect of both pcv and orf on gpnmb expression, the gpnmb expression levels was measured in pcv -infected and gfp-orf -transfected cells. the bona fide pcv infection was confirmed by the detection of pcv nucleic acid (figure ) . as shown in figures b,c lead to the down-regulation of gpnmb, the gfp-fused orf construct was transfected into pams and the gpnmb expression was measured at h or h post-transfection ( figure d) . consistently, orf overexpression also phenocopying pcv infection, which the gpnmb expression was significantly reduced at transcriptional and translational levels (figures e,f) . these results suggested that orf is the key viral protein that regulated host gpnmb levels. taken together, we demonstrated that pcv infection down-regulates gpnmb expression and the orf is the key viral protein. we have shown that orf regulates the gpnmb expression in host cells. in our previous study, by using yeast twohybrid assay, we revealed that orf interacts with gpnmb (lv et al., ) . to further elucidate how orf regulates gpnmb expressions, we employed the gst pull-down assay to assess the direct interaction between orf and gpnmb. the gst-fused orf or gst protein were treated with pulldown lysis buffer and then immobilized with glutathione beads, followed by the addition of cell lysates with flag-gpnmb. the gst pull-down assay clearly showed that the host gpnmb can be captured by gst-orf via a direct physical contact ( figure a ). to further explore the interaction between gpnmb and orf , the confocal fluorescence microscopy was used to determine the cellular localization of gpnmb. pams cells were co-transfected with pdsred-gpnmb and pegfp-orf . as shown in figure b , orf colocalized with gpnmb in the cytoplasm near by the cell nucleus. next, the co-ip experiments were performed to confirm the direct interaction between gpnmb and orf proteins in vitro. the results showed that flag-gpnmb could co-precipitate with gfp-orf ( figure c ). in addition, the co-ip assay also revealed the interactions between gpnmb and flag-orf ( figure d ). this further confirmed that orf interacts with gpnmb. taken together, these results demonstrated that orf protein colocalizes and interacts with gpnmb in pam cells. we have shown that pcv infection and orf transfection down-regulates gpnmb expression. however, the role of gpnmb in regulating pcv replication is still unknown. to determine whether host gpnmb affects pcv replication, a pk- cell line that stable overexpressing gpnmb (lenti-gpnmb) was generated. the overexpression of gpnmb was confirmed both at transcriptional and translational levels (figures a,b) . the gpnmb-overexpressed cells were infected with pcv at a moi of . . in gpnmb-overexpressed cells, pcv genome and orf rna expression were significantly decreased at and h post-infection, as compared with control (figures c,d) . these results indicated that gpnmb overexpression could reduce pcv replication and orf expression. to further confirm this observation, pk- cells that transfected with gfp-gpnmb plasmid were also infected with pcv at a moi of . . the overexpression of gpnmb was confirmed at transcriptional and translational levels (figures a,b) . consistently, the pcv genome and orf rna expression were also significantly decreased in gfp-gpnmb transfected cells (figures c,d) . together, these results indicated that gpnmb inhibits pcv replication and orf expression. we have revealed that overexpression of gpnmb reduces pcv replication and orf expression. to further investigate the anti-pcv potential of gpnmb, the gpnmb knockdown pk- cells (shgpnmb- , shgpnmb- , and shgpnmb- ) were generated. as shown in figures a,b , the highest knockdown efficiency was noticed in the shgpnmb- cells and thus this was used in following study. the shn and shgpnmb- cells were infected with pcv at a moi of . . at and h post-infection, the pcv viral mrna and orf rna were significantly increased, compared with that in the control (figures c,d) . these results indicated that the pcv replication level was higher in gpnmb deficient cells. together, these data collectively suggested that gpnmb plays vital roles in restricting pcv replication. it has been demonstrated that the pcv replication level is correlated with cyclin a expression level (tang et al., ) . in the present study, the high-throughput sequencing results revealed that the cell cycle pathways were changed in the orf -overexpressed cells (supplementary figure s ) . in our previous study, we have revealed that pcv orf protein down-regulate cyclin a expression and this was also confirmed in the present study (figures a,b ) (lv et al., ) . however, whether the gpnmb could regulate cyclin a expression was still unknown. to explore this, we measured the cyclin a level in gpnmb overexpression or knockdown cells. as shown in figures c-e , overexpression of gpnmb significantly boosted cyclin a expression. cyclin a controls g /s, s/g , and g /m phase cell cycle transitions, which are critical for initiation and progression of dna synthesis (yam et al., ) . thus, we measured the cell cycle in gpnmboverexpressed cells. as shown in figure f , the proportion of gpnmb-overexpressed cells found to be in s-phase was . %, whereas which for control cells were . % or . %, respectively. the results suggested that the overexpression of gpnmb could trigger a less proportion of cells to enter s-phase. to further confirm that gpnmb regulates cyclin a expression, we also tested cyclin a expression in gpnmb knockdown cells. as shown in figures a-c , downregulation of gpnmb significantly inhibited cyclin a expression. consistently, knockdown of gpnmb lead to more cells to enter s-phase ( figure d) . these results demonstrated that gpnmb positively regulates cyclin a expression. together with the observation that gpnmb inhibits pcv replication (figures - ) and cyclin a overexpression suppresses pcv replication (tang et al., ) , our finding implies that gpnmb inhibits pcv infection may through the up-regulation of cyclin a. pcv -associated disease (pcvad) affects most pig-producing regions and causes massive economic lose (matzinger et al., ) . increasing studies have shown that pcv subverts host cellular signals and pathways to benefit its replication. previous study has revealed that pcv orf protein can facilitate the proteasomal degradation of regulator of g protein signaling (rgs ) to promote il- and il- secretion (choi et al., ) . moreover, orf protein also has been reported competes with p in binding to pirh and mediates the deregulation of p homeostasis (karuppannan et al., ) . additionally, pcv orf has been found for stabilizing the concentration of ferritin heavy chain (fhc) to antagonizes host cell apoptosis (lv et al., ) . however, whether pcv orf is involved in these responses was still unknown. in the present study, the high-throughput sequencing was conducted and the functional classification and pathway enrichment analysis of degs were performed to elucidate this enigma. degs libraries analysis provided data regarding transcriptomic changes in pam cells transfected with orf or infected with pcv . we found more genes were changed in the orf -transfected cell compared with pcv infected cells. this could be explained by the fact that the transfection efficiency is higher that pcv infection. in previous study, by using yeast two-hybrid assay, we have identified five proteins potentially interact with orf , including gpnmb, cyp a , ywhab, znf , and srsf (lv et al., ) . in this study, the transcription analysis of cellular response to pcv infection and orf -transfection in pam cells also revealed that the gpnmb expression was affected by pcv infection (supplementary tables s , s ). next, we experimentally proved that pcv infection and orf transfection down-regulates gpnmb expression (figure ) . this is consistent with the transcription analysis done by other group, which also showed that the gpnmb mrna level was down-regulated after pcv infection . the gpnmb was an orf -interacting host factor as identified by yeast two-hybrid assay. in this study, we employed diffident approach to validate the interaction between orf and gpnmb. gst-pulldown assay demonstrated that gpnmb can bind to orf protein ( figure a) . the isolation of flag-gpnmb/pegfp-orf or flag-orf /gpnmb complexes suggested that gpnmb could bind to orf in vitro (figures c,d) . furthermore, confocal microscopy confirmed the co-localization of gpnmb and orf protein, confirming the interaction between gpnmb and orf ( figure b) . taken together, these results convincingly demonstrated that pcv orf interacts with host protein gpnmb. it has been demonstrated that pcv infection affects gpnmb expression, but whether the gpnmb can alter pcv replication remains unknown. in the present study, the effect of gpnmb on pcv replication and orf protein expression were investigated. to this aim, cell lines that stalely overexpressing or knockdown gpnmb were generated. several results collectively showed that gpnmb inhibited pcv replication and orf protein expression (figure ) . furthermore, the transient overexpression of gpnmb also inhibited pcv replication (figure ) . taken together, all these results demonstrated that gpnmb is an important cellular factor that restricts pcv replication. growing evidence identified gpnmb as an attractive therapeutic target for tumor and cancer (swanton and jones, ; he et al., ; laichalk and thorley-lawson, ; grey et al., ; balistreri et al., ) . here, we showed the gpnmb plays vital role in restricting pcv replication. however, whether the gpnmb could also inhibit other viruses needs further investigations. it has been generally accepted that viruses could regulate host cellular life cycle to favor their replication (tischer et al., ) . rotavirus replication correlated with s/g interphase arrest in cell cycle (gluck et al., ) . herpesviruses (flemington, ) , severe acute respiratory syndrome coronavirus (sars-cov) protein (yuan et al., ) , influenza a virus and its ns protein (jiang et al., ) , human respiratory syncytial virus and murine norovirus (mnv) (davies et al., ) can induce cell cycle arrest in the g /g phase. pcv replication is both s-and g /m-phase dependent, pcv infection induces cellular s phase accumulation via the suppression of cyclin a (tang et al., ) . in addition to the finding that the gpnmb interacted with pcv orf and restricted pcv replication, we surprisingly find that gpnmb enhanced cyclin a expression and triggered a less proportion of cells to enter s-phase (figures , ) . this is consistent with the previous study that knockdown of gpnmb suppresses the cell proliferation (oyewumi et al., ) . thus, it reasonable to speculate that the gpnmb inhibits pcv infection through the up-regulation of cyclin a. however, the molecular mechanism still needs further investigations. in addition, whether the pcv infection regulates cyclin a expression via the down-regulation of gpnmb should be dissected in further studies. in conclusion, in this study we characterized the gpnmb was down-regulated in pcv -infected and orf transfected pam cells by using high-throughput sequencing. further studies confirmed that pcv and orf decreased gpnmb expression and there was direct interaction between orf and gpnmb. in addition, we also revealed that gpnmb restricts pcv replication, regulates cyclin a expression and leads to a less proportion of cells enter s-phase. this study identified a novel host factor gpnmb that interacts with pcv orf protein and restricts pcv replication and may provide insights into the pcv -host interaction. kg, lx, mw, and yz designed the research. kg, mw, yh, yj, jl, px, zf, and rz performed the research. kg, lx, fx, and yz analyzed the data. fx and yz contributed to new reagents and analytic tools. kg, lx, mw, fx, and yz wrote the manuscript. the supplementary material for this article can be found online at: https://www.frontiersin.org/articles/ . /fmicb. . /full#supplementary-material oncogenic herpesvirus utilizes stress-induced cell cycle checkpoints for efficient lytic replication controlling the false discovery rate in behavior genetics research the orf protein of porcine circovirus type 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independent prognostic indicator of recurrence and a novel therapeutic target in breast cancer osteoactivin bioinformatic analysis: prediction of novel functions, structural features, and modes of action functional roles of osteoactivin in normal and disease processes strategies in subversion: de-regulation of the mammalian cell cycle by viral gene products correlation of the cyclin a expression level with porcine circovirus type propagation efficiency isolation and identification of porcine circovirus type yangling isolate and sequence analysis of the whole genomes replication of porcine circovirus: induction by glucosamine and cell cycle dependence p signaling modulation of cell cycle arrest and viral replication in porcine circovirus type infection cells genome-wide search for the genes accountable for the induced resistance to hiv- infection in activated cd + t cells: apparent transcriptional signatures, co-expression networks and possible cellular processes high-quality binary protein interaction map of the yeast interactome network g /g arrest and apoptosis induced by sars-cov b protein in transfected cells gpnmb/osteoactivin, an attractive target in cancer immunotherapy key: cord- -hoaxv e authors: jeong, gi uk; song, hanra; yoon, gun young; kim, doyoun; kwon, young-chan title: therapeutic strategies against covid- and structural characterization of sars-cov- : a review date: - - journal: front microbiol doi: . /fmicb. . sha: doc_id: cord_uid: hoaxv e the novel coronavirus, sars-cov- , or -ncov, which originated in wuhan, hubei province, china in december , is a grave threat to public health worldwide. a total of , , confirmed cases of coronavirus disease (covid- ) and , deaths were reported globally up to may , . however, approved antiviral agents for the treatment of patients with covid- remain unavailable. drug repurposing of approved antivirals against other viruses such as hiv or ebola virus is one of the most practical strategies to develop effective antiviral agents against sars-cov- . a combination of repurposed drugs can improve the efficacy of treatment, and structure-based drug design can be employed to specifically target sars-cov- . this review discusses therapeutic strategies using promising antiviral agents against sars-cov- . in addition, structural characterization of potentially therapeutic viral or host cellular targets associated with covid- have been discussed to refine structure-based drug design strategies. in late december , a newly identified coronavirus strain capable of crossing the species barrier and infecting humans was first reported in wuhan, hubei province, china, and was provisionally termed novel coronavirus zhu et al., ) . this novel virus was later designated as severe acute respiratory syndrome coronavirus (sars-cov- ), owing to its genetic similarity with other coronavirus strains (gorbalenya et al., ) . it is known to cause coronavirus disease , characterized by influenza-like mild or moderate respiratory symptoms including dry cough, fever, headache, and pneumonia, as well as severe lung injury and multi-organ failure, which eventually lead to death huang c. et al., ) . the world health organization (who) officially declared covid- as a pandemic on march , due to the rapid global dissemination of sars-cov- . according to the who, a total of , , confirmed cases of covid- and , deaths were recorded up to may , in over countries. moreover, effective antiviral therapeutic agents or vaccines are not yet available for covid- . the repurposing of existing drugs designed for other viruses is the most practical strategy to treat patients with covid- because they have already been tested for their safety. although de novo development of antivirals is a time-, cost-, and effort-intensive endeavor, it is important to generate specific antivirals for sars-cov- that directly target the viral or host proviral factors (cascella et al., ; senanayake, ) . with increasing structural data of key proteins in both sars-cov- and the host, such as the spike glycoprotein (s), the main protease (m pro ), rna-dependent rna polymerase (rdrp), and human angiotensin-converting enzyme (hace ), the structure-based design of new drugs has emerged as the most promising antiviral strategy. in this review, we have summarized the promising therapeutic potential of pre-existing drugs against covid- . in addition, the structural characterization of potentially therapeutic viral or host cellular targets associated with covid- have been discussed to refine structure-based drug design strategies. sars-cov- is an enveloped, positive-sense, single-stranded rna virus and belongs to the genus betacoronavirus, which also includes sars-cov and mers-cov (andersen et al., ; lu et al., ; zhu et al., ) . the genome sequence of sars-cov- is more closely related to that of sars-cov ( % identity) than with that of mers-cov (∼ %) . notably, the s protein of sars-cov- and sars-cov are highly homologous with . % amino acid sequence identity . consequently, sars-cov- and sars-cov are believed to bind to the same host cell entry receptor hace zhou et al., ) instead of human dipeptidyl peptidase (hdpp ), which is used by mers-cov (raj et al., ) . sars-cov- has club-like spikes on its surface and a distinct replication strategy analogous to other coronaviruses. the life cycle and replication of sars-cov- is shown in figure . viral infection is initiated by the interaction between the s protein and hace , followed by subsequent endocytosis or membrane fusion. the s protein comprises two subunits: s and s . the s subunit contains the receptor binding domain (rbd) and binds to n-terminal hace , while the s subunit mediates virus-host membrane fusion. s proteins are cleaved by the host cell furin protease and transmembrane serine protease (tmprss ) at the s /s boundary and the s ′ position. proteolytic cleavage at the s /s boundary is thought to promote tmprss -dependent entry into the target cells (belouzard et al., ; hoffmann et al., ; walls et al., ) . after the release of the viral polycistronic rna into the cytoplasm, the replicase gene comprising open reading frames (orfs) a and ab is directly translated into either replicase polyprotein pp a (∼ kda, nsp - ) or pp ab (∼ kda, nsp - ) by a ribosomal− frameshift near the ′ -end of orf a and autoproteolytically cleaved into non-structural proteins (nsp - ) by two orf aencoded protease domains (brierley et al., ; herold et al., ; thiel et al., thiel et al., , harcourt et al., ; prentice et al., ; ziebuhr, ) . furthermore, the main protease m pro (also called cl pro ) and papain-like protease (pl pro ) participate in this extensive proteolytic cleavage. the large pp ab polyprotein has no < conserved cleavage sites that are mediated by m pro , which cleaves at leu-gln↓(ser, ala, gly) (arrow indicates the cleavage site) (ziebuhr et al., ; hegyi and ziebuhr, ) . positive-strand rna viruses usually form a cytoplasmic enzyme complex called replicase-transcriptase complex (rtc) that can mediate the synthesis of the full-length genome (replication) or discontinuous mrnas (transcription) (gorbalenya et al., ; pasternak et al., ; sawicki et al., ) . structural and accessory proteins are subsequently translated from these transcripts, and new viruses assemble by budding into the lumen of the endoplasmic reticulum-golgi intermediate compartment (ergic) and are eventually secreted (klumperman et al., ; hogue and machamer, ) . antivirals can be broadly divided into two categories: directacting antivirals (daa) and indirect-acting antivirals (iaa). daas directly target specific viral components, such as viral polymerase, or steps in the viral life cycle without affecting other host cellular processes. the development of daas can facilitate the treatment of patients with covid- . in contrast, iaas target host proviral factors and indirectly inhibit viral infection or replication by impeding the function or interaction of these factors. iaas have an advantage over daas because they are not susceptible to viral mutations, which are frequently found in rna viruses. however, iaas can alter the host cellular system and are not considered safe. therefore, daas targeting viral entry, proteases, and replication can serve as effective antivirals owing to their enhanced safety features. drug repurposing of preexisting antiviral agents is considered one of the most practical strategies because there is no available approved antiviral drug or vaccine for covid- . furthermore, the de novo development of drugs typically requires over $ billion usd and - years (cascella et al., ; senanayake, ) . drug repurposing of several approved antivirals against covid- has progressed into clinical trials (table ) . however, there is a potential risk of drug-resistant mutations with the use of daa. a combination of repurposed drugs can reduce the time, cost of treatment, and risk of drug-resistance, and increase therapeutic efficacy to facilitate progression into clinical trials (cheng et al., ) . moreover, due to the existence of crystal structures of viral and host cellular proteins associated with sars-cov- , such as s protein, m pro , rdrp, and hace , structure-based drug design can be performed to develop more effective drugs with reduced off-target toxicity (schomburg and rarey, ). the cryo-electron microscopy (cryoem) structure of the extracellular domain of the s protein of sars-cov- revealed a homotrimeric conformation (wrapp et al., ) . the binding of rbd-located in the s subunit-to hace on the host cell surface initiates interaction between the virus and the host cell; therefore, the switching conformation of rbd is considered an important event for viral entry (shang et al., ) . cryoem figure | viral life cycle of sars-cov- . interaction between the s protein of sars-cov- and hace initiates sars-cov- infection. following receptor binding, the virus enters the cell by acid-dependent proteolytic cleavage of the s protein by tmprss or other proteases. upon fusion of the viral and host cell membranes, viral genomic rna is released in the cytoplasm. the viral rna initiates translation of co-terminal polyproteins (pp a/ab) by− frameshifting. these polyproteins are subsequently cleaved into nonstructural proteins (nsps) by m pro and pl pro . several nsp proteins interact with nsp (rdrp) to form the replicase-transcriptase complex (rtc), which is responsible for the synthesis of full-length viral genome (replication) and sub-genomic rnas (transcription). the viral structural proteins are expressed and translocated into the endoplasmic reticulum (er). the nucleocapsid (n) protein-encapsidated genomic rna translocates with the structural proteins into the er-golgi intermediate compartment (ergic) for virion assembly. the newly synthesized virions are budded through the cell membrane and exocytosed. studies revealed that the rbd in two out of three s proteins binds to the n-terminal domain (ntd) of the neighboring protomer of the s protein. these inter-molecular interactions result in a down (closed) conformation, wherein the hace interaction interfaces are buried inside the structure. moreover, the rbd in the third s protein forms an up (open) conformation to facilitate binding with the n-terminal region of hace (figure a ) (wrapp et al., ) . the cryoem study of sars-cov- s showed that single rbd formed an open conformation in an asymmetric trimer. the structural comparisons between the s protein of sars-cov (pdb id crz) and sars-cov (pdb id vsb) showed that the major structural differences came from rbd in a closed conformation. although the rbd of s from sars-cov and sars-cov- were largely resembled, the sars-cov- rbd showed a higher binding affinity toward hace than sars-cov rbd shang et al., ) . the cyroem structure of full-length hace revealed a homodimeric conformation, with each monomer of hace binding to one rbd of the sars-cov- s protein ( figure b ) . the crystal structure of hace in complex with sars-cov- rbd (pdb id m j and vw ) showed that sars-cov- rbd binds to the n-terminal region of hace via s , q , t , f , d , k , h , e , e , d , y , q , l , l , m , y , q , n , k , d , and r residues of hace and k , v , g , y , y , l , f , y , a , g , e , f , n , y , q , g , q , t , n , g , v , and y residues of sars-cov- rbd ( figure c ) (shang et al., ; wrapp et al., ) . most of these interactions are mediated by α of hace ( figure c) ; moreover, an n-glycosylation chain at n of hace interacts with sars-cov- s protein (shang et al., ) . as mentioned earlier, the s /s junction and s ′ site of the s protein are cleaved by furin and tmprss , to enable efficient entry of sars-cov- into the host cell (figure a) . in addition to trypsin, cathepsin l, and elastase, tmprss is known to activate the s protein and induce virus-cell membrane fusion (matsuyama et al., ) . a recent study reported that tmprss is also essential for sars-cov- entry into target cells matsuyama et al., ) . the overall structure reveals that human ace forms a homodimer (orange and light-yellow) with b at (dark and light gray), which is located in the transmembrane region. the two sars-cov- rbds are shown as dark and light green surfaces. (c) the interaction interface between rbd and ace is shown (pdb id m j). the residues involved in the interaction between sars-cov- rbd and hace are represented with stick models in green and orange, respectively. alpha helix (α ) of hace is also labeled. (d) the overall structure of sars-cov- rbd in complex with its neutralizing antibody cr (pdb id w ). the fab regions of the heavy and light chains are shown in hot pink and pink, respectively. sars-cov- rbd is shown in green. (e) structural comparison of interfaces between sars-cov- rbd and nab or hace . the interaction interfaces with the light chain of cr , heavy chain of cr , and hace are shown in pink, hot pink, and orange, respectively. (f) hinge movement of hace upon binding of the enzyme inhibitor. the apo form (pdb id r ) and inhibitor-bound form (pdb id r l) are superimposed and shown in blue and red, respectively. accordingly, targeting proteins that participate in sars-cov- entry can be a potential therapeutic strategy. the use of neutralizing antibodies (nabs) against sars-cov- 's s protein is thought to be promising for the treatment of patients with covid- (pinto et al., ) . a nab-cr -known to target sars-cov rbd and prevent lung pathology, can also bind to sars-cov- rbd (ter meulen et al., ; tian et al., ) . the crystal structure of sars-cov- rbd in complex with cr revealed that cr forms a distinct interaction interface with sars-cov- rbd, and does not overlap with the interaction interface between hace and sars-cov- rbd (figures d,e) . although cr binds to sars-cov rbd and sars-cov- rbd with binding affinities (kd) of and nm, respectively, it is unable to neutralize sars-cov- in vitro largely due to its inability to form the interaction interface and its low binding affinity (pinto et al., ; yuan et al., ) . however, continuous efforts are being undertaken to identify potent nabs by collecting plasma from infected individuals, and this has shown significant progress. the p b- f from sars-cov infected patients have overlapping residues, g and y , with higher rbd binding affinity than ace /rbd ( . and . nm respectively) (ju et al., ) . furthermore, the interaction interface of c /rbd overlapped with the ace binding region, and b share similar binding structures with prominent neutralizing effects (barnes et al., ; wu et al., ) . also they showed recent concern of mutation in s (d g) that might increase sars-cov- 's transmission rate and has a rare chance to affect the rbd-binding mab c , because of the distance between the rbd region and d (barnes et al., ) . in addition to identifying nabs targeting sars-cov- 's s protein, a pilot trial to use recombinant soluble human ace in covid- patients has been initiated (clinicaltrial.gov #nct ). however, this trial was recently withdrawn as it was not approved by the center for drug evaluation (cde). because ace can counter the activation of renin-angiotensin-aldosterone system (raas) treatment with ace inhibitors, it can increase ace expression in some patients to compensate for the blocked ace activity (vaduganathan et al., ) . in some animal studies, treatment of raas inhibitor resulted in increased expression of ace in specific tissues (ferrario et al., ; soler et al., ) . in this regard, some researchers hypothesized that treatment of the raas inhibitor might enhance the accessibility of sars-cov- into cells and therefore increase the risk of severity in patients carrying covid- (fang et al., ; watkins, ) . however, a recent case population study showed that there was no correlation between use of raas inhibitors and increased risk of covid- (de abajo et al., ) . the ramipril, ace inhibitor showed cardiac protective effects without increased expression of ace (burchill et al., ) . these contradictory results suggested that clinical validations of raas inhibitors are needed to demonstrate its effectiveness toward covd- . the highresolution x-ray crystal structure of apo-hace and hace in complex with its enzymatic inhibitor mln- showed that inhibitor binding at the active site of hace can cause large hinge-bending movement (towler et al., ) (figure f) . furthermore, a structure-based drug discovery study showed that an enzymatic hace inhibitor can prevent sars-cov infection (huentelman et al., ) . therefore, hace inhibitors can potentially prevent sars-cov- infection. although the structure of human tmprss is not available yet, homology modeling and in silico docking studies have demonstrated the molecular mechanisms of camostat mesylate, nafamostat, and bromhexine hydrochloride in inhibiting tmprss (sonawane et al., ) . in this respect, active sitespecific inhibitors of tmprss can be used as potential antiviral agents against sars-cov- . the crystal structure of sars-cov m pro -a cysteine proteaseconsists of domains - . the catalytic processes of m pro are mediated by the non-canonical cys-his catalytic dyad located between domains i and ii (anand et al., (anand et al., , . the m pro protein is highly conserved among sars-cov, mers-cov, and sars-cov- , and it shares the common substrate recognition sequence consisting of lq(s,a,g) (ziebuhr et al., ; hegyi and ziebuhr, ; dai et al., ) . among them, the gln in p of the substrate is an important common feature required for their catalytic activity. human proteases with a similar substrate specificity to that of m pro do not exist; therefore, development of m pro inhibitors is a potential therapeutic strategy for targeting sars-cov- . sars-cov- m pro consists of three domains, analogous to that of m pro from other covs (figure a ) (dai et al., ; jin et al., ; zhang et al., b) . the crystal structure of m pro revealed that it forms homodimers (dimeric protomer) through interactions between domain ii of protomer a and n-terminal residues of protomer b (figure a ) (zhang et al., b) . homodimerization of m pro is required for its enzymatic activity. mutational studies on the dimeric interface, as well as crystal structure analysis, revealed that the interaction between two protomers is required to form the s pocket at the substrate binding site (figure b ) (anand et al., ; lim et al., ; zhang et al., b) . the substrate binding site of sars-cov- consists of s ′ -s -s -s pockets lined with, h , s , m , y , f , l , n , g , c , h , h , m , e , l , h , f , d , q , t , a , and q residues ( figure b ) (dai et al., ; jin et al., ; zhang et al., b) . notably, the s pocket of covs is typically hydrophobic and can accommodate the bulky p fragment (figure b) . several structure-based drug discovery studies have investigated the interaction of inhibitors in the substrate-binding pockets of sars-cov- m pro ( figure c ) (dai et al., ; jin et al., ; zhang et al., b) . a previous study for developing broad spectrum inhibitors targeting cov m pro showed that inhibitors of sars-cov- contain a (s)-γ-lactam ring at p position to mimic glutamine and occupy the s pocket of sars-cov- m pro (zhang et al., a) . a total of structures of sars-cov- m pro in both apo and inhibitor complex forms are available in the protein data bank (pdb) database (https://www.rcsb. org/) until april . zhang et al. ( b) have developed peptidomimetic α-ketoamide inhibitors targeting sars-cov- m pro . they also solved the crystal structure of m pro in complex with α-ketoamide b (pdb id y g) and showed the presence of a γ-lactam ring at p position and cyclopropyl at p position ( figure d) . the biochemical ic of sars-cov- , sars-cov, and mers-cov m pro were found to be . , . , and . µm, respectively (zhang et al., b) . simultaneously, dai et al. ( ) developed inhibitors with an aldehyde-substituted compound at warhead for occupying the s site and thus it covalently bonds with the catalytic cysteine of sars-cov- m pro (pdb id lze and mok) (dai et al., ) (figure e) . these compounds showed high inhibition activity with ic of and nm in vitro and reduced sars-cov- infection with figure | structure of sars-cov- viral m pro and its complex with inhibitors. (a) the crystal structure of sars-cov- m pro . m pro is a cysteine protease that consists of three domains and two protomers. protomer b is shown in darker colors than protomer a and each domain is shown in different colors (sky blue, split pea, and violet represent domains , , and , respectively). (b) substrate binding site of sars-cov- m pro . the substrate binding site of m pro is subdivided into s , s ′ , s , and s (shown in bold orange). the inhibitors bind to residues shown as yellow sticks (h , s , m , y , f , l , n , c , h , m , e ec of . and . µm in plaque reduction assay (dai et al., ) . the crystal structure of sars-cov- m pro in complex with the inhibitor compound n (pdb id bqy), previously designed to inhibit cov m pro , revealed that n occupies the substrate binding pocket and forms a covalent bond with catalytic c of sars-cov- m pro . consistently, the lactam ring at p position of n forms a hydrogen bond with h of sars-cov- m pro ( figure f ) (yang et al., ; jin et al., ) . x , a potential inhibitor of sars-cov- m pro , also occupies the substrate binding pocket; however, it does not form covalent bonds (pdb id w ) ( figure g ). in conclusion, m pro of sars-cov- is a key protein that participates in the proteolytic processing of polyproteins and shows no overlapping substrate specificity with any of the known human proteases. several potent inhibitors share common structural features, including covalent bond formation with catalytic cysteine and a lactam ring at p position. because most inhibitors occupy the substrate binding pocket of sars-cov- figure | cryoem structure of rdrp in complex with cofactors (nsp and nsp ), rna template, and remdesivir. (a) surface representation of the cryoem structure of sars-cov- rdrp in complex with its cofactors (two nsp and one nsp ) (pdb id m ). nsp and nsp are shown in gray and pink, respectively. the β-hairpin, niran, interface, thumb, palm, and finger of sars-cov- rdrp are shown in cyan, yellow, green, orange, purple, and blue, respectively. (b) a cartoon representation of the overall structure of sars-cov- rdrp in complex with the rna template and its inhibitor remdesivir (pdb id bv ). the rna template and primer strand are shown in blue and red, respectively. the red arrow indicated the direction of ntp entry. (c) magnified view of remdesivir monophosphate binding region. remdesivir covalently binds to the primer rna strand and interacts with the template rna. m pro , targeting this pocket could be an efficient and safe strategy in terms of toxicity. replication of sars-cov- genomic rna is mediated by a multiprotein complex consisting of several non-structural proteins, such as nsp , nsp , nsp , and nsp . the functional core of this multiprotein complex consists of rna-dependent rna polymerase (rdrp, also called nsp ) . sars-cov- rdrp plays an important role in the replication and transcription of viral genomic rna (figure ) and its catalytic residues are highly conserved among covs (venkataraman et al., ; . it is because of this that the nucleotide analog remdesivir (gs- , gilead) was treated to target rdrp of mers-cov, sars-cov, and sars-cov- (warren et al., ; holshue et al., ; wang m. et al., ) . although the viral rdrp is a core component of viral replication, nsp and nsp are still required for full-fill transcriptional activity of rdrp (zhai et al., ; venkataraman et al., ; kirchdoerfer and ward, ; gao et al., ) . the cryoem structure of nsp revealed an n-terminal β-hairpin (aa - ), extended nidovirus rdrpassociated nucleotidyl-transferase domain (niran, aa - ), interface domain (aa - ), and rdrp domain (aa - ) consisting of finger, palm, and thumb subdomains (gao et al., ; yin et al., ) (figure a ). structural studies have demonstrated that nsp can recognize the rna template in a sequence-independent manner, suggesting that the enzymatic activity of rdrp is largely sequence independent. the cryoem structure of sars-cov- rdrp in complex with an rna template or its small molecule inhibitor, remdesivir, (figure b ) revealed the molecular inhibitory mechanism of remdesivir (yin et al., ) . remdesivir monophosphate interacts with the primer strand and uridine of the template strand by base stacking and hydrogen bonding, respectively, at the center of the catalytic active site of rdrp (yin et al., ) (figure c) . the covalent incorporation of remdesivir monophosphate into the primer strand blocks the entry of nucleotide triphosphates to the active site, and terminates the transcriptional activity of rdrp (yin et al., ) (figure b ). other nucleotide analog compounds such as favipiravir, ribavirin, eidd- , and eidd- may exhibit a similar mechanism of action as remdesivir to inhibit rdrp with non-obligate rna chain termination (elfiky, ; sheahan et al., ; wang y. et al., ) . although the u.s. food and drug administration issued an emergency use authorization for remdesivir on may , for the treatment of suspected or laboratory-confirmed covid- in adults and children hospitalized with severe symptoms, the clinical efficacy of remdesivir against sars-cov- is not known yet. moreover, no significant clinical benefits of remdesivir against sars-cov- were observed in a recent randomized, double-blind, placebo-controlled, multicenter clinical trial (clinicaltrials.gov, nct ) . taken together, compounds that target sars-cov- rdrp are largely nucleotide analogs because of their ability to form covalent bonds with the viral template rna and block the catalytic active site of rdrp. zoonotic coronavirus outbreaks such as covid- can not only affect public health but also have a major impact on societies and the global economy. therefore, global cooperation among academic institutions, governments, and pharmaceutical companies is necessary to overcome covid- . despite intensive worldwide efforts undertaken by researchers to contain the spread of sars-cov- , covid- has attained pandemic status. considering that the development of an effective vaccine and new therapeutics are still in the early stages, repurposing fda-approved and well-characterized drugs might be a pragmatic approach. consequently, some of these drugs, such as remdesivir, have been approved for emergency use and some are being tested in clinical trials. in addition, combination treatment might be an approach which could achieve synergistic effects and reduce the risk of drug-resistant mutations. a few studies have shown that some pre-existing drugs are effective for the treatment of patients with covid- . in this review, we described the ongoing therapeutic strategies targeting various components of the sars-cov- life cycle ( table ). in addition, we provided structural insights into the mechanism of action of well-characterized drugs targeting the interaction between hace and the spike protein of sars-cov- for viral entry, as well as m pro and rdrp for viral replication. we believe that structural characterization can aid in developing an effective therapeutic strategy not only against covid- but also other viral outbreaks in the future. gj and hs conceived, designed, did the literature review, provided, and wrote the manuscript. gy assisted in the preparation and design. dk and y-ck conceived, designed, assisted in the literature, final review, and co-wrote the manuscript. all authors contributed to the article and approved the submitted version. structure of coronavirus main proteinase reveals combination of a chymotrypsin fold with an extra alpha-helical domain coronavirus main proteinase ( clpro) structure: basis for design of anti-sars drugs the proximal origin of sars-cov- structures of human antibodies bound to sars-cov- spike reveal common epitopes and recurrent features of antibodies activation of the sars coronavirus spike protein via sequential proteolytic cleavage at two distinct sites tmprss -inhibitors play a role in cell entry mechanism of covid- : an insight into camostat and nafamostat characterization of an efficient coronavirus ribosomal frameshifting signal: requirement for an rna pseudoknot combination renin-angiotensin system blockade and angiotensinconverting enzyme in experimental myocardial infarction: implications for future therapeutic directions features, evaluation and treatment coronavirus (covid- 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papain-like protease activity coronavirus puts drug repurposing on the fast track conservation of substrate specificities among coronavirus main proteases nucleotide sequence of the human coronavirus e rna polymerase locus sars-cov- cell entry depends on ace and tmprss and is blocked by a clinically proven protease inhibitor first case of novel coronavirus in the united states clinical features of patients infected with novel coronavirus in wuhan pharmacological therapeutics targeting rna-dependent rna polymerase, proteinase and spike protein: from mechanistic studies to clinical trials for covid- structure-based discovery of a novel angiotensin-converting enzyme inhibitor covid- pandemic; transmembrane protease serine (tmprss ) inhibitors as potential drugs structure of m pro from covid- virus and discovery of its inhibitors human neutralizing antibodies elicited by sars-cov- infection structure of the sars-cov nsp polymerase bound to nsp and nsp co-factors coronavirus m proteins accumulate in the golgi complex beyond the site of virion budding structure of the sars-cov- spike receptor-binding domain bound to the ace receptor dynamically-driven enhancement of the catalytic machinery of the sars c-like protease by the s -t -i /a mutations on the extra domain scutellaria baicalensis extract and baicalein inhibit replication of sars-cov- and its c-like protease in vitro genomic characterisation and epidemiology of novel coronavirus: implications for virus origins and receptor binding hiv protease inhibitors: a review of molecular selectivity and toxicity efficient activation of the severe acute respiratory syndrome coronavirus spike protein by the transmembrane protease tmprss enhanced isolation of sars-cov- by tmprss -expressing cells inhibition of sars-cov- infections in engineered human tissues using clinical-grade soluble human ace nidovirus transcription: how to make sense cross-neutralization of sars-cov- by a human monoclonal sars-cov antibody 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mutants viral replicase gene products suffice for coronavirus discontinuous transcription mechanisms and enzymes involved in sars coronavirus genome expression potent binding of novel coronavirus spike protein by a sars coronavirusspecific human monoclonal antibody ace x-ray structures reveal a large hinge-bending motion important for inhibitor binding and catalysis renin-angiotensin-aldosterone system inhibitors in patients with covid- rna dependent rna polymerases: insights from structure, function and evolution structure, function, and antigenicity of the sars-cov- spike glycoprotein remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus ( -ncov) in vitro remdesivir in adults with severe covid- : a randomised, doubleblind, placebo-controlled, multicentre trial therapeutic efficacy of the small molecule gs- against ebola virus in rhesus monkeys preventing a covid- pandemic cryo-em structure of the -ncov spike in the prefusion conformation a noncompeting pair of human neutralizing antibodies block covid- virus binding to its receptor ace inhibition of sars-cov- (previously -ncov) infection by a highly potent pan-coronavirus fusion inhibitor targeting its spike protein that harbors a high capacity to mediate membrane fusion evolution of the novel coronavirus from the ongoing wuhan outbreak and modeling of its spike protein for risk of human transmission nelfinavir inhibits replication of severe acute respiratory syndrome coronavirus in vitro structural basis for the recognition of sars-cov- by full-length human ace design of widespectrum inhibitors targeting coronavirus main proteases structural basis for inhibition of the rna-dependent rna polymerase from sars-cov- by remdesivir a highly conserved cryptic epitope in the receptor-binding domains of sars-cov- and sars-cov insights into sars-cov transcription and replication from the structure of the nsp -nsp hexadecamer α-ketoamides as broad-spectrum inhibitors of coronavirus and enterovirus replication: structure-based design, synthesis, and activity assessment crystal structure of sars-cov- main protease provides a basis for design of improved alpha-ketoamide inhibitors a pneumonia outbreak associated with a new coronavirus of probable bat origin a novel coronavirus from patients with pneumonia in china molecular biology of severe acute respiratory syndrome coronavirus virus-encoded proteinases and proteolytic processing in the nidovirales coronaviruses-drug discovery and therapeutic options the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © jeong, song, yoon, kim and kwon. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- -vd ctqro authors: hua, chen; zhu, yun; wu, congquan; si, lulu; wang, qian; sui, long; jiang, shibo title: the underlying mechanism of -hydroxyphthalic anhydride-modified bovine beta-lactoglobulin to block human papillomavirus entry into the host cell date: - - journal: front microbiol doi: . /fmicb. . sha: doc_id: cord_uid: vd ctqro we have previously demonstrated that -hydroxyphthalic anhydride ( hp)-modified bovine beta-lactoglobulin ( hp-β-lg) is highly effective in inhibiting entry of pseudovirus (psv) of high- and low-risk human papillomavirus (hpv) into the target cell. intravaginally applied hp-β-lg-containing vaginal gel could significantly inhibit hpv infection and reduce viral load in the cervical region. however, we still do not understand the underlying molecular mechanism by which hp-β-lg is able to inhibit hpv infection. here, though, we showed that hp-β-lg did not inactivate hpv psv, but rather blocked entry of hpv psv into the target cell via its interaction with virus, not cell. it bound to the positively charged region in the hpv l protein, suggesting that hp-β-lg binds to hpv l protein through the interaction between the negatively charged region in hp-β-lg and the positively charged region in hpv l protein, thus competitively blocking the binding of hpv to the receptor on the basement membrane in vaginal mucosa. although hp-modified chicken ovalbumin ( hp-ova) also carries high net negative charges, it exhibited no anti-hpv activity, suggesting that the interaction between hp-modified protein and hpv l protein relies on both electrostatic and matchable conformation of the binding sites in both proteins. when topically applied, hp-β-lg did not enter the host cell or blood circulation. these findings suggest that hp-β-lg targets hpv l protein and blocks hpv entry into the host cell, thus being safe and effective for topical application in the treatment of hpv infection. cervical cancer is the second most common cancer among women in china (chen et al., ) . almost all these cases result from the persistent infection of human papillomavirus (hpv) (bosch et al., ) . hpvs are a large viral family consisting of about different types (bernard et al., ) . among them, types have high oncogenic properties, and these are regarded as the high-risk types, such as hpv , hpv , and hpv . the high-risk types of hpv have relatively low self-clearance rate compared to the low-risk types of hpv (safaeian et al., ) . to prevent the infection of these highrisk hpvs and the occurrence of cervical cancer, several prophylactic vaccines, like cervarix, gardasil, and gardasil (deschuyteneer et al., ; mccormack, ) , have been developed and approved for marketing in many countries. however, no therapeutic vaccines or drugs have been approved for clinical use to treat hpv-infected patients. hpv is a non-enveloped, double-stranded dna virus. its capsid is made up of two proteins, major protein l and minor protein l . they are both involved in receptor binding and viral entry. analysis of the atomic structure of native t = hpv virus-like particle (vlp) reveals that l pentamers form the icosahedral shell of hpv (baker et al., ; li et al., ) . the l pentamer strongly binds to hpv receptors, such as heparan sulfate proteoglycans (hspgs), on the basement membrane to mediate viral entry and infection (kines et al., ) . the highly potent neutralizing antibodies elicited by hpv vaccines were found to bind to the l protein and prevent hpv infection (li et al., ) . thus, the l pentamer is regarded as the most important target for development of antiviral agents against hpv infection. viral entry inhibitors represent a class of antiviral agents that inhibit virus infection by blocking virus entry into the host cells. jiang et al. reported the first peptide-based hiv entry inhibitor, sj- , in (jiang et al., and the first hiv entry inhibitor-based anti-hiv drug, enfuvirtide, was approved by u.s. fda for clinical use in (dando and perry, ) . later, jiang and colleagues have reported that anhydride-modified proteins such as -hydroxyphthalic anhydride ( hp)-modified proteins are potent virus entry inhibitors against a number of enveloped viruses, such as hiv , herpes simplex virus (hsv) , and ebola virus (ebov) (li et al., ) . in , jiang's group reported that hp-modified bovine beta-lactoglobulin ( hpβ-lg) could also inhibit entry into the target cell of the pseudovirus (psv) of non-enveloped virus, hpv (high-risk hpv and hpv , and low-risk hpv ) (lu et al., ) . in the clinical trial, the topical application of vaginal gel containing hp-β-lg was proven to be very safe and highly effective in suppressing hpv infection and reducing viral load in vaginal mucosa (guo et al., a,b) . however, we still do not understand the molecular mechanism by which hp-β-lg inhibits hpv infection. in this study, we found that while hp-β-lg could not inactivate hpv psv, it was effective in inhibiting hpv psv entry into the host cell via its interaction with virus, not the cell. unlike β-lg, hp-β-lg could bind with l protein and the peptides derived from the positively charged region in the hpv l protein, suggesting that hp-β-lg inhibits hpv infection by binding, via its negatively charged region, to l protein, and thus competitively blocking hpv binding to the receptor on the basement membrane in vaginal mucosa, which then inhibits hpv from entering and replicating in the host cell. we have also shown that topically applied hp-β-lg does not enter the host cell and blood circulation. therefore, the hp-β-lg-containing formulations can be safely used in vagina for treatment of hpv infection and prevention of cervical cancer development. reagents -hydroxyphthalic anhydride (hp), , , -trinitrobenzenesulfonic acid (tnbs), human serum albumin (hsa), β-lactoglobulin (β-lg), chicken ovalbumin (ova), and bovine serum albumin (bsa) were purchased from sigma. horseradish peroxidase (hrp)-conjugated goat anti-human iga and ige antibodies were purchased from abcam (uk). hrp-conjugated rabbit anti-mouse igg antibody was purchased from dako (denmark). the hpv -and hpv- -l /l -expressing plasmids and luciferase pclucf plasmid were kindly provided by dr. john schiller at the laboratory of cellular oncology, national cancer institute, nih, md, usa. anhydride-modified proteins were produced as previously described (lu et al., ) . briefly, each kind of protein was dissolved with . m phosphate buffer (ph . ) at a final concentration of mg/ml. afterward, protein solutions were mixed with anhydrides (hp at m in dmso) to a final concentration of mm by the addition of five equal aliquots at -min intervals, while the ph was adjusted to . with m naoh after each mixing. all mixtures were kept at °c for two more hours and then dialyzed against pbs. the hpv pseudovirus was produced as previously described . briefly, t cells, which had been seeded in a -cm culture dish ( × cells) at h before transfection, were transfected with a mixture of hpv -l /l -expressing plasmid (p shell) and pclucf plasmid for hpv psv, or a mixture of hpv -l / l -expressing plasmid (p shell) and pclucf plasmid for hpv psv, using vigofect (vigorous biotechnology corp.). the cells were suspended in . ml of lysis buffer and incubated for h at °c with slow rotation. the lysate was cooled on ice for min, mixed with m nacl solutions to adjust the concentration of nacl to . m, and further kept on ice for min. then, the lysate was centrifuged at , × g for min at °c, and quantified for the concentration of l /l capsid protein levels. to remove aggregates, the pseudovirion stock was filtered before use for the neutralization assay. the frontiers in microbiology | www.frontiersin.org inhibitory activity of hp-β-lg against hpv psv entry into hela or hacat cells was detected as previously described (surviladze et al., ; kwak et al., ) . briefly, hela or heccatt cells were seeded at . × cells in μl of % fbs dmem (dmem- ) per well in a -well plate, followed by a culture at °c overnight. hp-β-lg or β-lg was serially diluted in dmem and incubated with μl of hpv psv (equal to ng l ). mixtures were added to hela or hacat cells and incubated at °c for h. after replacement of the culture supernatants with free medium and culture for additional h, cells were lysed for measurement of luciferase activity, according to the manufacturer's manual (promega, madison, wi, usa). hp-β-lg diluted in pbs was coated onto all wells of a -well polystyrene plate (corning, usa) at °c overnight. after blocking with protein-free blocking buffer (thermo fisher scientific, usa), hpv l (type or ) protein at μg/ml was added into the wells for μl/well, and the plate was incubated at °c for h. after washing with pbst three times, mouse anti-hpv l (type or ) antibody (abcam, uk) was added at a : , dilution. after incubation at °c for h and washing again, hrp-conjugated rabbit antimouse igg antibody (dako, denmark) was added for h. after washing and adding tetramethylbenzidine (sigma, usa), the absorbance was measured at nm. similar protocols were used for detection of hp-β-lg in the serum of rhesus monkeys and hp-β-lg-specific iga and ige in the vaginal swab eluates, which were leftover samples from the clinical trials for evaluating the in vivo safety and efficacy of the intravaginally applied hp-β-lg-containing vaginal gels (registry no.: chictr-trc- ) (guo et al., a,b) . measurement of the binding ability of -hydroxyphthalic anhydride modified bovine beta-lactoglobulin and beta-lactoglobulin to peptides two positively charged peptides of hpv l protein, l -i (residues - : lkakpkftlgkrkat) and l -ii (residues - : ststtakrkkrkl), were synthesized by a standard solid-phase fmoc method as previously reported (he et al., ) . the peptide solution ( mm) was prepared in mm phosphate buffer (ph . ) and added into the titration sample cells. hp-β-lg and β-lg were also dissolved in phosphate buffer to μm and then added to the titration needle. isothermal titration calorimetry (itc) analysis was carried out at constant temperature of °c with stirring speed of , rev/min. each titration volume was μl, and the titration interval was s for the first drop and s for the others. hela cells were plated in a -well plate (for . × /well) overnight. then, cells were incubated with hp-β-lg at a final concentration of μg/ml at . h before or , . , , , , , , , and h after addition of hpv psv (equal to ng l ), followed by an incubation at °c for h. after replacement of culture supernatant with fresh medium and an incubation for additional h, cells were lysed to determine the entry inhibition ratio, according to the manufacturer's manual (promega). hela cells in -well plates were preincubated with μl of hp-β-lg ( μm) or β-lg ( μm) at °c for h and then washed with dmem three times before addition of hpv psv. for the control, hp-β-lg-or β-lg-pretreated hela cells were not washed before hpv psv was added. hpv psv entry into hela cells was detected as described above. the plated hela cells and hp-β-lg mixture were both prechilled at °c for min. then, the cells were incubated with the hp-β-lg mixture at °c for h. after washing with cold pbs buffer twice, the cells were further incubated at °c for h. then the cells were lysed to determine luciferase activity, according to the luciferase assay system manual (promega, usa). the experiment on rhesus macaques was conducted under ethical guidelines and approved by the ethics committee of the institute of laboratory animal science, chinese academy of medical sciences and peking union medical college (approval number: ilas-vl- - ). in brief, the rhesus macaques were randomly divided into treatment group and control group. in the treatment group, vaginal gel containing hp-β-lg ( . mg per dose) was dispersed into carbomer gel and administered intravaginally or rectally. in the control group, only carbomer gel was applied. then, ml of blood was collected before or after injection at − h, h, min, h, h, h, h, h, and h. the serum was separated after condensation. to determine whether hp-β-lg could enter into hela or t cells, cells ( × ) were seeded onto coverslips in a -well plate. then, hp-β-lg ( . μg/ml) was added for h. then, the supernatant was replaced with dmem containing % fbs. after days, the cells were fixed by % paraformaldehyde (pfa; sigma-aldrich, st. louis, mo), perforated by . % triton x- , and blocked with % bsa (amresco, llc, solon, oh). next, cells were incubated overnight with anti- hp-β-lg mab or anti-β-actin mab ( : , ) at °c. after five washes, the cells were incubated with alexa fluor -labeled donkey anti-mouse igg ( , , , thermo fisher scientific, wilmington, de, usa) at room temperature for h. after five washes, the coverslips were sealed with prolong gold antifade reagent with , -diamidino- phenylindole (thermo fisher scientific) and scanned with the leica sp confocal microscope. frontiers in microbiology | www.frontiersin.org our previous studies have shown that -hydroxyphthalic anhydride-modified bovine beta-lactoglobulin ( hp-β-lg) is a promising hpv entry inhibitor against entry of psv of both high-risk and low-risk hpv types (lu et al., ) . this inhibitory activity was attributed to the interaction of the increased net negative charges on β-lg after -hydroxyphthalic anhydride modification (lu et al., ) . one may ask whether all proteins with increased net negative charges can inhibit hpv entry into the target cell. to answer this question, we compared the inhibitory activity of hp-β-lg with that of -hydroxyphthalic anhydride-modified human serum albumin ( hp-hsa) and -hydroxyphthalic anhydride-modified chicken ovalbumin ( hp-ova) against entry of hpv psv into hela cells, using the unmodified β-lg, hsa, and ova as controls. as shown in figure a , hp-β-lg exhibited potent anti-hpv activity in a dose-dependent manner with an ic (the half maximal inhibitory concentration) of . μg/ml, which is more potent than that of hp-hsa (ic : . μg/ml). hp-ova and all three unmodified proteins at the concentration as high as μg/ml showed no significant inhibitory activity against hpv psv entry into the target cell. these results confirm that not all, just some, proteins, such as β-lg and hsa, modified by -hydroxyphthalic anhydride gain the ability to inhibit hpv infection. in our previous studies, we proved the antiviral activity of hp-β-lg against entry of the high-risk hpv types and and low-risk hpv type into the target cell (lu et al., ) . aside from these three representative hpv types worldwide, another important high-risk type, hpv , is found with high incidence in china. to determine whether hp-β-lg is also effective against hpv , we constructed the hpv pseudovirus and tested its sensitivity to hp-β-lg (control: β-lg). as shown in figure b , hp-β-lg could also inhibit hpv psv entry into the target cell in a dose-dependent manner (ic of . μg/ml), confirming that hp-β-lg has broad-spectrum anti-hpv activity. considering that the hacat cells express abundant integrin and heparan sulfate proteoglycan (hspg), the receptors for hpv (aksoy et al., ; kumar et al., ) and act as target cells for hpv infection, we also evaluated the inhibitory activity of hp-β-lg against hpv and hpv psv entry into hacat cells. as shown in figures c,d frontiers in microbiology | www.frontiersin.org against entry of hpv and hpv psv into hacat cells, suggesting that hp-β-lg acts on the virus, rather than the target cell. bovine beta-lactoglobulin binds to the l protein and the peptides derived from the positively charged residue-enriched region in human papillomavirus l protein as we proposed before, hp-β-lg inhibited hpv psv entry into the target cell possibly through the binding of the negatively charged residues in hp-β-lg with the positively charged residues in a protein on the surface of the viral particle, thereby blocking the interaction between viral protein and receptor on the target cell (lu et al., ) . however, it is still unclear which protein on the virus surface may be involved in this interaction. the most abundant protein on the hpv particle is l protein. here we detected the interaction between hp-β-lg and hpv l protein by elisa. as shown in figures aa,ab, hp-β-lg could strongly bind to the l protein of both hpv and hpv , while the unmodified β-lg protein could not. the c-terminal positively charged region of hpv l was proved to regulate the binding of hpv to the receptor (sibbet et al., ; bousarghin et al., ) . this region was exposed on the viral surface, and involved in the binding to cell surface receptors or neutralizing antibodies. therefore, we synthesized two positively charged peptides in this region of l protein, l -i (residues - ) and l -ii (residues - ). the binding ability of hp-β-lg and β-lg to these peptides was measured by isothermal titration calorimetry (itc). as shown in figure b , β-lg had no obvious binding to l -i or l -ii peptide, while hp-β-lg showed high binding ability against both l -i (binding constant k α value equals . × /m) and l -ii (binding constant k α value equals . × /m). these results suggested that hp-β-lg may bind to the positively charged sites in the c-terminal region of l protein on the hpv surface to block the interaction between the viral particle and cell receptor. to determine whether hp-β-lg could inactivate hpv psv, we incubated hpv psv with hp-β-lg at room temperature for h and then removed hp-β-lg by peg- precipitation. as shown in figure a , the treated virus could still infect the target host cell with no reduced activity, compared the control virus (dmem-or β-lg-treated), suggesting that the binding between hp-β-lg and hpv psv may be noncovalent and reversible so that hp-β-lg cannot permanently inactive the virus. hpv entry into the host cell takes more than h, a relatively slow process compared to other viruses (aksoy et al., ) , which provides a long window period for an entry inhibitor to bind with the viral protein responsible for interaction with the receptor on the target cell and block viral entry. here, we performed a time-addition assay to pinpoint the window of functional hp-β-lg blocking of hpv entry into the target cell. as shown in figure b , hp-β-lg ( μg/ml) could fully inhibit hpv psv entry when it was added to the cells . h before and . , , , , and h after addition of hpv psv, respectively, while about , , and % of hpv psv entry were blocked when hp-β-lg was added to the cells , , and h after addition of hpv psv, respectively ( figure b ). this result suggests that during this relatively long period of entry process (aksoy et al., ) , the entry inhibitor hp-β-lg remains sufficiently active to inhibit hpv psv entry into the target cell. to confirm the functional stage of hp-β-lg in blocking viral entry, we used a temperature shift assay to slow down the viral infection process. at °c, hpv psv could bind to cell receptors, but could not fully enter the cytoplasm because the speeds of membrane fusion and cytoskeletal transformation are largely reduced at such low temperature. we incubated hpv psv and hela cells at °c for h so that hpv could bind to the cell surface. then we washed away the unbound hpv before shifting the cells to °c for further h of incubation. when hp-β-lg was added into the cells at the beginning and then removed by washing, no viral entry occurred (figure c ). this proved that hp-β-lg blocked the attachment between psv and cell receptor at the earliest stage of infection. to determine whether hp-β-lg could also bind to the receptor on the target cell surface to block hpv entry, we incubated hela cells with hp-β-lg (control: β-lg) at °c for h. the cells were washed with dmem to remove the unbound proteins, or not washed (as control), before addition of hpv psv. under the washing conditions, hpv psv entry into the hela cells was not blocked, while under the non-washing conditions, hpv psv entry into the hela cells was effectively blocked. no inhibition was observed in the β-lg control groups, no matter whether the β-lg-treated hela cells were lg was unable to inactivate hpv psv. hpv psv was incubated with hp-β-lg (control: β-lg) at room temperature for h and then separated by peg- to analyze its entry activity. the dmem-treated virus was taken as negative control. (b) hp-β-lg blocked hpv psv entry into the target cell. hela cells were incubated with hpv psv, while hp-β-lg ( μg/ml) was added at different time points (− . , , . , , , , , , , and h) to analyze the inhibitory activity. (c) hp-β-lg blocked hpv psv entry by targeting virus. temperature shift assay for anti-hpv activity of hp-β-lg (control: β-lg). diluted hp-β-lg or β-lg at μg/ml was mixed with hpv psv and prechilled at °c for min; then they were added to hela cells for h. after washing twice, the cells were further incubated at °c for h and then analyzed for fluorescent integrated density. (d) hp-β-lg inhibited hpv psv entry by targeting the virus, not the host cell. hela cells were incubated with hp-β-lg (control: β-lg) at °c for h with/without washing by dmem before addition of hpv psv. each sample was tested in triplicate, and the experiment was repeated twice. data from a representative experiment are presented as mean ± sd. asterisks represent significant differences. ***p < . . frontiers in microbiology | www.frontiersin.org washed or not washed ( figure d) . these results suggest that hp-β-lg inhibition of hpv psv entry into hela cells does not result from its binding to hpv's receptor on the host cell. bovine beta-lactoglobulin could not penetrate into the cell and the vaginally applied -hydroxyphthalic anhydride modified bovine beta-lactoglobulin did not enter into the blood circulation to determine whether intravaginally applied hp-β-lg could enter into epithelial cells, we incubated hela cells and t cells with hp-β-lg for h and then washed the cells three times using pbs. the cells were stained with mouse anti- hpβ-lg antibody or anti-β-actin antibody, as a control, and alexa fluor -labeled donkey anti-mouse igg. different from the anti-β-actin antibody-treated cells that showed strong fluorescence signals, anti- hp-β-lg antibody-treated cells displayed no significant fluorescence signals inside or outside the cells (figure ) . these results indicate that hp-β-lg could neither enter into the cell nor bind the cell surface proteins, including the receptor(s) for hpv. to investigate whether the topically applied hp-β-lg enters the blood circulation, a carbomer gel with or without hp-β-lg was topically administered in the vagina or anus of rhesus macaques. their blood samples were collected at different time points before and after gel application. the concentration of hp-β-lg in the blood samples and that in the gel (as positive control) were quantified with elisa using anti- hp-β-lg mab. as shown in figure , no hp-β-lg was detected in the blood samples of rhesus macaques vaginally or rectally administered with carbomer gel with or without hp-β-lg, confirming that the topically applied hp-β-lg does not enter the blood circulation. about - % of acute hpv infections become persistent, leading to various forms of cancer (shanmugasundaram and you, ) , such as the cervical cancer. although several multivalent prophylactic hpv vaccines have been used in clinics to prevent hpv infection and cervical cancer development, they have no effect against pre-existing hpv infection in middle-aged and elderly women, the population at high risk for cervical cancer. therefore, it is essential to develop effective and safe therapeutic agents for treatment of hpv infection in order to reduce the morbidity of cervical cancer. viral entry inhibitors have been proven effective and safe for the treatment of viral infections (jiang et al., ; dando and perry, ; lu et al., ; li et al., ; su et al., ) . we have previously reported that an hpv entry inhibitor, hp-β-lg, is highly effective in blocking the entry of hpv, both high-and low-risk types, into the host cell (lu et al., ) . in a randomized clinical trial, topical application of the vaginal gel containing hp-β-lg has shown excellent efficacy and safety to treat high-risk hpv infections (guo et al., a,b) . however, how hp-β-lg inhibits hpv entry into the target cell is still unclear. figure | hp-β-lg could not enter into the target cell. after incubation with hela and t cells, hp-β-lg in the cells was detected by immunofluorescence assay using anti- hp-β-lg mab (green). β-actin was used as positive control. cell nuclei were stained by , -diamidino- -phenylindole (blue). in our previous study, we demonstrated that the inhibitory activity of hp-β-lg on hpv psv entry into the target cell is closely correlated with the number of the positively charged lysine and arginine residues in hp-β-lg, as modified, i.e., the net negative charges on hp-β-lg (lu et al., ) , suggesting that the negatively charged residues on hp-β-lg play an important role in hp-β-lg-mediated inhibition of hpv infection. as shown in figure a , the unmodified bovine β-lg contains positively charged residues ( lys and arg) and negatively charged residues ( asp and glu) (qin et al., ) , thus having a net charge of − . in hp-β-lg, out of the lys and all arg were modified by hp at mm (lu et al., ) , resulting in the neutralization of of the positive charges. therefore, the net charge of hp-β-lg becomes − , making hp-β-lg be active in binding to a viral protein with high positive net charges and inhibiting virus infection. in this study, we demonstrated that hp-β-lg could strongly bind to the l protein on the hpv psv, while the unmodified β-lg protein could not (figure a) . using itc analysis, we showed that unlike β-lg, hp-β-lg could strongly interact with the positively charged residue-enriched c-terminal regions (residues - and - ) of l protein, which is responsible for the attachment of the hpv particle to the host cell, possibly through its interaction with the cellular receptor(s). one may argue that hp-β-lg may bind to any virus with high net positive charges on its surface protein and inhibit its infection. indeed, hp-β-lg can also inhibit infection of simian immunodeficiency virus (siv) (wyand et al., ) and hsv , but it is not effective against infection of mers-cov and vsv (unpublished data) . in this study, we showed that hp-modified hsa ( hp-hsa) was also effective, while hp-modified ova ( hp-ova) was not effective in inhibiting hpv psv entry (figure ) , even though hp-ova also contains high net negative charges on its surface. thus, aside from having high net negative charges, these results suggest that the active site in hp-β-lg for binding to the viral protein must possess a conformation that fits with that in the corresponding site in the viral protein (figure b) , enabling hp-β-lg to strongly and closely interact with the viral protein to block hpv entry into the host cell. indeed, the molecular docking analysis indicated that hp-β-lg could strongly bind to the positively charged region of hpv l protein, which is the binding site for the negatively charged hspg receptor for hpv ( figure b ). hp-β-lg has many more negative charges on the surface, and the interface between hp-β-lg and hpv-l is much larger than the hspg molecule. therefore, hp-β-lg can competitively bind to hpv l to block the binding of the hspg receptor. based on the results obtained from this study, we proposed a mechanistic model of hp-β-lg for inhibiting hpv infection in the cervical mucosa ( figure c) . the newly invaded hpv accesses the basal cell layer through micro lesions or breaks in the cervical epithelium and binds to the hspg receptor on the basement membrane through interaction between the positively charged region in l protein of hpv and the negatively charged region in hspg. this interaction results in a series of conformational changes in the capsid, which leads to protease digestion of l and exposure of its n terminus, thus initiating entry of hpv into to the epithelial cells (kines et al., ; pyeon et al., ) . after hpv infects the epithelial cell, it gradually matures along with the host cell cycles and moves toward the top layer of epithelia, followed by eventual release from the epidermal cells. the newly released a b figure | hp-β-lg could not enter into blood circulation when applied through the vagina or anus. (a) concentration of hp-β-lg in sera of monkeys through vaginal administration. (b) concentration of hp-β-lg in sera of monkeys through rectal administration. hp-β-lg was formulated in a carbomer gel ( . mg/each) and administered through the vagina or anus of rhesus macaques. their blood samples were collected at different time points before and after the application (− to h). hp-β-lg in the blood samples was analyzed by elisa. each sample was tested in triplicate, and the experiment was repeated twice. data from a representative experiment are presented as mean ± sd. the asterisks represent significant differences: *p < . ; **p < . ; ***p < . . frontiers in microbiology | www.frontiersin.org hpv particles access the basement membrane through micro lesions of the cervical epithelium and start another infection and replication cycle, gradually establishing persistent infection and promoting the development of cervical cancer. hp-β-lg that is topically applied in the vagina can bind to the l protein of the newly invaded or the newly released hpv and block the attachment of hpv to the basement membrane, thereby inhibiting the entry of hpv into the host cells for replication. after treatment with hp-β-lg for a certain period of time (e.g., months), the newly produced hpv-free epithelial cells in the lower part of the mucosal epithelium are expected to move upward and push the hpv-infected cells away from the vaginal mucosa (this movement is accelerated during the menstrual period), making all layers of mucosal epithelium free of hpv infection. considering that the presence of micro lesions or breaks in the cervical epithelium, possibly caused by sexual activity, is the requirement for hpv to access the hspg receptor on basement membrane (bousarghin et al., ) , combinational use of some biocompatible materials for mucosa wound healing, such as human collagen proteins (hua et al., ) , may have synergistic or complementary effects against hpv infection. the results from the time-of-addition assay, cell washout assay, and virus inactivation indicate that hp-β-lg could interact with the virions, rather than the cells, to effectively block the entry of hpv psv into the target cell, but could not inactivate the virions (figure ) . these biological properties of hp-β-lg make it an ideal anti-hpv agent for topical application to treat hpv infection because it can interact with hpv particles on the vaginal mucosa and block the virions binding to the receptor, e.g., hspg, on basement membrane. since hp-β-lg cannot enter into the cell or the blood circulation, it is not expected to cause systemic toxicity to human or induce harmful hp-β-lg-specific antibodies. therefore, the topical formulations containing hp-β-lg can be safely used to treat local hpv infection. the increased net negative charges on β-lactoglobulin after modification of the protein with -hydroxyphthalic anhydride. the structure of hp-β-lg was modeled by coot (emsley et al., ) using the crystal structure of β-lg (pdb entry: beb). the structures were shown as an electrostatic surface by pymol (alexander et al., ) . (b) predicted interactions between hpv l pentamer and hp-β-lg. crystal structure of hspg-bound hpv l pentamer (pdb entry: w o) was shown as electrostatic surface (dasgupta et al., ) , and the docking structure between hpv l and hp-β-lg was generated by autodock (trott and olson, ) . (c) schematic illustration showing how hp-β-lg inhibits hpv entry and infection. the newly invaded or newly released hpv particles access the basal layer through the mucosal lesion, or breaks, and bind to the hspg receptor on the basement membrane, resulting in the entry of hpv into and replication in mucosal epithelial cells. hp-β-lg can bind with hpv l protein, through the electrostatic interaction between the negatively charged region in hp-β-lg and the positively charged region in l protein, to block the attachment of hpv to the hspg receptor, resulting in inhibition of hpv infection in vaginal mucosa. all datasets generated for this study are included in the manuscript/supplementary files. the animal study was reviewed and approved by institute of laboratory animal science, chinese academy of medical sciences and peking union medical college (approval number: ilas-vl- - ). sj, lsu, and yz conceived and designed the experiments. ch, cw, lsi, and qw conducted the experiments. yz performed the computer modeling and analysis of the interaction between hp-β-lg and hpv l protein. ch, yz, lsu, and sj analyzed the data and wrote the manuscript. all authors have read and approved the final manuscript. this work was supported by an intramural fund of fudan-jinbo functional protein joint research center, fudan university. hpv infection of hacats is dependent on beta integrin, and alpha integrin processing hpv entry into cells bcl::cluster: a method for clustering biological molecules coupled with visualization in the pymol molecular graphics system structures of bovine and human papillomaviruses. analysis by cryoelectron microscopy and three-dimensional image reconstruction classification of papillomaviruses (pvs) based on pv types and proposal of taxonomic amendments the causal relation between human papillomavirus and cervical cancer positively charged sequences of human papillomavirus type capsid proteins are sufficient to mediate gene transfer into target cells via the heparan sulfate receptor cancer statistics in china structural basis of oligosaccharide receptor recognition by human papillomavirus molecular and structural characterization of the l virus-like particles that are used as vaccine antigens in cervarix, the as -adjuvanted hpv- and - cervical cancer vaccine features and development of coot safety evaluation of chemically modified beta-lactoglobulin administered intravaginally a randomized open-label clinical trial of an anti-hpv biological dressing (jb -bd) administered intravaginally to treat high-risk hpv infection. microbes infect conserved residue lys in the cavity of hiv- gp coiled-coil domain is critical for six-helix bundle stability and virus entry characterization by high-resolution crystal structure analysis of a triple-helix region of human collagen type iii with potent cell adhesion activity hiv- inhibition by a peptide the initial steps leading to papillomavirus infection occur on the basement membrane prior to cell surface binding intermediate heparan sulfate binding during hpv- infection in hacats impact of inhibitors and l antibodies upon the infectivity of diverse alpha and beta human papillomavirus types rational design of a triple-type human papillomavirus vaccine by compromising viral-type specificity the c-terminal arm of the human papillomavirus major capsid protein is immunogenic and involved in virus-host interaction chemically modified human serum albumin potently blocks entry of ebola pseudoviruses and viruslike particles chemically modified bovine beta-lactoglobulin inhibits human papillomavirus infection development of small-molecule hiv entry inhibitors specifically targeting gp or gp quadrivalent human papillomavirus (types , , , ) recombinant vaccine (gardasil®): a review of its use in the prevention of premalignant anogenital lesions, cervical and anal cancers, and genital warts bovine beta-lactoglobulin modified by -hydroxyphthalic anhydride blocks the cd cell receptor for hiv a herpesvirus inhibitor from bovine whey establishment of human papillomavirus infection requires cell cycle progression functional implications of structural differences between variants a and b of bovine beta-lactoglobulin determinants of incidence and clearance of high-risk human papillomavirus infections in rural rakai targeting persistent human papillomavirus infection alpha integrin is not the obligatory cell receptor for bovine papillomavirus type a peptidebased hiv- fusion inhibitor with two tail-anchors and palmitic acid exhibits substantially improved in vitro and ex vivo anti-hiv- activity and prolonged in vivo half-life essential roles for soluble virion-associated heparan sulfonated proteoglycans and growth factors in human papillomavirus infections autodock vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading effect of -hydroxyphthaloyl-beta-lactoglobulin on vaginal transmission of simian immunodeficiency virus in rhesus monkeys we thank dr. jing xue at the institute of laboratory animal science, chinese academy of medical sciences and peking union medical college for performing the experiments on rhesus macaques; dr. john schiller at the laboratory of cellular oncology, national cancer institute, nih, md, usa for providing plasmids for constructing hpv psv; and yuhong fu for assistance in preparing anhydrate-modified proteins. key: cord- -cxi cr authors: yoshida, asuka; kawabata, ryoko; honda, tomoyuki; tomonaga, keizo; sakaguchi, takemasa; irie, takashi title: ifn-β-inducing, unusual viral rna species produced by paramyxovirus infection accumulated into distinct cytoplasmic structures in an rna-type-dependent manner date: - - journal: front microbiol doi: . /fmicb. . sha: doc_id: cord_uid: cxi cr the interferon (ifn) system is one of the most important defensive responses of mammals against viruses, and is rapidly evoked when the pathogen-associated molecular patterns (pamps) of viruses are sensed. non-self, virus-derived rna species have been identified as the pamps of rna viruses. in the present study, we compared different types of ifn-β-inducing and -non-inducing viruses in the context of sendai virus infection. we found that some types of unusual viral rna species were produced by infections with ifn-β-inducing viruses and accumulated into distinct cytoplasmic structures in an rna-type-dependent manner. one of these structures was similar to the so-called antiviral stress granules (avsgs) formed by an infection with ifn-inducing viruses whose c proteins were knocked-out or mutated. non-encapsidated, unusual viral rna harboring the ′-terminal region of the viral genome as well as rig-i and typical sg markers accumulated in these granules. another was a non-sg-like inclusion formed by an infection with the cantell strain; a copyback-type di genome, but not an authentic viral genome, specifically accumulated in the inclusion, whereas rig-i and sg markers did not. the induction of ifn-β was closely associated with the production of these unusual rnas as well as the formation of the cytoplasmic structures. eukaryotic cells are equipped with various defense mechanisms to detect and respond to viral infections rapidly. the interferon (ifn) system is one of the most important natural defenses of mammalian cells in the early phase of viral infection. host cells sense the invasion of viruses by recognizing their pathogen-associated molecular patterns (pamps), including the structural characteristics of viral rnas that differentiate them from cellular rnas . viral rnas are detected by non-self rna sensors such as toll-like receptors (tlr), and a family of cytosolic rna helicases termed rig-i-like receptors (rlrs), including retinoic-acid inducible gene-i (rig-i), melanoma differentiation-associated gene (mda ), and laboratory of physiology and genetics gene (lgp ). this is followed by the subsequent induction of ifn-β (gitlin et al., ; kaisho and akira, ; saito et al., ) . autocrine or paracrine ifns bind to ifn receptors on the cell surface, leading to the expression of s of ifn-stimulated genes (isgs) through the jak/stat signaling pathway, which ultimately exerts various antiviral effects . translational arrest is one of the ifn responses of host cells triggered by viral infections. various eukaryotic translation initiation factor (eif ) kinases such as protein kinase r (pkr) are activated in response to ifns, and the accumulation of phosphorylated eif α inhibits the translation of both cellular and viral mrnas kedersha and anderson, ; holcik and sonenberg, ) . cytoplasmic stress granules (sgs), which are the foci of concentrated s translation preinitiation complexes and defined by certain marker rna binding proteins such as t-cell intracellular antigen- (tia- ), tia- -related protein (tiar), and ras-gap-sh domain-binding protein (g bp ), are formed under these conditions kedersha and anderson, ) . because they contain stable inert mrna, sgs are believed to serve as temporary sites at which mrnaprotein complexes are stored to pause active translation or be decayed in adjacent processing bodies (anderson and kedersha, ; balagopal and parker, ; buchan and parker, ) . a number of viruses have been shown to induce the formation of sgs in infected cells, and this may be related to the virus-induced shut-off of cellular protein translation. for example, hepatitis c virus, poliovirus, semliki forest virus, and mammalian orthoreovirus promote the shut-off of cellular proteins and the assembly of sgs at the early phase of infection, and this is inhibited as the infection progresses (mcinerney et al., ; white et al., ; qin et al., qin et al., , ariumi et al., ; white and lloyd, ; garaigorta et al., ; panas et al., ; ruggieri et al., ; fitzgerald and semler, ; pager et al., ; carroll et al., ) . two rna viruses, respiratory syncytial virus and coronavirus, are also known to utilize sgs as part of the machinery to inhibit host cellular protein translation (raaben et al., ; lindquist et al., lindquist et al., , . sgs were not detected during infection by influenza a virus (iav); however, a recombinant iav lacking non-structural protein (ns ), an inhibitor of pkr, efficiently induced the formation of sgs and the production of ifn-β in a pkr-dependent manner (khaperskyy et al., ; mok et al., ; onomoto et al., ) . in this case, sgs were suggested to play an important role as the sites of viral rna sensing and subsequent anti-viral responses, because rlrs localized together with viral nucleoproteins and rna as well as anti-viral proteins in sgs (onomoto et al., ; ng et al., ) . the rna species produced during the course of rna viral replication, such as mrna, dsrna, and -triphosphate ( -ppp) rna including leader, trailer, genome, and antigenome rnas as well as defective interfering (di) genomes, have been shown to trigger the production of type i ifns (yoneyama et al., ; hornung et al., ; pichlmair et al., ; strahle et al., ; hausmann et al., ; baum et al., ; baum and garcia-sastre, ; kato et al., ; marq et al., ; davis et al., ; bowzard et al., ; weber et al., ; runge et al., ; schmolke et al., ) . we and other groups have recently reported that recombinant viruses of sendai virus (sev), a prototype of the family paramyxoviridae, in which the c proteins are knocked-out or mutated, generate dsrna in infected cells at levels similar to the production of ifn-β (takeuchi et al., ; irie et al., ) . previous studies also reported that, in the cases of sev and iav, copyback (cb)-and internal deletion (id)-type di genomes, respectively, rather than full-length viral genomes, preferentially associated with rig-i and strongly induced the production of ifn-β (baum et al., ; baum and garcia-sastre, ) . in spite of the large number of studies conducted in this field, it remains unknown what kinds of viral rna species are recognized by rlrs and where the sites of recognition are in real infections by rna viruses. in the present study, we compared some types of ifn-β-non-inducing and ifn-β-highly inducing viruses in the context of sev infection. one of the biggest advantages of this study is that the comparison can be performed within the context of the same viral species. we found that some types of unusual rna species that were distinguishable according to specific detectability by the anti-dsrna antibody or fish analysis were produced during the viral replication of ifn-inducing sevs, but not ifn-non-inducing sev strains, and accumulated into distinct cytoplasmic structures in an rna-type-dependent manner. these unusual rnas exhibited distinct properties in infected cells in terms of encapsidation with the viral n protein and subcellular distribution with sg marker proteins and rlrs. our results suggest that rna-typedependent mechanisms recognize and accumulate virus-derived, ifn-β-inducible, unusual rnas into specific compartment to trigger the production of ifn-β, and that sev may evade detection by the host innate immune system by preventing the production of these rna species. llc-mk cells (macaque monkey kidney-derived cells, described in kiyotani et al., ) and hela cells (ccl- ; purchased from atcc) were maintained as described previously (irie et al., ) . all of the sevs, even the wt of strain z and hamamatsu (hmt), used in this study were recovered from cdna using a reverse genetics technique as described previously (kato et al., ; fujii et al., ) , except for the cantell (cnt; vr- ; atcc), fushimi (fsm), and nagoya (ngy) strains. the sev recombinants, c /c(-), c(-), and v(-), were kindly provided by kato (national institute of infectious diseases, japan; kato et al., ; kurotani et al., ) . all of the sevs as well as virulent and avirulent newcastle disease virus (ndv) miyadera and d strains (toyoda et al., ) , respectively, were propagated in embryonated chicken eggs. sev and ndv titers were determined by an immunofluorescent infectious focus assay in llc-mk cells and expressed as cell infectious units (cius)/ml, as described previously (kiyotani et al., ) . one-step growth kinetics of the viruses was determined as described previously (irie et al., ) . plasmids encoding the p, c, and v proteins of sev strain z in the pcaggs. mcs vector have been described previously (sakaguchi et al., (sakaguchi et al., , irie et al., b irie et al., , . the polyclonal antibodies (pabs) against whole virions of sev and ndv were described previously (kiyotani et al., ) . the pabs against the sev p and c proteins were kindly provided by kato. the monoclonal antibody (mab) against the sev n protein was kindly provided by suzuki (national institute of infectious disease, japan). the mab and pab against g bp (sc- , santa cruz biotechnology; and ab , abcam, respectively), pab against rig-i ( ; immuno-biological laboratories, japan), mab against tiar, and dsrna (# , cell signaling technology; and j , scicons, hungary, respectively) were used according to the protocols of the suppliers. hela cells cultured on glass coverslips were infected with the indicated viruses or transfected with the indicated plasmids. the culture medium was replaced by serum-free dmem h postinfection (p.i.) or post-transfection (p.t.), and cells were treated with sodium arsenite (naaso ; sigma) at a final concentration of . mm for min or with ifn-α ( , iu/ml; r&d systems) for h. hela cells cultured on glass coverslips were transfected with the indicated plasmids using fugene hd transfection reagent (promega) or infected with the indicated viruses at an moi of . at h p.t. or p.i., cells were fixed and immunostained as described previously using appropriate combinations of primary and secondary antibodies (irie et al., ) . to detect rig-i, and dsrna, the tyramide signal amplification (tsa) kit with hrp-goat anti-mouse igg and alexa fluor tyramide (molecular probes) were used to increase the detection sensitivity. coverslips were mounted on glass slides with the slowfade gold antifade reagent with or without dapi (molecular probes) and observed using an lsm confocal microscope (carl zeiss). hela cells were infected with the indicated viruses at an moi of . at h p.i., total rna was prepared using the high pure rna isolation kit (roche diagnostics). viral rna in the working viral stocks was prepared using the high pure viral rna kit (roche diagnostics). quantitative (q) rt-pcr was performed as described previously (irie et al., (irie et al., , . qrt-pcr samples were analyzed using the eco real-time pcr system (illumina). for direct comparison, all of the indicated samples were analyzed in the same experiments. to detect the genome-length viral rnas and cbdi genomes of sev stocks, qrt-pcr was performed using the primer sets of sevz + sevz , as described previously (irie et al., a (irie et al., , , and cbdidetect , - , + cbdidetect , - , , which were complementary to the regions of the indicated positions of sev genome rna, as described recently by baum et al. ( ) . hela cells were lysed in ripa buffer ( . % np- , mm tris-hcl [ph . ], mm nacl) after h of infection by the indicated viruses or min of the arsenite treatment, and the insoluble fraction was removed by high-speed centrifugation. the viral proteins in the lysates were removed by three consecutive immunoprecipitation steps using anti-sev or anti-ndv pabs and protein g sepharose beads (ge healthcare life sciences). supernatants were harvested from the final immunoprecipitation samples, and were then subjected to rna preparation, as described above. hela cells cultured on glass coverslips were transfected with μg of the indicated rna samples prepared above, together with . μg of an empty puc vector, using the fugene hd transfection reagent. cantell samples ( . × ciu/ml) that were ∼ -fold serially diluted were inoculated into the allantoic cavity of -days-old embryonated chicken eggs, and incubated for h at • c. allantoic fluid was harvested from the eggs, and the titers of each fluid stock were determined as described above. rna samples were prepared from μl of each fluid stock, and the ratios of the cbdi genomes to viral genomes were determined by qrt-pcr as described above. hela cells cultured on glass coverslips were infected with the indicated viruses. at h p.i., cells were fixed with % paraformaldehyde solution in pbs, and then subjected to fish analysis using the fish tag rna green kit with alexa fluor dye (molecular probes) according to the protocol of the supplier. the rna probe was designed to be complementary to the region of , - , in sev genome rna. after fish, some samples were further subjected to fluorescent immunodetection as described above using the indicated antibodies. final samples were observed using an lsm confocal microscope. we first examined whether g bp -positive granular structures were formed during infections by a series of c knockedout and mutated sev recombinants and the parental z strain by immunofluorescence microscopy (figure ; supplementary figure s ). treating hela cells with sodium arsenite (naaso ) caused the formation of granular structures in the cytosol, which were figure | subcellular distribution of g bp and tiar in hela cells treated with or without sodium arsenite (a), and infected with a series of c-deficient rsevs, dy , dy , d y, c /c(-), and c(-), and a v-deficient rsev, v(-) (b). hela cells treated with ethanol (etoh) or sodium arsenite (naaso ) or infected with the indicated virus were immunostained with anti-g bp mab, anti-tiar mab, and anti-sev pab. (c) the g bp foci in the samples of (b) were observed under a fluorescent microscope, and the number of g bp granule-positive cells was counted and is presented as percentages against the number of sev antigen-positive cells (closed bars). the relative amounts of ifn-β mrna in cells infected with the indicated viruses were determined by qrt-pcr, and normalized to those of β-actin mrna (open bars). (d) similar experiments were performed for a series of c-mutated sev recombinants, and the results are presented as bar graphs, similarly to (c). defined as sgs based on the expression of related proteins such as g bp and tiar. g bp and tiar are well-established sg-associated proteins that are typically and diffusely present throughout the cytoplasm and dominantly present in the nucleus, respectively. however, treating cells with arsenite markedly changed the localization to form sgs containing these proteins in nearly all cells ( figure a) . when cells were infected with c(-), g bp -positive granular structures were observed in almost % of sev antigen-positive cells, and were considered to be sg-like structures since tiar was also detected in the majority of the granules (figures b,c) . in this situation, tiar was mostly in the cytoplasmic structures, whereas a larger part of tiar was still observed in the nucleus in the arsenite-treated cells. this difference is probably due to the different exposure time to the stimuli: min. for the treatment with arsenite and h for the infection. in contrast, the percentages were only % or less in cells infected with the parental z-wt as well as the dy , dy , and d y recombinants, which lacked the smaller c proteins, y , y , and both y and y , respectively ( figure c; supplementary figure s a ). an infection by c /c(-) and v(-), lacking the larger c proteins, c and c, and the v protein, respectively, resulted in a slight increase in the number of granules ( figure c ; supplementary figure s a ). of note, unlike the viruses reported previously, such as ns -deficient iav and vesicular stomatitis virus (mok et al., ; onomoto et al., ; dinh et al., ) , the fluorescence of the sev antigen was not colocalized with that of the representative sg marker g bp in the granules (figure b ; supplementary figure s ). ifn-β mrna levels in the infected cells were also compared between the viruses ( figure c) . strong correlations were observed between ifn-β mrna levels and the percentages of granular structure-forming cells against infected cells. similar strong correlations were observed for a series of c mutant viruses that possessed single-to triple-amino-acid substitutions of highly conserved, charged amino acids within the c proteins, which diminished their ability to antagonize the host ifn system to various degrees (figure d ; supplementary figure s b ; irie et al., ) . the marked difference observed in granular formation and ifn-β mrna levels among the viruses was not due to their different growing abilities. the one-step growth kinetics of all the recombinants used above was previously reported to be similar to that of the parental z-wt, except for c(-) and c /c(-), the titers of which were - logs lower than that of z-wt throughout the time course, and viral protein synthesis in the infected cells did not significantly differ among the viruses examined (kurotani et al., ; irie et al., a irie et al., , . treatment of hela cells with ifn-α did not induce g bp -positive granules, indicating that the formation of sglike structures observed by sev infection was triggered by sev infection, but not by sev-induced type i ifns (supplementary figure s c) . taken together, these results strongly suggested that a relationship may exist between the formation of g bp -positive granules and the induction of ifn-β in the c recombinants, and also that c proteins may suppress the formation of granules. we examined whether expression of the c protein alone and infection of the non-granule-forming sev could inhibit the formation of granules in cells treated with arsenite or infected with ndv (figure ; supplementary figure s ). ndv, another prototypic paramyxovirus, which was considered to be an ifn-β-inducing virus, could induce g bp -positive granules in nearly all cells infected with virulent as well as avirulent strains (miyadera and d strains, respectively; figure b ). the c protein alone failed to inhibit the formation of granules in cells treated with arsenite (figure a) as well as infected with ndv ( figure b) , although the number of granules was slightly reduced in the c-expressing cells treated with arsenite compared to that observed in the neighboring non-c-expressing cells (figure a) . in addition to c, the other p gene products, p and v proteins, also failed to inhibit the formation of both types of granule (supplementary figure s ) . infection by sev-z-wt and c-deficient recombinant c(-) also failed to inhibit the arsenite-induced formation of granules (figures c,d) . small granules dispersed in the cytoplasm were induced by arsenite in both cases of infection by wt and c(-). of note, the g bp -positive granules induced by arsenite and viruses, such as c(-) and ndv, differed in size; the granules induced by the infection were apparently larger than those induced by arsenite (figures and ) , implying a possible difference in cellular pathways leading to these two types of granular structure. these results demonstrated that sev did not have the ability to inhibit the formation of both types of granules. a marked difference was noted in the abilities of the sev strains to induce ifn-β. although most of the sev strains including z have been characterized by their strong ability to counteract the innate immune system, the cnt strain has been widely used as a virus that induces high levels of ifn-β (baum et al., ; tapia et al., ) . therefore, we compared the abilities of some sev strains to induce ifn-β and sg-like structures (figure ; supplementary figure s ). in all cases, g bp -positive granular structures were only detected in less than % of sev antigen-positive cells (figure ; supplementary figure s a ). ifn-β mrna was not highly induced in cells infected with the sev strains, except for cnt, whereas cnt induced ifn-β mrna at a level that was -fold higher than that by z (figure ) . regarding the sev strains, unlike that observed in the c recombinants, a correlation was not observed between ifn-β mrna levels and the percentage of granular structure-forming cells against infected cells (figure ) . the different levels of ifn-β mrna induced by the strains could not be attributed to differences in viral growth (supplementary figure s b) . these results suggested that the formation of g bp -positive granules was not necessarily required to sense the sev cnt infection, followed by the production of ifn-β, unlike the c recombinants. we attempted to identify the reason for the difference in granular formation between these two types of ifn-β-inducing virus, c(-) and cnt. to address this issue, we first examined the ability of total rna prepared from virus-infected as well as arsenite-treated cells to induce g bp -positive granules ( figure a; supplementary figure s a ). total rna samples prepared from cells infected with any of the ifn-β-inducing viruses, sev-cnt, c(-), and ndv, were able to induce the granules as well as ifn-β in hela cells despite no formation of the granules by the cnt infection, whereas those from cells infected with the ifn-β-non-inducing sev-z-wt or treated with arsenite were not (figure a) . we then examined the content rates of cbdi genomes, a potent ligand for rig-i, against viral genome-length rnas in the rna samples used above ( figure b ). the cnt sample contained cbdi genomes at a level that was ∼ , -fold higher than that in the z-wt sample, although the sample of c(-) contained similar levels to the z-wt sample ( figure b) . we further examined the effect of removal of encapsidated viral rna species, such as viral full-length and di genomes, by three consecutive rounds of immunoprecipitation using antisera against the whole virions of sev and ndv prior to the preparation of rna samples (supplementary figure s b) . total rna prepared from the postimmunoprecipitation samples of c(-) and ndv was still able to induce the granules as well as ifn-β, but that of cnt lost these abilities (figure c ). since the naked cbdi genomes have been reported readily to form an ideal structure as the rig-i ligands of -triphosphated, blunt-ended dsrna (kolakofsky, ) , these results indicated that the major ifn-β-inducing viral rna species produced in the cells infected with cnt was encapsidated cbdi genomes, whereas those for sev- c(-) and ndv were not. the results above suggested that there may be at least two types of ifn-β-inducing viral rna species with a marked difference in the formation of sg-like granules. to elucidate this difference, we examined the subcellular distribution of unusual viral rna species produced by c(-) and cnt (figures and ) . we and other groups previously reported a strong correlation between the production of dsrna detected by the anti-dsrna antibody j (j -dsrna) and the induction of ifn-β in infections of the c-mutated sev recombinants and ndv (takeuchi et al., ; irie et al., ) . therefore, we first examined the production and subcellular distribution of j -dsrna in the cells infected with the ifn-β-inducing viruses by immunofluorescence microscopy (figure ) . dsrna fluorescent signals were absent in cells infected with ifn-non-inducing z-wt as well as in those with the ifn-β-inducing strain cnt ( figure a ). in contrast, dsrna fluorescent signals were clearly observed in the cytoplasm of cells infected with ifn-β-inducing sev- c(-) and ndv with dispersed and granular distributions, respectively ( figure b) . a previous study reported that ns -deficient iav infections caused rig-i to form granular aggregates that contained sg markers as well as viral rna (onomoto et al., ) . therefore, the cells infected with c(-) and ndv were co-stained with anti-rig-i and anti-g bp antibodies ( figure c ). similar to iav, rig-i almost perfectly colocalized with the virus-induced g bp -positive granules in both cases of infection ( figure c) ; however, unlike iav, these granules did not colocalize with the viral antigen or viral j -dsrna ( figure b, arrowheads) , as shown in figures and . together with the results of figure , j -dsrna appeared to be not or less encapsidated by viral nucleoproteins because the access of the antibody to and the formation of dsrna by tightly encapsidated viral rna, such as viral genomes, was unlikely. we further attempted to visualize the subcellular distribution of the unusual viral rna species that were not fully encapsidated, unlike the genome-length viral rnas, by fish analysis (figure ) . infected cell samples were prepared without a protease treatment to exclude fully encapsidated viral rna species, and were then stained with an rna probe complementary to the , - , region of the (-)-sense viral genome rna, designed by referring to two wellcharacterized cbdi genomes of sev (calain et al., ; -gil et al., ) . fluorescence-positive cytoplasmic inclusions were observed in the c(-)-as well as in the cntinfected samples, while an apparent signal was not detected in z-wt-infected or uninfected samples ( figure a ). when the z-wt-infected sample was treated with proteinase k, apparent signals were observed throughout the cytoplasm ( figure a) . these results strongly suggested that fully encapsidated viral genomes were not detected, whereas nonor partially encapsidated viral rna species were detectable in this system. the fish-positive inclusions observed in cntinfected cells were no longer detected in the cells infected with the cnt-lowdi (figure a) , which contained ∼ -fold fewer cbdi genomes than the original cnt sample ( figure b) . together with the results of figure , the fish signals observed in the cnt-infected cells were considered as the cbdi genomes. the fish samples of c(-) and cnt-infected cells were further immunostained to examine the subcellular colocalization of fish signals, the sev n protein, rig-i, and g bp (figures b-d, respectively) . the sev n protein was detected in the fish-positive inclusions of cnt-infected cells, suggesting that the rna species detected in the cnt samples were only partially, not fully, encapsidated, unlike the fully encapsidated, full-length, intact viral genomes ( figure b ). in contrast, the n protein was not detected in the inclusions of c(-)-infected cells, which suggested that the rna species detected in the c(-) samples were not encapsidated ( figure b) . the fish-positive inclusions of cnt-infected cells were not apparently colocalized with rig-i or g bp , whereas most of those in the c(-)-infected cells colocalized obviously with rig-i and g bp , unlike the j -dsrna (figures c,d) . these results indicated that unusual viral rna species harboring the -region of (-)-sense sev genome rna, which was not produced during infection by ifn-β-non-inducing z-wt, was produced in infections by , and were selectively formed into distinct cytoplasmic inclusions in an rna-type-dependent manner. the inclusions in the c(-)-infected cells could be identified as avsgs, and may be the site to detect viral rna by rig-i, whereas those in cnt-infected cells were not avsgs, but as-yet-unidentified structures. although a number of studies have examined host innate immune responses against pathogenic microbes including rna viruses, the virus-derived rna species that serve as pamps in real viral infections and the sites at which pamps are recognized by rlrs have yet to be clarified in detail. two important insights were reported recently. regarding the real pamps in infections by rna viruses, the cb and id types of di genomes were identified as the ligands of rig-i in sev-and iav-infected cells, respectively, both of which could form ideal structures as the rig-i ligands (baum et al., ; baum and garcia-sastre, ; martinez-gil et al., ) . the sg-like structures have been suggested to serve as the sites at which the rlrs encounter viral rna and subsequently activate the ifn signaling pathways in infections by rna viruses (onomoto et al., ; yoo et al., ) . in order to establish what and where viral rna species were detected by rlrs, in the present study, we compared two types of ifnβ-inducing sev, a recombinant c(-) and a strain cnt, with a non-ifn-β-inducing z strain, in terms of the formation of sglike granules and the production of unusual viral rna species. a major advantage of our study is that the comparison can be performed within the context of the same viral species. several types of unusual viral rna species were found to be generated in cells infected with the ifn-inducing sevs but not those with the ifn-non-inducing sevs (summarized in table ). one was a dsrna (j -dsrna) that was detected by the anti-dsrna antibody, j (type i in table ) . as for sev, the generation of j -dsrna may have been restricted by the c proteins because it was only detected in cells infected with c-deficient or mutated recombinants, but not in those with intact sevs (figure ; takeuchi et al., ; irie et al., ) . we and other groups demonstrated that j -dsrna activated pkr, and this was followed by the phosphorylation of eif and the production of ifn-β, both of which resulted in antiviral effects in the host cells. the activation of pkr has been reported to induce the formation of sg-like structures during infections by some rna viruses, such as iav and measles virus (mev; mok et al., ; onomoto et al., ; okonski and samuel, ) , and this also appears to be the case for the sev c recombinants. unlike these viruses, the sg-like structures formed during c(-) infections did not include the j -dsrna, which was dispersed in the cytoplasm, although they contained rig-i (figure ) . this may lead to the assertion that the sg-like structures induced during infections by c recombinants are not the sites at which to detect sev infections. however, the sg-like structures formed by c(-) were revealed to contain another type of unusual viral rna species by fish analysis, in which an rna probe targeting the nt region of the -end of the (-)-sense sev genome was used (type ii in table ; figure ). this now strongly suggests that the sg-like structures found in the c(-) infection are defined as avsgs. unlike the iav infection, the type i and ii rna species seemed to be not or less encapsidated, given that they were not colocalized with viral antigens and were not removed from the infected cell lysates by immunoprecipitation using anti-sev antibody (figures and - ) . sev trailer rna, which is transcribed from the -ends of (+)-sense antigenome rna, was previously reported to interact with tiar to inhibit apoptosis and the formation of sgs induced by infection (iseni et al., ) . the c(-) virus was shown to induce apoptosis more quickly and severely in infected cells than the wt virus (irie et al., ) . although appearing to contain the nonencapsidated -end of a (-)-sense genome rna, at least in the part that is concordant with the trailer rna, it is unlikely that the type ii rna observed in the c(-)-infected cells has the ability of the trailer rna to inhibit apoptosis and sg formation. although details of the type i and ii rnas remain to be solved, given the cytoplasmic replication of sev rna without forming inclusion bodies and the differences of the rna species in subcellular distribution and reactivity with the j and fish probe, the type i dsrna somewhat unwound actively or incidentally into the type ii rna might be accumulated into the avsgs. although the sg-like structures and j -dsrna were not detected during cnt infections, fish-positive non-granularshaped inclusions were observed (type iii in table ; figure ). these cnt inclusions were markedly different from those of c(-). the inclusions did not include rig-i or g bp , but contained the n protein (figure ) , suggesting that the inclusions were not avsgs, and that the type iii rna was at least partially encapsidated. the cnt stock used in the present study contained a larger amount of cbdi genomes than the other viral stocks ( figure b) . the sev cbdi genomes have been shown to be partially and/or more loosely encapsidated than intact genomes (kolakofsky, ; strahle et al., ) . the sev cbdi genomes were recently identified as strong ligands for rig-i (baum et al., ; tapia et al., ) . indeed, the cnt-lowdi had lost the ability to induce ifn-β and the inclusions had not been found in the infected cells (figures b and ) . taken together, the type iii rna was identified as the cbdi genome. these results indicated that the process of detecting infections and the subsequent induction of ifn-β differed largely between c(-) and cnt: avsg-dependent and -independent mechanisms for c(-) and cnt, respectively. most viruses have been shown to possess the ability to antagonize host ifn pathways in order to avoid activating host antiviral actions, and this strategy is mostly based on a counteraction against the molecules involved in these pathways (versteeg and garcia-sastre, ) . however, a recent study reported that encephalomyocarditis virus (emcv) has the ability to disrupt sgs by cleaving g bp in order to avoid the innate immune detection of its infection and subsequent induction of ifn-β (ng et al., ) , suggesting that another effective strategy for viral evasion from the ifn system by preventing avsg formation exists. generation of the unusual viral rna species triggering the production of ifn-β seems to be suppressed during intact rna viral replication. similarly to sev, another paramyxovirus mev c protein was recently shown to impair the production of j -dsrna and the activation of pkr coupled with the formation of sg-like structures (okonski and samuel, ; pfaller et al., ) . unlike the case of sev, in which the knockout of c resulted in the production of j -dsrna (figures and ), but not cbdi genomes, the j -dsrna produced during the infection by a c-deficient mev recombinant was reported to be a cbdi genome (pfaller et al., ) . although the cbdi genomes were more dominantly produced by sev-cnt than by the other strains and c-recombinants tested ( figure b and data not shown), this unique property of cnt might be attributed to its c protein that possibly have a functional difference with those of the other sevs. the c proteins of both sev and mev have been shown to play critical roles in modulating viral rna synthesis and maintaining its integrity by potentially stabilizing the ribonucleoprotein (rnp)-polymerase complex (tapparel et al., ; reutter et al., ; bankamp et al., ; irie et al., a irie et al., , ito et al., ) . dysfunctions in the c proteins may result in a disturbance in integrity, leading to the production of the unusual, ifn-β-inducing rna species. the results of the present study indicate that several types of ifn-β-inducible, unusual viral rna species may be produced during sev infections and included in avsg-like and non-avsg-like cytoplasmic inclusions, which suggests that rna-typedependent mechanisms recognize and accumulate such unusual viral rnas in specific compartments. in addition, the production of these unusual rna species may 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for stress granule localization, core protein lambda interaction, and de novo virus replication the ' untranslated regions of influenza genomic sequences are 'ppp-independent ligands for rig-i induction of stress granule-like structures in vesicular stomatitis virus-infected cells poliovirus infection induces the colocalization of cellular protein srp with tia- , a cytoplasmic stress granule protein involvement of the leader sequence in sendai virus pathogenesis revealed by recovery of a pathogenic field isolate from cdna hepatitis c virus (hcv) induces formation of stress granules whose proteins regulate hcv rna replication and virus assembly and egress essential role of mda- in type i ifn responses to polyriboinosinic:polyribocytidylic acid and encephalomyocarditis picornavirus rig-i and dsrna-induced ifnbeta activation translational control in stress and apoptosis '-triphosphate rna is the ligand for rig-i inhibition of interferon regulatory factor activation by paramyxovirus v protein conserved charged amino acids within sendai virus c protein play multiple roles in the evasion of innate immune responses paramyxovirus sendai virus c proteins are essential for maintenance of negative-sense rna genome in virus particles recruitment of alix/aip to the plasma membrane by sendai virus c protein facilitates budding of virus-like particles sendai virus c proteins regulate viral genome and antigenome synthesis to dictate the negative genome polarity clustered basic amino acids of the small sendai virus c protein y are critical to its ran gtpase-mediated nuclear localization sendai virus trailer rna binds tiar, a cellular protein involved in virus-induced apoptosis measles virus nonstructural c protein modulates viral rna polymerase activity by interacting with host protein shcbp toll-like receptor function and signaling the paramyxovirus, sendai virus, v protein encodes a luxury function required for viral pathogenesis initiation of sendai virus multiplication from transfected cdna or rna with negative or positive sense rig-i-like receptors: cytoplasmic sensors for non-self rna stress granules: sites of mrna triage that regulate mrna stability and translatability influenza a virus inhibits cytoplasmic stress granule formation immediate protection of mice from lethal wild-type sendai virus (hvj) infections by a temperaturesensitive mutant, hvjpi, possessing homologous interfering capacity isolation and characterization of sendai virus di-rnas sendai virus c proteins are categorically nonessential gene products but silencing their expression severely impairs viral replication and pathogenesis respiratory syncytial virus induces host rna stress granules to facilitate viral replication activation of protein kinase r is required for induction of stress granules by respiratory syncytial virus but dispensable for viral replication short double-stranded rnas with an overhanging ' ppp-nucleotide, as found in arenavirus genomes, act as rig-i decoys a sendai virus-derived rna agonist of rig-i as a virus vaccine adjuvant importance of eif alpha phosphorylation and stress granule assembly in alphavirus translation regulation the ns protein of influenza a virus interacts with cellular processing bodies and stress granules through rna-associated protein (rap ) during virus infection encephalomyocarditis virus disrupts stress granules, the critical platform for triggering antiviral innate immune responses stress granule formation induced by measles virus is protein kinase pkr dependent and impaired by rna adenosine deaminase adar critical role of an antiviral stress granule containing rig-i and pkr in viral detection and innate immunity modulation of hepatitis c virus rna abundance and virus release by dispersion of processing bodies and enrichment of stress granules sequestration of g bp coupled with efficient translation inhibits stress granules in semliki forest virus infection measles virus c protein impairs production of defective copyback double-stranded viral rna and activation of protein kinase r rig-i-mediated antiviral responses to single-stranded rna bearing '-phosphates mammalian orthoreovirus escape from host translational shutoff correlates with stress granule disruption and is independent of eif alpha phosphorylation and pkr mammalian orthoreovirus particles induce and are recruited into stress granules at early times postinfection mouse hepatitis coronavirus replication induces host translational shutoff and mrna decay, with concomitant formation of stress granules and processing bodies mutations in the measles virus c protein that up regulate viral rna synthesis dynamic oscillation of translation and stress granule formation mark the cellular response to virus infection in vivo ligands of mda and rig-i in measles virus-infected cells regulation of innate antiviral defenses through a shared repressor domain in rig-i and lgp analysis of interaction of sendai virus v protein and melanoma differentiation-associated gene aip /alix is a binding partner of sendai virus c protein and facilitates virus budding rig-i detects mrna of intracellular salmonella enterica serovar typhimurium during bacterial infection sendai virus defective-interfering genomes and the activation of interferon-beta activation of the beta interferon promoter by unnatural sendai virus infection requires rig-i and is inhibited by viral c proteins sendai virus c protein plays a role in restricting pkr activation by limiting the generation of intracellular double-stranded rna defective viral genomes arising in vivo provide critical danger signals for the triggering of lung antiviral immunity inhibition of sendai virus genome replication due to promoterincreased selectivity: a possible role for the accessory c proteins structural comparison of the cleavage-activation site of the fusion glycoprotein between virulent and avirulent strains of newcastle disease virus viral tricks to grid-lock the type i interferon system incoming rna virus nucleocapsids containing a '-triphosphorylated genome activate rig-i and antiviral signaling inhibition of cytoplasmic mrna stress granule formation by a viral proteinase poliovirus unlinks tia aggregation and mrna stress granule formation the rna helicase rig-i has an essential function in double-stranded rna-induced innate antiviral responses dhx enhances rig-i signaling by facilitating pkrmediated antiviral stress granule formation we thank the staff of the analysis center of life science, hiroshima university, for the use of their facilities. we also thank dr. k. takeuchi (university of tsukuba) for fruitful discussions. this work was supported by jsps kakenhi (grant numbers and ). the supplementary material for this article can be found online at: http://journal.frontiersin.org/article/ . /fmicb. . key: cord- -gsvswr v authors: hedblom, grant a.; reiland, holly a.; sylte, matthew j.; johnson, timothy j.; baumler, david j. title: segmented filamentous bacteria – metabolism meets immunity date: - - journal: front microbiol doi: . /fmicb. . sha: doc_id: cord_uid: gsvswr v segmented filamentous bacteria (sfb) are a group of host-adapted, commensal organisms that attach to the ileal epithelium of vertebrate and invertebrate hosts. a genetic relative of the genus clostridium, these morphologically unique bacteria display a replication and differentiation lifecycle initiated by epithelial tissue binding and filamentation. sfb intimately bind to the surface of absorptive intestinal epithelium without inducing an inflammatory response. rather, their presence impacts the generation of innate and differentiation of acquired immunity, which impact the clearance of extracellular bacterial or fungal pathogens in the gastrointestinal and respiratory tracts. sfb have recently garnered attention due to their role in promoting adaptive and innate immunity in mice and rats through the differentiation and maturation of th cells in the intestinal tract and production of immunoglobulin a (iga). sfb are the first commensal bacteria identified that impact the maturation and development of th cells in mice. recently, microbiome studies have revealed the presence of candidatus arthromitus (occasionally designated as candidatus savagella), a proposed candidate species of sfb, in higher proportions in higher-performing flocks as compared to matched lower-performing flocks, suggesting that sfb may serve to establish a healthy gut and protect commercial turkeys from pathogens resulting in morbidity and decreased performance. in this review we seek to describe the life cycle, host specificity, and genetic capabilities of sfb, such as bacterial metabolism, and how these factors influence the host immunity and microbiome. although the role of sfb to induce antigen-specific th cells in poultry is unknown, they may play an important role in modulating the immune response in the intestinal tract to promote resistance against some infectious diseases and promote food-safety. this review demonstrates the importance of studying and further characterizing commensal, host-specific bacteria in food-producing animals and their importance to animal health. the distal gastrointestinal tract of all animals is colonized by a diverse array of bacterial, fungal, and protozoan species. an animal host maintains a mutualistic relationship with its microbial inhabitants in which the microbes provide protection from pathogenic bacteria through competing for ecological niches and fostering a stable environment for the development of host immunity (bäckhed et al., ) . many vertebrate intestines (such as mice, rats, chickens, humans, and turkeys) harbor commensal organisms named segmented filamentous bacteria (sfb) that bind specifically to the host intestinal epithelium. these organisms are closely related to the genus clostridium, and appear as long, segmented filaments that bind tightly to the host epithelium via a specialized structure (chase and erlandsen, ) . these bacteria were initially detected through microscopic examination of the gastrointestinal epithelium of mice (davis and savage, ) . sfb drew the attention of researchers due to their unique morphology, life cycle and binding location (schnupf et al., ) . since their discovery, a large body of research has been generated to characterize these bacteria and understand their role in the host-microbiome relationship. through examining mouse and rat models, it has been revealed that these bacteria play an important role in adaptive and innate immunity of the host. segmented filamentous bacteria are gram-positive, sporeforming bacteria that possess the capability to develop into long filaments, which are divided via the production of transverse septa (ericsson et al., ) . these bacteria exist in a developmental or vegetative form, which are characterized by the presence of intrasegmental bodies and spores, respectively (figure ) . sfb produce intrasegmental bodies, which can appear as either a spore or a holdfast. the distinction between these morphologies indicates that the holdfast form serves as the vegetative form, while the spore serves as the dormant form (chase and erlandsen, ) . segmented filamentous bacteria selectively attach to the ileal epithelium of the host species via the production of rounded, nipple-like projections called holdfasts. the holdfast serves as an anchoring mechanism and point of filament elongation, attaching to host enterocytes in the mucous membrane of the figure | gram stain of candidatus arthromitus from turkey ileum containing intrasegmental bodies ( , × magnification) (reiland, ) . epithelium, without penetrating the host cell wall (sanford, ) . this structure leads to the displacement and destruction of the intestinal microvilli surrounding the point of attachment (snellen and savage, ) , and leads to alterations in the electron density of both the host cell plasma membrane and apical cytoplasm (ericsson et al., ) . once attached, actin polymerization occurs directly underneath the holdfast structure and creates a pedestal-like formation similar to the adherence mechanism of salmonella enterica serovar typhimurium (jepson et al., ; ericsson et al., ) . though this attachment mechanism resembles that of pathogenic bacteria, it does not damage the host cell and causes no inflammatory response in the lamina propria (caselli et al., ) . once attached to the host epithelium, the filament begins to extend from the distal end, releasing additional holdfasts and spores from the maturing filament (chase and erlandsen, ) . the life cycle of an sfb filament is assumed to be about - days based on the rapid shedding of the intestinal epithelium of rodents, and longitudinal studies have shown that sfb appear in juvenile mice that are around days in age (davis and savage, ) . at this stage of development, sfb proliferate to become a dominant gut microbe and then recede in mature vertebrates to lower levels. during the early stages of colonization, sfb are transiently colonized with rod-shaped bacteria (schnupf et al., ) . it is believed that the spread of sfb then occurs via vertical transmission of spores from parents to offspring, as these bacteria are widely considered to be obligate anaerobes and have also appeared in the intestinal tissue of weaning mice (schnupf et al., ) . colonization occurs in mouse and rat hosts at the onset of the weaning process and has been found to be the same in arbitrary human microbiome studies (ericsson et al., ) . studies performed on human subjects ages months to years old revealed that % of individuals carry sfb in their gut from ages - months, % carry sfb from ages - months, and only . % carry sfb from ages - (yin et al., ) . the age-related drop in sfb intestinal carriage may be pharmaceutically reversed. for example, transient feeding mice rapamycin enhanced their lifespan, while dramatically increasing the prevalence of sfb in the small intestine (bitto et al., ) . in chickens, sfb colonization peaked at approximately weeks of age, and decreased as they aged to weeks of age. the decrease was inversely proportional to the amount of intestinal iga present (liao et al., ) . it is unknown whether increasing the prevalence of sfb in the intestinal tracts of adults is beneficial, or may result in autoimmunity (e.g., rheumatoid arthritis) (wu et al., ) . segmented filamentous bacteria spores germinate in the host's gut to produce teardrop-shaped, single-celled bacteria referred to as intracellular offspring (schnupf et al., ) . amongst intestinal commensals and symbionts, sfb are unique because they penetrate the intestinal mucus layer and intimately associate with host cells without invading the host (chase and erlandsen, ; sanford, ) . it is assumed that the intracellular offspring use flagella to reach the apical surface of polarized epithelium (kuwahara et al., ) . though cellular flagella have not been observed microscopically, an analysis of a sfb genome revealed a full set of chemotaxis and flagellin biosynthesis genes, strongly suggesting the presence of flagella in the early stages of spore maturation (kuwahara et al., ) . intracellular offspring attach to absorptive epithelial cells via their holdfast and induce condensed actin rearrangements underneath the point of attachment while displacing some of the neighboring microvilli structures (chase and erlandsen, ) . before luminal attachment, the nucleoid region of the intracellular offspring appears condensed, suggesting reduced amounts of transcription. however, when the intracellular offspring attaches to the host, the nucleoid region decondenses and allows for genomic transcription (chase and erlandsen, ) . holdfast attachment to enterocytes in the terminal ilea causes the intracellular offspring to increase in size, reaching up to µm in length before bacterial division commences through transverse septum formation (chase and erlandsen, ) . filaments continue to grow and divide from their distal end, reaching their maximum length of around - µm (martin et al., ; schnupf et al., ) . once the filament reaches its maximum length, a second round of symmetric division begins from the distal end to divide each original segment in half into secondary undifferentiated cells. these secondary cells range in length from to . µm, forming segments containing - cells (chase and erlandsen, ) . after elongation, the filament will often separate from the holdfast segment and enter into the ileum. these secondary segments then undergo differentiation form a mother cell and a daughter cell (klaasen et al., ) . differentiation of these filaments appears to be more pronounced in the presence of slightly aerobic conditions, when oxygen concentrations range from to . % environmental oxygen (schupf et al., ) . once differentiated, the mother cell engulfs and houses the daughter cell, where the daughter cell undergoes division into two intracellular offspring (klaasen et al., ) . the intracellular offspring contained within the mother cell are then subject to two fates, dispersal from the filament or sporulation (davis and savage, ; klaasen et al., ) . if favorable growth conditions are present, the septa that separate individual mother cells are degraded to form a tube in which the intracellular offspring are dispersed into the host's intestinal tract. these released offspring are then allowed to colonize additional host tissue and undergo filamentation and differentiation, thus completing the sfb lifecycle (martin et al., ; schnupf et al., ) . when an unfavorable or hostile environment is presented, the two intracellular offspring produce a single spore coat that covers both of the cells. once coated with the layer of peptidoglycan, the spore matures into a complete endospore inside of the mother cell, and is released from the filament (martin et al., ; schnupf et al., ) . these spores lack the ability to colonize the host until favorable environmental conditions, such as appropriate concentrations of oxygen are presented, and shed in the host's feces. once shed, the spores can be transmitted to another host via horizontal transmission (davis and savage, ) . though it was previously proposed that the entire sfb lifecycle occurred while attached to the host tissue (chase and erlandsen, ) , filaments containing intracellular offspring have not been observed in published tem and sem images of filaments attached to enterocytes (caselli et al., ) . alternatively, filaments containing differentiated intracellular offspring only appeared as detached and free-floating, indicating that maturation and differentiation of segments occur independently from host tissues. these filaments are separated from the holdfast segment, which then penetrates the epithelium until it undergoes endocytosis, phagocytosis, or transcytosis (caselli et al., ) . the ingestion of the holdfast segment presents a great number of bacterial antigens to antigen presenting cells and lymphocytes contained within the ileal epithelium (caselli et al., ) . in rare cases, a small number of segments may remain attached to the holdfast, but these segments often exhibit irregular morphologies and present enlarged intrasegmental junctions (caselli et al., ) . the genome of a rat-isolated sfb and a number of mice sfb isolates have recently been sequenced and published (kuwahara et al., ; prakash et al., ; sczesnak et al., ; pamp et al., ) . these sfb genomes are highly similar but do contain several species-specific genes of unknown function that may be involved in the species-specificity of sfb colonization (prakash et al., ) . all sequenced sfb have displayed a small genome size of . - . mbp, low g+c content ( . %), and around , - , protein encoding genes (prakash et al., ) . sfb possess a highly reduced genome similar to their genetic relatives within the genus clostridium (ericsson et al., ) . the biosynthetic pathways of most amino acids, vitamins and cofactors (such as b , b , and b , pyridoxine, nicotinamide, pantothenate, and biotin) are incomplete or absent altogether in sfb genomes (kuwahara et al., ; sczesnak et al., ; pamp et al., ) . sfb are also unable to synthesize nucleotides independently; instead they utilize alternative pathways that rely on the uptake of nucleotide bases (prakash et al., ) . to obtain nucleotides, amino acids, and peptides from the environment, sfb genomes contains genes encoding two extracellular nucleases as well as a list of proteases and peptidases, of which are membrane associated and to that are thought to be secreted (kuwahara et al., ; sczesnak et al., ) . in addition, sfb genomes contain numerous open reading frames (orfs) thought to encode a large number of transporters and permeases for small molecules and ions (such as amino acids, oligopeptide, dipeptides, manganese, zinc, iron, and phosphate) compared to other organisms with small genomes (sczesnak et al., ) . a particularly strong requirement for iron uptake was noted by sczesnak et al., since six different orfs for iron transporters are found in the mouse genome as well as three orfs for ferric iron regulator family proteins (sczesnak et al., ) . sfb also have several orfs for phosphotransferase systems predicted for uptake of sugars such as mannose, cellobiose, mannitol, and fructose (prakash et al., ; sczesnak et al., ) . finally, the sfb genomes contain genes for the non-oxidative pentose phosphate pathway and a complete glycolysis pathway to convert glucose to pyruvate, but are deficient for genes encoding almost all components of the krebs cycle, which is required for aerobic respiration (prakash et al., ; sczesnak et al., ) . however, sfb can tolerate small concentrations of oxygen and counteract oxidative stress, as sfb genomes contain genes predicted for two catalases, a peroxidase (rubrerythrin), and an arginase, which might limit nitric oxide production through catabolism of arginine (kuwahara et al., ; pamp et al., ) . these protective mechanisms are likely essential, given the replicative niche of sfb at the surface of the small intestinal epithelium where the oxygen tension is estimated to be around . % (he et al., ) . there are several factors that have been discovered about sfb that explain their auxotrophic nature. the genome of sfb isolated from a rat host (rat-yit) contains putative genes predicted to encode for proteases and for peptidases along with many other genes through to be involved with sporulation and germination (prakash et al., ) . peroxidase and catalase genes were also found, which explains the potential for sfb to exist in microaerophilic environments (prakash et al., ) . the genomes of sfb sequenced from mice and rat hosts revealed several clustered regularly interspaced palindromic repeat (crispr) loci, which serve as a prokaryotic defense mechanism, indicating that sfb genomes may have had exposure to invading dna throughout their evolutionary history (prakash et al., ) . flagellar, pilus, and chemotactic genes have been found in sfb genomes that suggest motility, which explains the organism's ability to penetrate the mucus layer lining of intestinal epithelial cells (prakash et al., ; schnupf et al., ) we have generated a representation of the metabolic and physiological predicted capabilities inferred from the genome contents of publicly sequenced genomes of sfbs (figure ). an extensive body of work has evidenced the presence of sfb in a large number of animal species, such as horses, cattle, pigs, turkeys, chickens and even humans (ericsson et al., ) . in nearly all of the mammalian species studied for the presence of sfb, the bacteria selectively colonize the ileum of the host, with the exception of fish species that lack a welldefined ileum (ericsson et al., ) . in poultry, sfb colonizes ileum (figure ) , the cecal tonsil or cecum (figure ; rahimi et al., ; bohorquez et al., ; liao et al., ) . attempts to colonize an animal species with sfb from another species have been unsuccessful. when germ-free mice and rats were inoculated with ileal homogenates containing sfb from both species, animals became colonized with sfb from their own species, indicating that sfb are host-specific and host selective (tannock et al., ) . host-specificity of sfb may be due to differences in the sequences of flagella genes flic and flicc , which show greater variability than flic and flic (chen et al., ) . it is not known with which host proteins the sfb flagella proteins interact to become adherent, however, sfb flagellar proteins may induce th cells by signaling through toll-like receptor (tlr ) in a subset of cd c hi cd b hi intestinal dendritic cells (uematsu and akira, ). for most animal species, holdfast cells have the capability to attach to goblet cells, m-cells, absorptive enterocytes, and cellular junctions of the ileal epithelium (meyerholtz et al., ) , whereas less is known for poultry. species to species variance exists in the preferred cell of attachment. for example, sfb in rats and figure | metabolic features of candidatus arthromitus through inference of genome contents from annotated genomes through use of rapid annotation using subsystem technology (reiland, ) . frontiers in microbiology | www.frontiersin.org pigs attach to follicle-associated epithelial cells (over peyer's patches) and absorptive ileal villi (tannock et al., ) . in mice and horse, attachment occurs primarily to follicle-associated epithelial cells and mainly to absorptive ileal villi for rabbits, cattle and canines . sfb are unique amongst intestinal commensals and symbionts because they penetrate the intestinal mucus layer and intimately associate with host cells, but do not invade the host (sanford, ) . the exact mechanism of host specificity remains unclear, but it is believed that initial binding of the holdfast segment to the host epithelium serves as a ligand-receptor interaction, triggering a response from the host (kuwahara et al., ) . sfb binding elicits actin polymerization and condensation at the point of attachment (chase and erlandsen, ) , suggesting a specific host response. host specificity of sfb suggests that these bacteria have coevolved with their hosts to promote the commensal relationship that exists between the two species. though initially considered to be common commensal members of the host microbiota, recent research suggests that sfb serve an important role in modulating host microbiome and immunity. sfb are unique amongst intestinal commensals and symbionts because they penetrate the intestinal mucus layer and intimately associate with host cells without invading the host (chase and erlandsen, ; lee et al., ; mortha et al., ; atarashi et al., ; bunker et al., ; farkas et al., ; furusawa et al., ; sadler et al., ) . the holdfast binding of sfb to the ileal mucosa does not elicit a strong inflammatory response (klaasen et al., ) . several interactions between the host and sfb have been investigated, indicating almost entirely positive associations between the sfb and the host, with rare exceptions. intestinal colonization of sfb in rainbow trout may produce a fatal disease called rainbow trout gastroenteritis (rtge), where sfb expand to large numbers in the intestinal tract and cause enterocytes to detach (del-pozo et al., ) . sfb were not always present near to all intestinal rtge lesions, suggesting that sfb affects intestinal barrier function and other bacteria may not be crucial for producing rtge lesions. experimental infection of susceptible rainbow trout stock with feces from rtge animals induced colonization and characteristic histopathological lesions containing sfb and an unidentified gram-negative coccus, suggesting that sfb, in part, may be the etiological cause of rtge (mccarthy et al., ) . recent advances have identified different methods to culture sfb in vitro, accelerating research to study the interaction of sfb with different members of the intestinal microbiota (ericsson et al., ; schupf et al., ) . a large amount of research has demonstrated the indispensable role that sfb play in the maturation of the host gut immune barrier, inducing both innate and adaptive immune responses (ivanov et al., ; sonnenberg et al., ; mortha et al., ; atarashi et al., ; bunker et al., ; farkas et al., ; furusawa et al., ; edelblum et al., ; schnupf et al., ) . immune modulation of sfb may extend beyond the intestinal tract in the blood, or to other mucosal barriers (mcaleer et al., ) . mice colonized with sfb were more resistant to sepsis secondary to experimental cecal ligation and puncture injury (cabrera-perez et al., ) . oral vancomycin treatment to mice diminished the number of intestinal grampositive bacteria (including sfb), which negatively impacted anti-fungal th immunity in the respiratory tract (mcaleer et al., ) . these data suggest that the composition of intestinal microbiota, especially sfb, is vital for impacting immunity to bacterial and fungal pathogens beyond the intestinal tract. segmented filamentous bacteria are best known for their ability to induce the differentiation of naïve cd + t cells to form antigen-specific th cd + cells (schnupf et al., ) in the terminal ileum of mice . ivanov et al. first demonstrated that conventionally raised b mice purchased form taconic farms were highly colonized with sfb; these bacteria were absent from conventional raised mice purchased from the jackson laboratory (ivanov et al., ) . introduction of sfb to b mice from the jackson laboratory induced il- a and il- production from intestinal cd + t cells, which became refractory to colitis induced by the intestinal pathogen citrobacter rodentium (ivanov et al., ) . il- is a cytokine that enhances production of antimicrobial peptides from intestinal epithelial cell and prevent bacterial pathogens from inducing attaching and effacing lesions (schupf et al., ) . sfb are not the only bacteria capable of inducing th cd + t cells in the intestinal tract of animals. virulent shiga-toxin producing e. coli (o ) and citrobacter rodentium induced a th response in the murine intestinal tract, and was dependent on bacterial adherence to host cells (atarashi et al., ) , as well as the commensal bifidobacterium adolescentis (tan et al., ) . th cells are a subset of cd + t cells that are distinguished by the expression of t cell receptor cd , nuclear transcription factor rar-related orphan receptor gamma t (rorγt) and production of interleukins il- a, il- f, il- , and il- (schnupf et al., ) . other immune cells present in the intestinal lamina propria are capable of secreting il- a and il- or express rorγt [e.g., innate lymphoid cells type (ilc ) and lymphoid tissue inducer-like cells (lti)], but these cells lack expression of cd (sonnenberg et al., ; mortha et al., ) . however, ilc and lti are not dependent on sfb for their induction. in the intestinal epithelium, il- a and il- f help to modulate neutrophil chemotaxis through producing cxcl chemokines via binding interactions with il- receptors il- ra and il- rc. il- a and il- f additionally aid in regulating the activation and differentiation of host neutrophils, and stimulate the production of host-defense peptides (schnupf et al., ) . systemic depletion of neutrophils in mice caused increased production of il- a and ileal sfb colonization (flannigan et al., ) . thus, neutrophil recruitment may lessen il- a and chemokine production, and serve as a negative feedback loop to limit sfb colonization. the regulation of these immunestimulatory compounds and cell types is essential in combatting intestinal colonization and infection from microorganisms. th cells provide colonization resistance to other pathogenic bacteria present at mucosal barriers, such as escherichia coli in the intestinal tract (zheng et al., ; edelblum et al., ) and respiratory fungi (mcaleer et al., ) . in newborn or germ-free mice, the presence of th cells in the lamina propria is rare, appearing only after colonization by microbes (gaboriau-routhiau et al., ) . the role of sfb in th cell production was initially demonstrated when mice were inoculated with mouse, rat, and human microbiota containing bacterial spores similar to that of the genus clostridium. only the experimental mice inoculated with a mouse-derived bacteria were shown to produce th cells in response to colonization. mice colonized with rat-and human-derived bacteria produced much less of a th response when compared to the mousederived microbiome treatment, indicating host-specific bacteria (such as sfb) as the causative agent of the immune response (gaboriau-routhiau et al., ; chung et al., ) . this association was also confirmed when s rrna sequencing was performed on the gut microbiome of mice presenting ileal th cells, revealing the presence of sfb (ivanov et al., ) . in experiments testing the reactivity of mouse lamina propria against a sfb expression library, two proteins of unknown function elicited a th cell response (yang et al., ) . it was predicted that these unknown proteins may serve as cell surface proteins, potentially elucidating the role that sfb attachment may serve in stimulating host immunity (yang et al., ) . proteins from sfb, secreted or bacterial-associated, are believed to interact with host cells and modulate immunity include adpribosyltransferases and a myosin-cross reactive antigen . the exact antigen presenting cell responsible for immune modulation by sbf is controversial, but it appears that sfb antigens presented to both intestinal macrophages or cd + intestinal dendritic cells (goto et al., ) are involved. because of the intimal relationship of sfb with intestinal epithelium, it is possible that metabolites from sfb may also impact the differentiation of th cells. intestinal macrophages, and not intestinal dendritic cells, appear to be vital for generating sfb-specific th responses in the murine ileum . analysis of the t cell receptor repertoire of th cells recognize peptide antigens produced by sfb (yang et al., ) . the addition of the th -indcing bacterial pathogen listeria monocytogenes failed to impact induction of th cells in sfb colonized mice (yang et al., ) , suggesting that the match of t-cell effector function with antigen specificity is driven by the type of bacteria that produce the antigen. th cell differentiation is additionally mediated through the production of serum amyloid a (saa) and reactive oxygen species (ros) produced in response to sfb binding. production of saa in the host epithelium is initiated by sfb binding and subsequent actin rearrangements, leading to a signal amplification via il- and ilc , both of which aid in th cell differentiation (schnupf et al., ) . saa also stimulates intestinal antigen presenting cells to secrete il- , which assists in th activation and survival (schnupf et al., ) , but il- also has as an antagonistic effect on development of th immunity (shih et al., ) . ros produced as a consequence of sfb binding to enterocytes helps to create a chemical environment that promotes th differentiation, as demonstrated in mice treated with ros scavenging compounds having lower amounts of th cells in vivo (atarashi et al., ) . sfb flagellar proteins may induce th cells by signaling through tlr in a subset of cd c hi cd b hi intestinal dendritic cells (uematsu and akira, ). the flagellar binding motifs that are targeted by tlr appear to be highly conserved in sfb and is nearly absent from other similar clostridium species, suggesting a specific role for sfb to modulate th immunity (prakash et al., ) . although sfb-induced th immunity may benefit the animal host, there are long-term consequences. sfb-induced th immunity is linked to the development of autoimmunity in susceptible breeds of mice (lee et al., ; yang et al., ; teng et al., ) by inducing differentiation and egress of t follicular cells from peyer's patches . it is unknown whether natural colonization by sfb in poultry is capable of promoting autoimmunity, but it must be considered if sfb, or its antigens are utilized as immunomodulators for food-producing animals. segmented filamentous bacteria mono-associated mice display rapid growth and development of peyer's patches, and sfb can also stimulate the formation of lymphoid follicles and tertiary lymphoid tissues in the host (lecuyer et al., ) . this activation of the host's intestinal immunity causes a drastic increase in fecal concentrations of secretory immunoglobulin a (siga), as the number and activity of iga secreting b-cells rises (klaasen et al., ) . germ-free mice monoassociated with sfb triggers the production of iga serum levels equivalent to that of specific pathogen free, sfb-negative mice (klaasen et al., ) . the expansion and stimulation of germinal centers present in peyer's patches is not entirely unique to sfb and has been seen to occur in other commensal bacteria such as morganella morganii and bacteroides distasonis, however, the response from sfb is much greater than these other organisms (schnupf et al., ) . both t-cell dependent (b- cell) and t-cell independent (b- cell) production of siga occurs in sfb mono-associated mice (schupf et al., ) . the amount of sfb-specific iga produced by the host in response to bacterial colonization is as high as . % of the total iga of the organism (talham et al., ) . iga transmitted by nursing mice to suckling pups has been shown to inhibit sfb colonization, and only after weaning do sfb populations begin to increase, coinciding with the time in which sfb colonization is typically recognized in mice (jiang et al., ) . the induction of siga by sfb may serve as a negative feedback mechanism to prevent overcolonization by sfb and dysbiosis in older animals (ohashi et al., ; liao et al., ) . light turkey syndrome (lts) is growing problem facing commercial turkey production in the united states. lts is a condition in which turkey flocks fail to meet their genetic potential weight, yielding birds that are - pounds below the industry standard for average flock weight (danzeisen et al., ) . birds affected by lts display symptoms similar to poult enteritis complex (pec), a disease in which birds experience weight loss, diarrhea, lethargy, and depression (mor et al., ) . however, lts is dissimilar to pec in that birds do not experience watery and pale intestinal contents or distended ceca, indicating differences between the syndromes (morishita et al., ) . the causative agent of pec is suspected to be microbial in nature, as inoculation of healthy birds with fecal homogenates derived from birds experiencing pec produced light weight poults when compared to un-inoculated birds, but a single responsible microbe has yet to be determined (mor et al., ) . similarly, inoculating healthy birds with fecal homogenates derived from turkeys with lts produced birds that were lighter than the control groups (mor et al., ) . the two conditions are not dependent on each other, as lts can occur in the absence of pec (danzeisen et al., ) . there exist a number of potential factors that may lead to the development of lts, such as colonization by pathogenic bacteria, viral infection, stunting of immune system development, inhibited nutrient absorption, and alterations to gut microbiome (danzeisen et al., ) . typically, lts/pes affects birds less than weeks of age (morishita et al., ) . a higher number of different pathogenic organisms are found in these younger birds than in birds aged - weeks. virus strains such as astrovirus, reovirus, and rotavirus types were detected in the host, and are not associated with poult mortality. coronavirus, which is commonly associated with mortality, was not detected in lts/pes poults (morishita et al., ) . in an attempt to understand the microbial basis of lts, danzeisen et al. performed s rrna microbiome analysis of low-performing and high-performing (based upon flock weights) flocks to determine the role of microbial succession in promoting digestive health samples were sequenced to discern the presence and abundance of dominant otus present in higher-performing flocks as compared to lowerperforming flocks (danzeisen et al., ) . after analysis it was determined that at the age of - weeks, higher-performing turkey flocks harbored significantly higher proportions of clostridium bartlettii and candidatus division arthromitus, a sfb (danzeisen et al., ) . previous studies regarding sfb colonization of poultry have identified sfb as a causative agent in intestinal disease (goodwin et al., ) , but sfb was later ruled out as a causative agent (sell et al., ) . also, sfb belong to several microbial taxa and are not considered a homogeneous group (thompson et al., ) . as demonstrated in mice and rat models, sfb have been proven to be potent stimulators of host immunity and ileal health. though the role of sfb (in particular candidatus arthromitus) in the digestive health of turkeys is not quite understood, the evidence provided by danzeisen et al. suggests that epithelial binding of these bacteria may promote early digestive health (danzeisen et al., ) . the potential for candidatus arthromitus to serve as an immunostimulatory probiotic makes it an organism of great interest to poultry researchers, as the turkey production industry is in need of alternatives to promote animal health in this age of restricted use of antibiotics in food-producing animals and increasing antimicrobial resistance. since their initial characterization in the s, sfb have transitioned from being considered an interesting and unique member of the gut microbiome with a unique morphology, to serving as a model organism to study immunomodulatory symbiotic bacteria and their effects on the host. the hostspecific binding mechanism employed by these bacteria to attach to ileal epithelium is similar to that of enteric pathogens. unlike enteric pathogens, sfb do not harm the host epithelium and instead live in a commensal, if not mutualistic manner. intimate binding to the host mucosal epithelium allows sfb to receive nutrients from the host, satisfying their auxotrophic requirements, while delivering antigens to the host. epithelial binding also initiates several immune responses from the host. as demonstrated in mice and rat models, sfb have been shown to stimulate the maturation of the host's th and iga responses, improving the ability of the host to protect against invading pathogens. additionally, sfb compete with other members of the intestinal microbiota by modulating access to nutrients and occupying available ecological niches. the fitness-bolstering effects produced by sfb in mouse models are well-understood, but little is known about the roles these bacteria play in the other vertebrate animals. it has been suggested through microbiome analyses of turkeys that sfb, specifically candidatus arthromitus, may provide a protective role in preventing the onset of the enteric condition lts, the cause of which is not well understood. the role of sfb in turkeys must be better elucidated to determine the beneficial effects these bacteria have in disease prevention and in ileal health. developing an understanding the role that commensal microorganisms, like sfb, play in the overall function of the gut microbiome will aid in our understanding the interplay between microbiome and host, providing insights into digestive health and the development of immunity. hr and gh contributed equally to the writing of the manuscript. ms, tj, and db also helped to write the manuscript. we would like to thank usda-afri award # - - and also the department of food science and nutrition and the college of food, agricultural and natural resource sciences at the university of minnesota for providing the funds needed to perform and complete this study. th cell induction by adhesion of microbes to 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autoimmune arthritis by promoting differentiation and migration of peyer's patch t follicular helper cells immune-modulating gut symbionts are not "candidatus arthromitus immune responses of tlr + lamina propria dendritic cells in enterobacterial infection gut-residing segmented filamentous bacteria drive autoimmune arthritis via t helper cells focused specificity of intestinal th cells towards commensal bacterial antigens comparative analysis of the distribution of segmented filamentous bacteria in humans, mice and chickens interleukin mediates early host defense against attaching and effacing bacterial pathogens we thank judi stasko (usda ars national animal disease center) for technical assistance with scanning electron microscopy. key: cord- -gy bvot authors: chen, i-yin; moriyama, miyu; chang, ming-fu; ichinohe, takeshi title: severe acute respiratory syndrome coronavirus viroporin a activates the nlrp inflammasome date: - - journal: front microbiol doi: . /fmicb. . sha: doc_id: cord_uid: gy bvot nod-like receptor family, pyrin domain-containing (nlrp ) regulates the secretion of proinflammatory cytokines interleukin beta (il- β) and il- . we previously showed that influenza virus m or encephalomyocarditis virus (emcv) b proteins stimulate il- β secretion following activation of the nlrp inflammasome. however, the mechanism by which severe acute respiratory syndrome coronavirus (sars-cov) activates the nlrp inflammasome remains unknown. here, we provide direct evidence that sars-cov a protein activates the nlrp inflammasome in lipopolysaccharide-primed macrophages. sars-cov a was sufficient to cause the nlrp inflammasome activation. the ion channel activity of the a protein was essential for a-mediated il- β secretion. while cells uninfected or infected with a lentivirus expressing a a protein defective in ion channel activity expressed nlrp uniformly throughout the cytoplasm, nlrp was redistributed to the perinuclear space in cells infected with a lentivirus expressing the a protein. k(+) efflux and mitochondrial reactive oxygen species were important for sars-cov a-induced nlrp inflammasome activation. these results highlight the importance of viroporins, transmembrane pore-forming viral proteins, in virus-induced nlrp inflammasome activation. severe acute respiratory syndrome coronavirus (sars-cov), a member of the genus betacoronavirus within the family coronaviridae, is an enveloped virus with a single-stranded positive-sense rna genome of approximately kb in length. the two-thirds of the genome encodes large polyprotein precursors, open reading frame (orf) and orf b, which are proteolytically cleaved to generate non-structural proteins (tan et al., ) . the one-third of the genome encodes four structural proteins, spike (s), envelope (e), matrix (m) and nucleocapsid (n), and non-structural proteins, along with a set of accessory proteins ( a, b, , a, b, a, b, and b) (perlman and dandekar, ; tan et al., ) . sars-cov is the etiological agent of sars (drosten et al., ; fouchier et al., ; ksiazek et al., ; kuiken et al., ; peiris et al., ) . at least , laboratory-confirmed cases of human infection, with a fatality rate of . %, were reported to the world health organization from november to july . high levels of proinflammatory cytokines, including tumor necrosis factor (tnf)-α, interleukin (il)- β, and il- , were detected in autopsy tissues from sars patients (he et al., ) . although dysregulation of inflammatory cytokines may be involved in lung injury and the pathogenesis of sars-cov, the underlying molecular mechanisms are not fully understood. the innate immune systems utilizes pattern recognition receptors (prrs) to detect pathogen-associated molecular patterns (medzhitov, ; kawai and akira, ) . recognition of virus infection plays an important role in limiting virus replication at the early stages of infection. nod-like receptor family, pyrin domain-containing (nlrp ) is activated by a wide variety of stimuli, including virus infection (bauernfeind et al., ) . four models describing activation of the nlrp inflammasome have been proposed thus far (hornung and latz, ; schroder et al., ; tschopp and schroder, ) . first, the disturbances in intracellular ionic concentrations, including k + efflux and ca + influx, play an important role (fernandes-alnemri et al., ; petrilli et al., ; arlehamn et al., ; ichinohe et al., ; ito et al., ; murakami et al., ; munoz-planillo et al., ) . second, cathepsin b and l, which are specific lysosomal cysteine proteases, are though to play a role after phagocytosis of cholesterol crystals (duewell et al., ) , fibrillar peptide amyloid-beta , silica crystals, and aluminum salts . third is the release of reactive oxygen species (ros) or mitochondrial dna from damaged mitochondria (zhou et al., , nakahira et al., ; shimada et al., ) . finally, viral rna or rna cleavage products generated by rnase l activate the nlrp inflammasome via the dexd/h-box helicase, dhx (allen et al., ; mitoma et al., ; chen et al., ; chakrabarti et al., ) . upon activation, the nlrp is recruited to the mitochondria via association with mitochondrial antiviral signaling (mavs) or mitofusin expressed on the outer mitochondrial membrane subramanian et al., ) ; these molecules then recruit the apoptosis-associated speck-like protein containing a caspase recruitment domain (asc) and pro-caspase- to form the nlrp inflammasome. this event activates the downstream molecule, caspase- , which catalyzes the proteolytic processing of pro-il- β and pro-il- into their active forms and stimulates their secretion (kayagaki et al., ; shi et al., ) . it is increasingly evident that nlrp detects rna viruses by sensing the cellular damage or distress induced by viroporins (ichinohe et al., ; ito et al., ; triantafilou et al., ; nieto-torres et al., ) , transmembrane pore-forming proteins, encoded by certain rna viruses; these proteins alter membrane permeability to ions by forming membrane channels (tan et al., ; chen and ichinohe, ) . a recent study shows that the sars-cov e protein, which comprise only amino acids, forms ca + -permeable ion channels and activates the nlrp inflammasome (nieto-torres et al., ) . although the e and a proteins of sars-cov, which comprise amino acids and contain three transmembrane domains (zeng et al., ; lu et al., ) , are thought to act as na + /k + and k + channels, respectively (wilson et al., ; lu et al., ; torres et al., ; parthasarathy et al., ; pervushin et al., ; wang et al., ) , the role of the a protein in activating the nlrp inflammasome remains unknown. here, we examined the role of the a protein in activating the nlrp inflammasome. six-week-old female c bl/ mice were purchased from the jackson laboratory. all animal experiments were approved by the animal committees of the institute of medical science (the university of tokyo). bone marrow-derived macrophages (bmms) were prepared as described previously (ichinohe et al., ) . in brief, bone marrow was obtained from the tibia and femur by flushing with dulbecco's modified eagle's medium (dmem; nacalai tesque). bone marrow cells were cultured for days in dmem supplemented with % l cell supernatant containing macrophage colony-stimulating factor, % heat-inactivated fetal bovine serum (fbs), and l-glutamine ( mm) at • c/ % co . hek ft cells (a human embryonic kidney cell line) and hela cells (a human epithelial carcinoma cell line) were maintained in dmem supplemented with % fbs, penicillin ( units/ml), and streptomycin ( µg/ml) (nacalai tesque). mdck cells (madin-darby canine kidney cells) and ht- cells (a human fibrosarcoma cell line) were grown in eagle's minimal essential medium (e-mem; nacalai tesque) supplemented with % fbs, penicillin ( units/ml), and streptomycin ( µg/ml) (nacalai tesque). influenza a virus strain a/pr (h n ) was grown at • c for days in the allantoic cavities of -day-old fertile chicken eggs (ichinohe et al., ) . the viral titer was quantified in a standard plaque assay using mdck cells (pang et al., ) . plasmids cdnas encoding the e and m proteins of sars-cov frankfurt strain (matsuyama et al., ) were obtained by reverse transcription and pcr of total rna extracted from sars-covinfected vero cells, followed by pcr amplification using specific primers. pcdna . d- a-v his was provided by ming-fu chang (national taiwan university college of medicine, taipei, taiwan). to generate the plasmids plenti -e-v his, plenti - a-v his, and plenti-m-v his, cdna fragments of e, a, and m were amplified from pcdna . d-e-v his, pcdna . d- a-v his, and pcdna . d-m-v his using specific primer sets and then ligated into plenti -topo vectors (invitrogen). to generate plasmids pca -flag-e, pca -flag- a, and pca flag-m, pca -ha-e, pca -ha- a, and pca -ha-m, cdna fragments of e, a, and m were amplified from pcdna . d-e-v his, pcdna . d- a-v his, and pcdna . d-m-v his using specific primer sets, digested with ecor i and not i, and subcloned into the ecor i-not i sites of the pca -flag-asc plasmid or pca -ha-m plasmid, respectively (ito et al., ) . to construct plasmids expressing the e mutant v f, the mutated e fragments were amplified by inverse pcr with wildtype e-containing plasmids and specific primer sets. the pcr products were cleaved by dpn i, ligated in a ligase-and t kinase-containing reaction and then transformed into dh α competent cells (toyobo). to construct plasmids expressing the a mutant a-cs, fragments were amplified from wildtype a-containing plasmids using a-specific primer sets and transformed as described above. hek ft cells were seeded in -well cluster plates and transfected with µg plenti -e/ a/m-v his, plenti-gfp (green fluorescent protein), or plenti-m using polyethylenimine (pei) max. at h post-transfection, the cells were lysed with ripa buffer ( mm tris-hcl, % np- , . % sodium dodecyl sulfate (sds), mm nacl and mm edta). and the lysates were subjected to sds-polyacrylamide gel electrophoresis (page) followed by electroblotting onto polyvinylidene difluoride (pvdf) membranes. the membranes were incubated over night with mouse anti-v -tag (r - , invitrogen), mouse anti-influenza a virus m ( c , abcam), mouse anti-gfp (gf , nacalai tesque), or rabbit antitubulin (dm a, santa cruz) antibodies, followed by horseradish peroxide-conjugated anti-mouse igg (jackson immuno research laboratories) or anti-rabbit igg (invitrogen). after washing times with washing buffer ( . % tween- /pbs), the membranes were exposed using chemi-lumi one super (nacalai tesque), and the chemiluminescent signals were captured by an imagequant las- mini apparatus (ge healthcare). to generate lentiviruses expressing v -tagged sars-cov e, a, and m proteins, the full-length cdna encoding each viral protein was cloned into the plenti . /v -topo vector (invitrogen) using the following primers: sars-cov e forward, -caccatgtactcattcgtttcgga- , and reverse, -gaccagaagatcaggaactc- ; sars-cov a forward, caccatggatttgtttatgagatt- , and reverse, -caaaggcacgctagtagtcg- ; sars-cov m forward, -caccatggcagacaacggtactat- , and reverse, -ctgtactagcaaagcaatat- . sub-confluent monolayers of hek ft cells seeded in a collagen-coated dish ( cm in diameter) were transfected with µg of plenti . /v -topo vector expressing each viral protein or egfp together with virapower packaging mix (invitrogen) using lipofectamine (invitrogen). the supernatants containing lentiviruses were harvested and filtered through a . µm filter (millipore) at - h post-transfection (ito et al., ) . the lentiviral titer was then quantified using ht- cells as described previously . bone marrow-derived macrophages were plated at a density of × in -well plate and infected with a/pr influenza virus or lentivirus at a multiplicity of infection (moi) of or . for h, respectively. then, bmms were stimulated with µg/ml of lps and cultured for additional h in complete media. supernatants were collected at h post-infection and centrifuged to remove cell debris. the amount of il- β in the supernatants was measured in an enzyme-linked immunosorbent assay (elisa) using paired antibodies (ebioscience) (ichinohe et al., . to clarify the cellular localization of the wild-type and mutant a proteins of sars-cov, hela cells were cultured on coverslips and transfected with µg of pca -flag- a or pcd -flag- a-cs together with . µg of er-mcherry or dsred-golgi (ito et al., ) . at h post-transfection, cells were fixed with % paraformaldehyde and permeabilized with % triton x- /pbs. after washing with pbs and blocking with % bsa/pbs, the cells were incubated with a mouse anti-flag antibody (m , sigma) followed by incubation with alexa fluor -conjugated goat anti-mouse igg (h+l) (life technologies). to observe the cellular distribution of nlrp in the e-or a-expressing cells, hela cells were cultured on coverslips and transfected with µg of pca -ha-e, pca -ha-ev f, pca -ha- a, pca -ha- a-cs, or pca control vector together with . µg of pca -nlrp . at h post-transfection, cells were fixed and permeabilized with % paraformaldehyde and % triton x- /pbs. after washing and blocking, the cells were incubated with rabbit anti-ha ( , mbl) and mouse anti-nlrp (cryo- ; adipogen) antibodies, followed by alexa fluor -conjugated goat anti-rabbit igg (h+l) and alexa fluor -conjugated goat anti-mouse igg (h+l) (life technologies). fluorescent signals were observed by confocal microscopy (a r + , nikon). statistical significance was tested using a two-tailed student's t-test. p-values < . were considered statistically significant. we previously demonstrated that the influenza virus m protein (a proton-selective ion channel), its h g mutant (which has lost its proton selectivity and enables the transport of other cations such as na + and k + ), and the emcv b protein (a ca + channel) stimulates nlrp inflammasome-mediated il- β secretion (ichinohe et al., ; ito et al., ) . in addition, the sars-cov e protein acts as a ca + -permeable ion channels that activates the nlrp inflammasome (nieto- torres et al., ) . the fact that a protein of sars-cov acts as viroporin prompted us to examine whether it also triggers inflammasome activation. thus, we first generated lentivirus plasmids expressing v -tagged proteins and confirmed their expression in hek ft cells by immunoblot analysis (figures a-c) . we next transduced lipopolysaccharide (lps)-primed bmms with the lentiviruses expressing the sars-cov e, a, m, influenza virus m , or emcv b proteins. consistent with previous reports (ichinohe et al., figure d) . similarly, the lentiviruses expressing the sars-cov e or a proteins stimulated il- β release from lps-primed bmms ( figure d) . furthermore, il- β secretion from lpsprimed bmms co-infected with e-and a-expressing lentiviruses was significantly higher than that from sars-cov e-expressing lentivirus-infected cells ( figure e) . these data indicated that the expression of sars-cov viroporin a is sufficient to stimulate il- β secretion by lps-primed bmms. previous studies demonstrated that the n-terminal amino acids of the sars-cov e protein are important for ion channel formation, and that mutations n a and v f [located in the transmembrane domain (from amino acid residues - )] prevent ion conductivity (wilson et al., ; torres et al., ; verdia-baguena et al., ) . in addition, the sars-cov a protein contains a cysteine-rich domain (amino acid residues - ) that is involved in the formation of a homodimer to generate the ion channel (lu et al., ; chan et al., ) . thus, mutation of the cysteine-rich domain blocks the ion conductivity by the a protein (chan et al., ) . to this end, we substituted amino acids cys- , cys- , and cys- within the cysteine-rich domain of the sars-cov a protein with serine to generate a lentivirus expressing the ion channel activity-loss mutant, a-cs (chan et al., ; figure a) . to test whether the ion channel activity of the sars-cov a protein is required to stimulate secretion of il- β, we transduced lpsprimed bmms with lentiviruses expressing the sars-cov e, v f, a, a-cs, or m proteins. consistent with a previous report (nieto -torres et al., ) , we found that the v f mutant lentivirus failed to stimulate il- β release from bmms ( figure b) . notably, the a-cs mutant completely abrogated il- β secretion (figure b) , suggesting that the ion channel activity of the a protein is required for sars-cov a-induced il- β secretion. figure | nlrp inflammasome activation by sars-cov a. hela cells were transfected with the expression plasmid encoding nlrp and that encoding ha-tagged sars-cov a, a-cs, e, or v f, and by with a confocal microscope. scale bars, µm. data are representative of at least three independent experiments. next, we determined the subcellular localization of the sars-cov a protein using confocal microscopy. when the sars-cov cell-free supernatants were collected at h (lentiviruses) or h (atp) post-infection or stimulation, and analyzed for il- β by elisa. data are representative of at least three independent experiments, and indicate the mean ± sd; * * p < . and * * * p < . . a protein was expressed in hela cells, we observed two main distribution patterns. consistent with previous reports (yu et al., ; yuan et al., ) , the a protein localized to the golgi apparatus ( figure a ). in addition, the a proteins concentrated in spot structures, which mainly localized to the endoplasmic reticulum (er) (figure b ). by contrast, the a-cs mutant was concentrated in the golgi apparatus rather than in the er and did not form spot structures (figures a,b) . we next examined the intracellular localization of nlrp . activation of the nlrp inflammasome led to a redistribution from the cytosol to the perinuclear space, a process considered as a hallmark of nlrp activation (zhou et al., ; ito et al., ; johnson et al., ; moriyama et al., ) . although cells expressing the ion channel activity-loss mutants a-cs or v f uniformly expressed nlrp throughout the cytoplasm, it was redistributed to the perinuclear region in sars-cov a-or e-expressing cells (figure ) . together, these data provide evidence that the ion channel activity of the sars-cov a protein is essential for triggering the nlrp inflammasome. both k + efflux and ros production are involved in the il- β release induced by the sars-cov a protein finally, we investigated the mechanism by which sars-cov a triggers nlrp inflammasome activation. a previous study showed that the a protein of sars-cov acts as a k + channel (lu et al., ) . in addition, k + efflux is a well-known activator of the nlrp inflammasome (mariathasan et al., ; petrilli et al., ) . these observations prompted us to examine whether k + efflux is required for a-mediated il- β secretion. to this end, bmms in k + -rich medium were infected with influenza a virus or lentiviruses expressing the sars-cov e or a proteins. in agreement with a previous result (ichinohe et al., ) , we found that il- β secretion caused by influenza virus was completely blocked when the extracellular k + concentration was increased to mm ( figure a) . the inhibitory effect of the k + -rich medium was also observed when cells were stimulated with lentiviruses expressing the sars-cov e or a proteins ( figure b ). since mitochondrial ros are important for nlrp inflammasome activation (nakahira et al., ; zhou et al., ) , we next stimulated bmms with extracellular atp or lentiviruses expressing the sars-cov e or a proteins in the presence or absence of the antioxidant, mito-tempo, a scavenger that is specific for mitochondrial ros trnka et al., ) . as reported previously (nakahira et al., ; ito et al., ) , treatment of bmms with mito-tempo completely blocked il- β secretion in response to atp ( figure a) . similarly, il- β release induced by the sars-cov e and a proteins was significantly inhibited by mito-tempo ( figure b) . these observations indicate that the sars-cov a protein disrupts intracellular ionic concentrations and causes mitochondrial damages, thereby activating the nlrp inflammasome. in summary, we found that the ion channel activity of sars-cov a protein is essential for activation of the nlrp inflammasome. in addition, both k + efflux and mitochondrial ros production are required for sars-cov a-mediated il- β secretion. thus far, several models have been proposed to explain nlrp inflammasome activation by rna viruses. first, viral rna or rna cleavage products generated by rnase l activate the nlrp inflammasome via the dexd/h-box helicase, dhx (allen et al., ; mitoma et al., ; chen et al., ; chakrabarti et al., ) . second, viroporins encoded by rna viruses activates the nlrp inflammasome (ichinohe et al., ; ito et al., ; triantafilou et al., ; nieto-torres et al., ) . in the case of influenza virus, the proton-selective m ion channel in the acidic trans-golgi network activates the nlrp inflammasome (ichinohe et al., ) . interestingly, an m mutant in which histidine was substituted with glycine at position (h g), causing loss of proton selectivity, enables transport of other cations (i.e., na + and k + ), thereby leading to enhanced secretion of il- β from lps-primed bmms and dendritic cells when compared with the wild-type m protein. in addition, the b proteins of emcv, poliovirus, enterovirus (ev ), and human rhinovirus (a member of the picornaviridae family) triggers nlrp inflammasome activation by inducing ca + flux from the er and golgi compartments (ito et al., ; triantafilou et al., ) . furthermore, hepatitis c virus stimulates nlrp inflammasome-mediated il- β production though its p viroporin (negash et al., ; farag et al., ) . third, a recent study has demonstrated that the d protein of ev directly interacts with nlrp to facilitate the assembly of nlrp inflammasome complex (wang et al., ) . in the case of sars-cov, the viroporin e forms forms ca +permeable ion channels and activates the nlrp inflammasome (nieto-torres et al., ) . in addition, another viroporin a was found to induce nlrp inflammasome activation (yue et al., ) . although alanine substitution at cys- , which is required for dimer or tetramer formation (lu et al., ) , still allows activation of the nlrp inflammasome by interacting with caspase- (yue et al., ) , the ion channel activity-loss mutant a-cs (cys-to-ser substitution at positions cys- , cys- , and cys- ) (chan et al., ) completely abrogated il- β secretion from lps-primed bmms, suggesting that the a protein of sars-cov has the ability to induce the nlrp inflammasome activation by multiple mechanisms. previous studies show that the a protein of sars-cov is localized to the plasma membrane (minakshi and padhan, ) and acts as a k + channel (lu et al., ) , thereby (presumably) stimulating the k + efflux at the plasma membrane. indeed, we found that il- β secretion caused by the a protein was significantly inhibited when the extracellular k + concentration increased to mm. although it remains unclear whether another viroporin a of sars-cov (castano-rodriguez et al., ) activates the nlrp inflammasome, these data highlights the importance of viroporins in sars-cov-induced nlrp inflammasome activation. a better understanding of the mechanism that governs the nlrp inflammasome will facilitate the development of more effective interventions for the treatment of infectious diseases and increase our understanding of viral pathogenesis. the nlrp inflammasome mediates in vivo innate immunity to influenza a virus through recognition of viral rna the role of potassium in inflammasome activation by bacteria inflammasomes: current understanding and open questions role of severe acute respiratory syndrome coronavirus viroporins e, a, and a in replication and pathogenesis rnase l activates the nlrp inflammasome during viral infections the ion channel activity of the sars-coronavirus a protein is linked to its pro-apoptotic function response of host inflammasomes to viral infection hcv genomic rna activates the nlrp inflammasome in human myeloid cells identification of a novel coronavirus in patients with severe acute respiratory syndrome nlrp inflammasomes are required for atherogenesis and activated by cholesterol crystals the p viroporin of the hepatitis c virus contributes to liver inflammation by stimulating production of interleukin- beta the pyroptosome: a supramolecular assembly of asc dimers mediating inflammatory cell death via caspase- activation aetiology: koch's postulates fulfilled for sars virus the nalp inflammasome is involved in the innate immune response to amyloid-beta expression of elevated levels of pro-inflammatory cytokines in sars-cov-infected ace + cells in sars patients: relation to the acute lung injury and pathogenesis of sars silica crystals and aluminum salts activate the nalp inflammasome through phagosomal destabilization critical functions of priming and lysosomal damage for nlrp activation inflammasome recognition of influenza virus is essential for adaptive immune responses influenza virus activates inflammasomes via its intracellular m ion channel mitochondrial protein mitofusin is required for nlrp inflammasome activation after rna virus infection encephalomyocarditis virus viroporin b activates nlrp inflammasome a mitochondria-targeted triphenylphosphoniumconjugated nitroxide functions as a radioprotector/mitigator herpes simplex virus infection induces activation and subsequent inhibition of the ifi and nlrp inflammasomes the role of pattern-recognition receptors in innate immunity: update on toll-like receptors caspase- cleaves gasdermin d for non-canonical inflammasome signalling a novel coronavirus associated with severe acute respiratory syndrome newly discovered coronavirus as the primary cause of severe acute respiratory syndrome severe acute respiratory syndrome-associated coronavirus a protein forms an ion channel and modulates virus release cryopyrin activates the inflammasome in response to toxins and atp protease-mediated enhancement of severe acute respiratory syndrome coronavirus infection toll-like receptors and innate immunity the yxxphi motif within the severe acute respiratory syndrome coronavirus (sars-cov) a protein is crucial for its intracellular transport the dhx rna helicase senses cytosolic rna and activates the nlrp inflammasome the rna-and trim -binding domains of influenza virus ns protein are essential for suppression of nlrp inflammasome-mediated il- beta secretion k(+) efflux is the common trigger of nlrp inflammasome activation by bacterial toxins and particulate matter critical role for calcium mobilization in activation of the nlrp inflammasome autophagy proteins regulate innate immune responses by inhibiting the release of mitochondrial dna mediated by the nalp inflammasome il- beta production through the nlrp inflammasome by hepatic macrophages links hepatitis c virus infection with liver inflammation and disease severe acute respiratory syndrome coronavirus e protein transports calcium ions and activates the nlrp inflammasome il- r signaling in dendritic cells replaces pattern-recognition receptors in promoting cd (+) t cell responses to influenza a virus structural flexibility of the pentameric sars coronavirus envelope protein ion channel coronavirus as a possible cause of severe acute respiratory syndrome immunopathogenesis of coronavirus infections: implications for sars structure and inhibition of the sars coronavirus envelope protein ion channel activation of the nalp inflammasome is triggered by low intracellular potassium concentration the nlrp inflammasome: a sensor for metabolic danger? cleavage of gsdmd by inflammatory caspases determines pyroptotic cell death oxidized mitochondrial dna activates the nlrp inflammasome during apoptosis the adaptor mavs promotes nlrp mitochondrial localization and inflammasome activation characterization of viral proteins encoded by the sars-coronavirus genome conductance and amantadine binding of a pore formed by a lysine-flanked transmembrane domain of sars coronavirus envelope protein rhinovirus-induced calcium flux triggers nlrp and nlrc activation in bronchial cells antioxidant properties of mitotempol and its hydroxylamine nlrp inflammasome activation: the convergence of multiple signalling pathways on ros production? coronavirus e protein forms ion channels with functionally and structurally-involved membrane lipids viral proteins function as ion channels ev d protein binds with nlrp and enhances the assembly of inflammasome complex sars coronavirus e protein forms cation-selective ion channels identification of a novel protein a from severe acute respiratory syndrome coronavirus subcellular localization and membrane association of sars-cov a protein sars-coronavirus open reading frame- a drives multimodal necrotic cell death characterization of the a protein of sars-associated coronavirus in infected vero e cells and sars patients thioredoxininteracting protein links oxidative stress to inflammasome activation a role for mitochondria in nlrp inflammasome activation i-yc and ti designed the study and wrote the manuscript. i-yc and mm performed the experiments. i-yc, mm, and ti analyzed the data. m-fc provided reagents and advice. all authors reviewed the manuscript. we thank dr. matsuyama (national institute of infectious diseases, tokyo, japan) for providing the total rna extracted from sars-cov-infected vero cells. the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © chen, moriyama, chang and ichinohe. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- -w mvk x authors: nasir, arshan; caetano-anollés, gustavo title: identification of capsid/coat related protein folds and their utility for virus classification date: - - journal: front microbiol doi: . /fmicb. . sha: doc_id: cord_uid: w mvk x the viral supergroup includes the entire collection of known and unknown viruses that roam our planet and infect life forms. the supergroup is remarkably diverse both in its genetics and morphology and has historically remained difficult to study and classify. the accumulation of protein structure data in the past few years now provides an excellent opportunity to re-examine the classification and evolution of viruses. here we scan completely sequenced viral proteomes from all genome types and identify protein folds involved in the formation of viral capsids and virion architectures. viruses encoding similar capsid/coat related folds were pooled into lineages, after benchmarking against published literature. remarkably, the in silico exercise reproduced all previously described members of known structure-based viral lineages, along with several proposals for new additions, suggesting it could be a useful supplement to experimental approaches and to aid qualitative assessment of viral diversity in metagenome samples. the last few years have dramatically increased our knowledge about viral systematics and evolution. the discoveries of "giant" viruses (la scola et al., ; arslan et al., ; philippe et al., ; legendre et al., legendre et al., , and their virophages (la scola et al., ; desnues et al., ; gaia et al., ; levasseur et al., ) along with accumulation of large-scale protein structure and function data enabled testing hypotheses regarding the origin, classification, and evolution of the viral supergroup. this led to data-driven hypotheses of viral evolution (koonin et al., ; and new schemes for classifying viruses, different from traditional classification approaches that use genome features (baltimore, ) or host/geographical preferences (king et al., ) . for example, bamford and coworkers proposed to define novel viral lineages based on the three-dimensional ( d) structural similarities of major viral capsid/coat proteins and virion assembly pathways (abrescia et al., ) . under this classification, the many known viral families infecting distantly related hosts were pooled into four major viral lineages, the picornavirus-like lineage, the prd /adenovirus-like lineage, the hk -like lineage, and the btv-like lineage (abrescia et al., ) . these lineages were mainly described for icosahedral viruses however helical and enveloped viruses are also believed to fall into a limited number of lineages (abrescia et al., ) . interestingly, member viruses of the prd /adenovirus and hk -like lineages infect species in all three domains of cellular life, archaea, bacteria, and eukarya (woese et al., ) . stark differences in membrane composition and cellular biology exist among cellular domains that likely hinder horizontal transfer of viruses between domains of life (nasir et al., . thus, the structural and genetic similarities of viruses infecting the three cellular domains suggest they likely originated prior to the origin of modern diversified cells (benson et al., ; krupovič and bamford, ) . this scenario is also supported by our recent phylogenomic exploration of the origin of viral and cellular proteomes . while the member viruses within a lineage exhibit strong d structural similarities in capsid/coat fold architectures (or principles in constructing a functional virion) regardless of the viral replicon (i.e., dna or rna) and/or infected host type, the lineages however are believed to be unrelated to each other indicating the polyphyletic origin of viruses (bamford, ) . the conservation of protein structure over long evolutionary distances (chothia and lesk, ; caetano-anolles and caetano-anolles, ; illergård et al., ; abroi and gough, ; caetano-anollés and nasir, ; lundin et al., ) forms the backbone of structure-based viral classification (abrescia et al., (abrescia et al., , . this concept is especially applicable to viral capsid proteins as there is strong evolutionary pressure to (abrescia et al., ; , or keyword searches. where available ( out of ), the distribution (%) in the proteomes of archaea (a), , bacteria (b), and eukarya (e) are also given along with assignment to one of the four experimentally defined lineages (abrescia et al., ) or to novel or "other/unclassified" category (see text). fsfs highlighted in bold were not detected in any of the studied , viral proteomes in . maintain the overall morphology of the virus particle (abrescia et al., ) . moreover, the capsid is the only feature that distinguishes plasmids, integrated viral genomes, and other "naked" genetic elements from bona fide viruses (abrescia et al., ) . for these reasons, the capsid has been termed the virus "self " (bamford, ) and viruses have been referred to as "capsid-encoding organisms" (in comparison to ribosomeencoding cellular organisms) (raoult and forterre, ) . the idea is strengthened by the fact that only a limited number of virion morphotypes may be considered geometrically and energetically favorable (bamford et al., ) . indeed, a quick glance of the structural classification of proteins (scop) database (andreeva et al., ; fox et al., ) reveals only fold superfamilies (fsfs) corresponding to keywords "capsid" or "coat" (table ) . remarkably, these fsfs are either very rare or completely absent in cellular proteomes . these observations identify the capsid as a reliable marker for improving or revising the current taxonomy of viruses. the availability of the scop database, a "gold standard" in the structural classification of proteins, and development of algorithms required to scan viral proteins against hidden markov model (hmm) libraries of known protein structures (gough et al., ; gough and chothia, ) now enable us to computationally detect the "type" of capsid fold present in viruses. here we survey capsid/coat related fsfs in the proteomes of , completely-sequenced viruses (corresponding to all seven known replicon types) with the broad objectives of characterizing each known viral lineage (benchmarked against abrescia et al., ) and suggesting novel members for existing lineages (or even novel lineages). remarkably, our computational exercise recovered the previously experimentally defined viral lineages along with proposals for new additions, suggesting it could be a reliable supplement to experimental approaches for rapid identification of viral lineages, for example, in metagenomic samples. accurate assignment of viruses into known lineages will be especially invaluable for novel viruses for which little is known and experimental characterization is technically challenging. importantly, and despite the great genetic diversity and host biases observed among modern viruses (nasir et al., ; koonin et al., ) , virion construction principles appear generally and relatively more conserved in evolution than viral gene sequences or host-associated preferences and present a more viable classification approach for modern viruses (bamford et al., ; bamford, , ) , in addition to providing insights about viral origins and evolution forterre, ) . capsid/coat related fsfs were first extracted from scop ver. . (last updated february ) using keywords "capsid" and "coat." this yielded capsid and coat related fsfs (table ) . because, keyword search is directly dependent on how fsfs are described in scop (e.g., procapsid), this likely missed several genuine capsid/coat related fsfs. therefore, we mapped the protein data bank (pdb) codes corresponding to the four experimentally-defined viral lineages (abrescia et al., ) to scop . to get their fsf descriptions. four pdb entries were not present in scop . ( yue, c b, bbd, and vvf) and thus were not considered. the remaining pdb entries corresponded to new fsfs, out of which four (b. . , b. . , e. . , and i. . ) were new additions to the list (i.e., were not detected earlier by keyword search). the list was further refined by looking for scop relatives for each fsf (i.e., other fsfs part of the same fold). as a result, four more fsfs b. . , b. . , b. . , and b. . were added to the list, as scop relatives of b. . . the final list included fsfs ( keywords, scop relatives, and from abrescia et al., ) , out of which were detected in our sampled viral and cellular proteomes (highlighted in boldface in table ). throughout the manuscript, fsfs are named using scop concise classification strings (ccs) for quick identification. for example, b. . fsf belongs to scop class "b" (i.e., all-beta proteins), fold no. , and fsf no. in that fold and class. viral and cellular proteome data and fsf assignments were taken from . fsf information was available for , viruses belonging to , dsdna, ssdna, dsrna, plus-ssrna, minus-ssrna, ssrna-rt, and dsdna-rt viruses and , cellular organisms belonging to archaea, , bacteria, and eukarya. viral and cellular proteomes that gave a significant hit (e < . ) to any of the capsid/coat related fsfs (table ) were kept for taxonomic assignment and manual inspection. a total of , manually curated and verified proteins tagged to "virion" keyword in uniprotkb keywords category "cellular component" were downloaded from http://www.uniprot.org/ keywords/ (november , ) . these proteins corresponded to all known viral replicons including dsdna (n = , ), ssdna ( ), dsrna ( ), plus-ssrna, ( ), minus-ssrna ( , ), dsdna-rt ( ), ssrna-rt ( ), and in addition, satellite viruses ( ), unclassified virophages, phages, and viruses ( ), and deltaviruses ( ). these proteins were scanned against superfamily hmms (gough et al., ; gough and chothia, ) for recognition of fsf domains using a stringent e-value cutoff < . . we examined how the known capsid/coat related fsfs, identified via scop or from literature, corresponded to experimentally defined viral lineages (abrescia et al., ) and examined their distribution in the , viral (corresponding to seven viral replicons) and , cellular (archaea, bacteria, and eukarya) proteomes. the picornavirus-like lineage is characterized by the "jellyroll" or "β-barrel" fold, which is commonly seen in rna viruses (abrescia et al., ) . it is the largest defined viral lineage, including members from plus-ssrna (bromoviridae, caliciviridae, comoviridae, dicistroviridae, luteoviridae, nodaviridae, picornaviridae, sequiviridae, tetraviridae, tombusviridae, tymoviridae), dsrna (birnaviridae), ssdna (microviridae, parvoviridae), and dsdna (papillomaviridae, and polyomaviridae) viruses but no minus-ssrna and retrotranscribing viruses, according to (abrescia et al., ) . of these, comoviridae and sequiviridae are now classified under secoviridae, which constitutes one of the five families in the viral order picornavirales (other families being dicistroviridae, iflaviridae, marnaviridae, and picornaviridae). the "jelly-roll" fold has a topology of eight β-strands organized into two antiparallel sheets and is represented by the "nucleoplasmin-like vp (viral coat and capsid proteins)" fold (b. . ) in scop. the b. fold in the scop hierarchy includes children fsfs (that are not necessarily related in evolution according to scop definitions): (i) "phm/pngase f" fsf (b. . ) involved in oxidation-reduction metabolic processes (not detected in any of our sampled viral proteomes), (ii) "group ii dsdna viruses vp" fsf (b. . ), which is the "double β-barrel" fold signature of the prd /adenovirus-like lineage (read below), (iii) "nucleoplasmin-like core domain" fsf (b. . ) involved in the assembly of nucleosomes in cells, and (iv-vii) fsfs b. . , b. . , b. . , and b. . (figure ) that define the picornavirus-like lineage and are individually described below. the "positive stranded ssrna viruses" fsf (b. . ) was detected mostly in rna viruses including plus-ssrna ( families), dsrna (birnaviridae), and the novel addition of minus-ssrna (ophioviridae) viruses (table , figure for virion morphotypes). thus, our computational approach extended the picornavirus-like lineage to also include minus-ssrna viruses. experimental work will be required to confirm if these viruses truly belong to this lineage. other novel additions included polemoviruses and sobemoviruses (plus-ssrna viruses that are yet to be assigned to a viral family), eight unclassified plus-ssrna viruses, hepeviridae (family of plus-ssrna viruses that includes the human and avian hepatitis e viruses), iflaviridae and marnaviridae (thus completing the detection of all five picornavirales families in our computational assignments) and one dsdna virus belonging to myoviridae (prochlorococcus phage p-ssm ). interestingly, myoviridae possess the so-called "hk " capsid/coat related fold also seen in eukaryotic herpesviridae. together they constitute the hk -like lineage (read below) and are believed to be unrelated to the picornavirus-like lineage. thus, assignment of fsf b. . to myoviridae could either be a false hit or suggests that the two lineages could (in fact) be distantly related. for example, unlike other dsrna viruses that constitute the btv-like lineage (read below), birnaviridae share genomic (birghan et al., ) and structural similarities (coulibaly et al., ) with plus-ssrna viruses. based on our assignments, birnaviridae fall into the picornavirus-like lineage and possess the b. . fsf hallmark of plus-ssrna viruses. however, the arrangement of the major capsid protein in birnaviruses is similar to the other members of the btv-like lineage (abrescia et al., ) casting doubts on its accurate affiliation. perhaps, mixing of ancestral viruses of the picronavirus-like and btv-like lineages led to modern birnaviruses (coulibaly et al., ) or alternatively represent the evolutionary link between the two lineages. the "ssdna viruses" fsf (b. . ) was detected in many ssdna viruses of the microviridae and parvoviridae families. the capsid and spike proteins (f and g) of bacteriophage phix (microviridae) possess the "jelly-roll" fold and were reliably matched to b. . (figure ). in addition, b. . was also detected in an unclassified ssdna virus dragonflyassociated microphage possibly linking this virus to the lineage. microviridae also possess another capsid/coat related fsf "scaffolding protein gpd of bacteriophage procapsid" (a. . ) that acts as a molecular chaperone and becomes part of the external scaffold of viral procapsid, which is later removed to release the mature virion (figure , dokland et al., ) . although a. . is not part of the mature virion, it was uniquely figure | pdb structures corresponding to fsfs of four experimentally defined viral lineages (abrescia et al., ) detected in microviridae and thus could still serve as marker to identify micorviridae members together with b. . . the "group i dsdna viruses" fsf (b. . ) includes coat and l proteins from polyomaviruses and papillomaviruses, both established members of the picornavirus-like lineage. finally, the "satellite viruses" fsf (b. . ) was detected in the circoviruslike genome rw_b virus (ssdna). it seems that the coat protein of this virus resembles the "jelly-roll" coat proteins of satellite viruses (e.g., satellite panicum mosaic virus), which harbor a typical "jelly-roll" fold but with up to - additional β-strands (ban et al., ) . thus, this fsf could be considered another specialized form of the "jelly-roll" fold. own (read below). importantly, the picornavirus-like lineage now includes viruses with all replicon types except two groups of retrotranscribing viruses and supports the idea that viruses with different replicons can share strong structural and molecular properties (bamford, ) . the exercise also revealed that structural relatives of the "jelly-roll" fold are found in cells (e.g., histone chaperones and metabolic folds dutta et al., ; liu et al., ; cheng and brooks, ) and thus it may not be a unique viral hallmark. however, none of the five putative picornavirus-like lineage associated fsfs (a. . , b. . , b. . , b. . , and b. . ) were detected in any of the archaeal proteomes while b. . was detected in roughly % of bacterial and % of eukaryotic proteomes, and b. . in only % of eukaryotic proteomes indicating rare presence in cellular proteomes ( table ) . these rare occurrences could (possibly) be episodes of virus-to-cell horizontal gene transfer (hgt) during infection (akita et al., ; sutter et al., ) . indeed, b. . , a hallmark of plus-sense rna viruses, was relatively widespread among eukaryotes ( % spread) consistent with previous knowledge that rna viral infections are common in eukaryotic species but absent in archaea and extremely rare in bacteria (nasir et al., koonin et al., ) . importantly, the host range of the picornavirus-like lineage is apparently restricted to bacteria and eukarya (not accounting for myoviridae that also infects archaea) and % of the viral families listed in (abrescia et al., ) were detected along with several new novel additions indicating the success of our computational survey. the prd /adenovirus-like lineage includes dsdna viruses that infect species in the three cellular domains of life. the prototype members include the human adenoviruses (adenoviridae), paramecium bursaria chlorella viruses (phycodnaviridae), the bacteriophage prd (tectiviridae), and the archaeal sulfolobus turreted icosahedral virus (turriviridae). the lineage is characterized by the "double jelly-roll" fold, which likely formed by the duplication of the "jelly-roll" fold (krupovič and bamford, ) . however, the "jelly-roll" and "double jelly-roll" folds are utilized differently in assembling capsids and hence form two distinct lineages (krupovič and bamford, ) . capsids of viruses belonging to the prd /adenovirus lineage are assembled in trimers consisting of two β-barrels arranged around a pseudo six-fold axis. the "double β-barrel" fold corresponds to "group ii dsdna viruses vp" fsf (b. . ) (figure ) and was detected in adenoviridae, asco/asfarviridae, iridoviridae, marseilleviridae, mimiviridae, phycodnaviridae, tectiviridae, and two unclassified dsdna viruses (micromonas pusilla virus t and ostreococcus lucimarinus virus olv ). notable exceptions from (abrescia et al., ) were of poxviridae, corticoviridae, and turriviridae. however, the "double βbarrel" protein domain in poxviruses only facilitates virion formation and does not become part of the capsid. in turn, the corresponding pdb entries ( bbd and vvf) for turriviridae and corticoviridae (identified from abrescia et al., ) were not part of scop and were thus missed by scop-based superfamily hmms (gough et al., ; gough and chothia, ) . thus, their absence is likely not due to failure of our approach but due to incomplete coverage of pdb in scop. new additions of asco/asfarviridae and mimiviridae were confirmed independently (krupovič and bamford, ) . the "double β-barrel" is apparently a virus hallmark and was detected in only % of eukaryotic proteomes ( table ) , suggesting it was likely acquired in the few cellular proteomes from their viruses by hgt. the hk -like lineage includes tailed viruses belonging to archaeal and bacterial caudovirales (myoviridae, podoviridae, and siphoviridae) and the eukaryotic herpesviridae (figure ) . the hk fold corresponds to two major fsfs, the "major capsid protein gp " (d. . ) from bacteriophage hk and the "major capsid protein vp " (e. . ) from herpes simplex virus (figure ) . it has been verified that the "floor" domain of herpesvirus vp and hk gp have similar structural organization and are evolutionarily related (baker et al., ) . moreover, a small tail similar to that of podoviridae has been detected in the herpesvirus capsid, further supporting their inclusion in the hk -like lineage (schmid et al., ) . in addition, the "head decoration protein d" (gpd, major capsid protein d) fsf (b. . ) was also detected exclusively in siphoviridae and one unclassified caudovirales (providencia phage redjac). the b. . is a "beta-clip" fold that forms an incomplete barrel somewhat similar to the "jelly-roll" structure (figure ) . its main function is to decorate the head shell and stabilize the capsid (yang et al., ) . there were no additional scop relatives for either d. . or e. . , the two major markers for the hk -like lineage. however, b. . had six scop relatives: (i) "afp iii-like domain" (b. . ), a type of antifreezing protein possessing a compact fold composed of beta-strands (davies et al., ) (found in cells but not detected in any virus) (ii) "urease, beta-subunit" (b. . ), a subunit of urease enzyme known to hydrolyze urea into carbon dioxide and ammonia (takishima et al., ) (again detected in cells but not in any virus) (iii) "dutpase-like" (b. . ), a metabolic enzyme near-universal in cells and also detected in a wide array of viruses, (iv) "tlp , baculovirus telokin-like protein" (b. . ), a virus-specific protein of unknown function expressed late in baculoviruses (raynes et al., ) , (v) "moea c-terminal domain-like" (b. . ), a widespread protein in cells (but not in viruses) that is involved in molybdopterin cofactor synthesis (xiang et al., ) , and (vi) the "set domain" (b. . ) found in both cells and viruses and involved in a range of metabolic and transport processes. the wide distribution of b. fold in cellular proteins (and its association with cell-related processes) suggests this fold was co-opted numerous times in evolution. perhaps, its unique presence in some viral proteins (i.e., b. . and b. . ) could be due to convergent evolution. fsf b. . however was exclusively detected in caudovirales and was completely absent in archaea and eukarya ( %) and only detected in out of , sampled bacterial proteomes ( . %) ( (canchaya et al., ) or possibly relics of ancient co-existence of viruses in primordial cells . indeed, the hk -like lineage is the second lineage after the prd /adenovirus lineage that includes viral members infecting all three cellular domains of life (abrescia et al., ) suggesting it originated prior to (or concurrently with) the diversification of cellular life (bamford, ; benson et al., ) . this lineage included three families of dsrna viruses, cystoviridae, reoviridae, and totiviridae (abrescia et al., ) . members of these families encode both an outer and inner capsid core. the inner core is evolutionarily conserved and is required within the host cell to avoid apoptotic response against foreign dsrna genomes (grimes et al., ) . the major core protein vp , which forms the inner shell of the bluetongue virus capsid, characterizes this lineage. about monomers of vp are packed with icosahedral symmetry following a rather unique pattern of subunit assembly. this arrangement was also detected in the saccharomyces cerevisiae virus l-a (totiviridae) (castón et al., ) and pseudomonas phage phi (cystoviridae) viruses (huiskonen et al., ) suggesting the architecture may be unique to dsrna viruses (abrescia et al., ) . vp is a multidomain protein containing different fsfs (figure ) . we discovered that "a virus capsid protein alpha-helical domain" (a. . ), "reovirus inner layer core protein p " (e. . ), and "l-a virus major coat protein" (e. . ) fsfs likely correspond to vp -like architectures, while the "outer capsid protein sigma " fsf (d. . ) was associated with the outer core of the reoviridae capsid. these fsfs were detected in the members of reoviridae and totiviridae (but not cystoviridae). birnaviruses, which also encode a dsrna genome, were classified in the picornavirus-like lineage because current knowledge dictates that they exhibit stronger affinity with the "jelly-roll" fold harboring viruses (abrescia et al., ) . another capsid/coat related fsf detected in reoviridae is the "viral protein domain" (b. . ). this protein is part of capsids in reoviridae (grimes et al., ; mathieu et al., ; vp and vp basak et al., ) but is also present in minus-ssrna (orthomyxoviridae) (rosenthal et al., ; ha et al., ) and plus-ssrna (members of nidovirales) viruses. structurally, the domain exhibits similarity to the "jelly-roll" fold. thus, "jelly-roll"-like fold structures are seen in each of the four major structural lineages and also in some cellular proteins. consistent with the signature folds of the prd /adenovirus and hk -like lineages, none of the five fsfs described here (a. . , e. . , e. . , d. . , and b. . ) had any scop relatives and their presence in cellular proteomes was near negligible ( table ) . the host range of the btv-like lineage is restricted to eukaryotic organisms ( table ) . the ssrna-rt (retroviridae) and dsdna-rt (caulimoviridae and hepadnaviridae) (figure ) viruses were not part of any of the four lineages in either (abrescia et al., ) or our initial assignments (see above). retrotranscribing viruses are typically enveloped and their proteins are difficult to crystalize for structural studies. the capsid protein fold from retroviridae contains an n-terminal domain ( -helix bundle) involved in core formation and a c-terminal domain ( -helix bundle) involved in capsid dimerization (jin et al., ; campos-olivas et al., ) . these domains correspond to the "retrovirus capsid protein, n-terminal core domain" (a. . ) and the "retrovirus capsid dimerization domain-like" (a. . ) fsfs (figure ) and were detected in many viruses belonging to retroviridae (e.g., human immunodeficiency virus- ). in contrast, the capsid fold from hepadnaviridae (e.g., hepatitis b virus) is also helical ( helices) and obeys a t = icosahedral symmetry. this fold corresponds to the "hepatitis b viral capsid (hbcag)" fsf (a. . ) (figure ) and was detected in members of hepadnaviridae. it has been hypothesized that the c-terminal domain of hiv- capsid protein shows significant similarities to the hbv capsid protein suggesting that the two lineages could be evolutionarily related (zlotnick et al., ) . we note that the capsid fold of hepadnaviridae is arranged in an array-like structure where two long helices form a hairpin that dimerizes into a -helical bundle closely resembling the -helical bundle of retroviridae capsid (a. . ). however, retroviral fsfs (a. . and a. . ) did not group with the capsid fsf from hepadnaviridae (a. . ) according to scop classification. search against the dali server (holm and rosenstrom, ) also failed to detect any apparent structural homology between the two domains (wynne et al., ) . therefore, more work is required to establish if the capsids from retrotranscribing viruses form independent lineages or just one (i.e., retrotranscribing-like lineage?). however, capsids from both retroviridae and hepadnaviridae are helical and this is in sharp contrast to the β-sheet rich capsids typically found in other lineages. while the hepadnaviridae a. . fsf was completely absent in all cellular proteomes, the two retroviridae fsfs (a. . and a. . ) were exclusively detected in and % eukaryotic proteomes but none of the prokaryotic proteomes (table ) . again, virus-to-cell hgt cannot be ruled out considering retrotranscribing viruses are hitherto unknown to infect prokaryotes (nasir et al., koonin et al., ) . both a. . and a. . had no additional scop relatives. fsf a. . had two additional relatives: (i) "acp-like" (a. . ), and (ii) "colicin e immunity proteins" (a. . ), the former detected both in cells and viruses while the latter only in bacteria. because the three fsfs are unique to retrotranscribing viruses, they can serve as useful markers to fish retrotranscribing viruses from virome metagenome samples. other enveloped viruses such as flaviviridae are also hard to classify based on core capsid proteins. the virions of flaviviruses are composed of three proteins, c, e, and m (figure ) . the aggregation of c protein forms the nucleocapsid, which encloses the plus-ssrna genome of flaviviruses. this protein belongs to the "flavivirus capsid protein c" fsf (a. . , the sole member of the fold) that was not detected in any other family besides flaviviridae or in any of the sampled cellular proteomes (table , figure ) again indicating its reliability in characterizing viruses. however, there is indication that instead of the nucleocapsid core, surface glycoproteins involved in membrane fusion may be more similar to other enveloped viruses and could be better markers for taxonomy characterization (abrescia et al., ) . in addition to the fsfs described above that were benchmarked against previous work (abrescia et al., ) , several other capsid/coat related fsfs unique to some viral families were also detected. for example, another candidate for novel viral lineage could be the "rna bacteriophage capsid protein" fsf (d. . ) (figure ) that was detected in several rna viruses of bacteria (leviviridae) (figure ) . structurally, the d. . fsf is composed of -stranded β-sheet followed by two α-helices. it was not detected in any other viral family beside leviviridae (and only in . % eukaryotic proteomes) and thus could be used to characterize leviviruses (leviviridae-like lineage? also speculated to be a new lineage by abrescia et al., ) . similarly, the "nucleocapsid protein dimerization domain" fsf (d. . ) was detected in coronoviridae and arteriviridae, belonging to viral order nidovirales (cavanagh, ) and in none of the cellular proteomes. coronoviridae and arteriviridae are common pathogens of animals and humans (e.g., sars). structurally, the domain is composed of a dimer of mixed α and β secondary structures (figure ) . this fsf could therefore be used as bait to fish out additional members of nidovirales, especially useful in quick identification of re-emergence of known coronaviruses. in turn, fsf b. . represents the n-terminal domains (n and n ) of the gp minor coat protein of ssdna bacteriophages belonging to inoviridae. structurally, the domain resembles the β-barrel fold and is primarily involved in phage infection of e. coli. another domain detected exclusively in inoviridae is the "inovirus (filamentous phage) major coat protein" fsf (h. . ), which exhibits a "pseudo-fold" comprising of oligomers of short identical α-helices (figure ) . together, the major and minor coat proteins (negligible presence in cellular proteomes, table ) can perhaps characterize inoviruses (i.e., inovirus-like lineage?). finally, "tmv-like viral coat proteins" fsf (a. . ) was detected in several plus-ssrna viruses of plants that exhibit "linear" morphology (e.g., benyviruses, potyviridae, and virgaviridae). structurally, the domain is described as a -helical bundle by scop (figure ) . interestingly, the major capsid proteins of archaeal linear viruses (lipothrixiviridae and rudiviridae) are also characterized by unique -helix bundles at their c-terminus. however, the arrangement of helices between virgaviridae and archaeal viruses differs along with other genomic differences (prangishvili and krupovic, ; suggesting perhaps that archaeal linear viruses evolved independently from bacterial and eukaryal linear viruses. this leaves us with fsfs i. . , j. . , j. . , and i. . , for which no hits were detected in our set of sampled viral proteomes and relatively little information were available from both the scop and superfamily databases ( table , highlighted in boldface). fsf i. . is the low-resolution protein structure of "bacteriophage hk procapsid (prohead ii), " as defined by scop. thus, it could be pooled along with other fsfs that define the hk -like viral lineage, albeit with caution. in turn, fsf j. . is the "hepatitis c virus n-terminal capsid protein fragment - ." it is a synthetic structure that is yet to be published. whereas, j. . is the "ilarvirus coat protein n-terminal fragment" of alfalfa mosaic virus ysmv (plus-ssrna, bromoviridae). thus, it could be tentatively assigned to the picornavirus-like lineage. finally, fsf i. . is defined as "reovirus components" in scop. this fsf includes minor core protein lambda , outer capsid protein mu , and reovirus core proteins. this could perhaps also supplement member identification of btv-like viral lineage. as final check, we retrieved protein entries tagged to the "virion" keyword of "cellular component" category in uniprotkb (see methods). these proteins were broadly defined as "viral protein detected in the virion" and included several capsid, envelope, matrix, and tegument proteins in addition to proteins directly involved in capsid assembly and virion formation. the list also included several proteins that are packaged into viral capsids for successful replication of viral replicon inside cells, such as the rna-dependent-rna polymerase of minus-ssrna viruses and enzymes responsible for host cell membrane degradation during virus entry. these proteins therefore broadly point to an interesting set of proteins that are related to virions of viruses but not necessarily relevant for viral taxonomy. this is showcased by the fact that a total of fsfs were detected in these proteins indicating the diversity and breadth of the biological process of virion synthesis (table s ). a total of (out of , with the exclusion of the bacteriophage procapsid fsf a. . ) fsfs detected in our sampled proteomes ( table ) (table s ). in addition, the list included several ancient and widespread protein folds such as the p-loop containing ntp hydrolase and sam-dependent methyltransferases, among other proteins, that were (near)universal in cellular proteomes. in fact, and of these fsfs were detected in > % prokaryotic and eukaryotic proteomes, respectively, indicating a similar use of d structural designs in cellular organisms, perhaps for processes other than virion synthesis or revealing a strong link to the co-existence of viral and cellular ancestors . despite a significant number of mostly cellular proteins that share structural similarities to steps involved in capsid assembly and virion synthesis, the paucity of capsid-like shells in cells however remains surprising (read below). our computational approach enabled a quick scan of thousands of viral proteins against structure libraries and recovered the experimentally defined four major capsid-based viral lineages (abrescia et al., ) along with proposals for new structurebased lineage additions. only very few members were missing. this could be a result of using a stringent criterion in assigning fsfs to viral proteins (i.e., e < . ) or alternatively absence of corresponding entries of the rcsb pdb database (rose et al., ) in scop. importantly, results show that viruses with different replicons and proteome histories have capsids that are structurally very similar and that hmm-based assignment (gough et al., ; gough and chothia, ) reproduced the well-known viral lineages. moreover, only a limited number of unique capsid/coat related structures (n = ), mostly unique to a particular viral family or group, exist in the virosphere that can characterize viruses belonging to - known groups (table ) . because the discovery of unique protein folds has slowed down considerably in the past five years (e.g., , folds in scop . updated vs. , folds in scop . updated february ), we speculate that the capsid/coat related viral protein folds identified in our study is not far from the true diversity of virion structural components in nature. the recent drive in metagenome and virome sequencing will no doubt aid in isolating new viruses harboring novel capsid/coat related folds. however, based on the observation that capsid/coat proteins repeat in viruses, we speculate that between and viral lineages exist in nature and the real number is likely closer to the lower bound. remarkably, the majority of virus capsid/coat-related fsfs are either completely absent or rare in cellular organisms with exceptions likely representing virus-tocell hgt . these observations identify the capsid structure as a useful marker for defining viruses, functionally analogous and effective as s rrna for the detection of prokaryotic dna/rna in metagenome samples. three limitations of the computational approach however must be noted: first, some capsid/coat protein folds characterize large groups of viruses (e.g., several plus-ssrna virus families characterized by the "jelly-roll" fold) indicating low resolution in pinpointing the quantity and nature of viruses present in samples, while the others are unique to one family (e.g., leviviridae or retro-transcribing viruses) thus indicating significant utility in recognizing specific viral groups. thus, the quality of analysis is expected to vary from sample to sample. second, only a qualitative assessment of viral diversity (e.g., whether retrotranscribing viruses are likely to be present in samples or not?) seems possible utilizing capsid as taxonomic marker. this is however still cheaper than either shotgun sequencing of all nucleic acids present in metagenome samples or a hybridizationcapture approach of pulling down nucleic acids homologous to known viruses (wylie et al., ) . both approaches are costprohibitive for large number of samples simply because viruses possess replicons of at least seven types and exhibit high levels of sequence polymorphisms. third, morphological similarities in viruses can also result from convergent evolution, especially because there are only a limited number of "economical" ways to pack viral genomes. these arguments have been discussed elsewhere and were considered to be less likely (abrescia et al., ) . for example, in addition to sharing the same capsid fold in similar arrangement, some viral lineages also share common atpases that package the viral genome into the capsid. thus, additional properties favor vertical inheritance of the well-defined lineages (abrescia et al., ) . moreover, protein domains grouped into common fsfs are recognized by the existence of a conserved backbone formed by unique interactions between amino acid side chains. the odds of originating the same backbone independently and multiple times in evolution are considered to be very small ( . - % in gough, ) indicating convergence an exception and divergence the rule when evaluating similarities in structures (abrescia et al., ) . nevertheless, the four new candidate structure-based lineages proposed by our study should be considered putative since vertical origin of member viruses within these new lineages remains to be established. however, because fsfs identified by our study are exclusive to viral families described, they are still invaluable markers for recognition of viral families present in unknown samples (e.g., retrovirus identification via three marker fsfs, figure ) . importantly, the presence of an fsf is not the sole criterion to classify a viral family into a lineage. it needs to be supported by the use of the capsid fold in similar organization and other genomic evidence (where available). thus, it is important to consider both structural (capsid) and non-structural (polymerases and hydrolases) proteins when studying viral evolution (e.g., in . viral capsid-like architectures are relatively rare in cells. cheng and brooks iii recently calculated distances of structural relatives of viral capsid proteins to capsid-like proteins in cells for a large number of folds (cheng and brooks, ) . using a stringent criterion (distance < . ), they concluded that the majority of capsid-like cellular proteins possessed variants of the "jelly-roll" fold and that these proteins were part of multi-domain proteins, which likely restricted their assembly into capsidlike structures (cheng and brooks, ) . notable exceptions however are of bacterial carboxysomes that show morphological resemblance to viral capsids but utilize folds not detected in extant viral proteomes (yeates et al., (yeates et al., , and archaeal protein nanocompartments that store metabolic enzymes and utilize protein fold exhibiting strong homology to the hk fold (sutter et al., ) . one obvious shortcoming is the lack of classification for enveloped viruses, lack of viral representatives in the rcsb pdb database, and current biases toward sequencing economically and industrially important viruses (delwart, ) . these shortcomings however will naturally be overcome with the completion of ongoing and planned (meta)-genome sequencing projects. we expect that increased sequencing of novel viruses, from atypical habitats and hosts, a logical outcome of recent trends toward metagenomics and environmental sampling, can considerably bridge this gap in the near future. we therefore conclude that while the proposal of capsid structure-based viral classification seems promising, more work is required to establish boundaries within the virosphere. remarkably, the hmm-based computational exercise impressively complements the experimental-based research and can be used to quickly determine the nature of newly discovered viruses and will aid in the qualitative assessment of viral diversity in metagenome samples. an and gc contributed equally to this work. we thank kyung mo kim, jay mittenthal, matthew hudson, patrick forterre, and jian ma for their support and valuable input that significantly improved the study. work presented in this manuscript is part of an's doctoral dissertation. an would like to thank the chateaubriand fellowship from the french government, dissertation completion fellowship from the graduate college of the university of illinois, and faculty development program fellowship from the comsats institute of information technology, islamabad, pakistan for their financial support. the supplementary material for this article can be found online at: http://journal.frontiersin.org/article/ . /fmicb. . /full#supplementary-material table s | list of fsfs identified via uniprotkb keyword search. fsfs are identified both by scop ids and ccs. for each fsf, the total number of hits detected by hmm search and its percentage representation out of archaeal (a), , bacterial (b), and eukaryotic (e) proteomes are also given 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not comply with these terms. key: cord- -xic wxh authors: sato, hiroki; yoneda, misako; honda, tomoyuki; kai, chieko title: morbillivirus receptors and tropism: multiple pathways for infection date: - - journal: front microbiol doi: . /fmicb. . sha: doc_id: cord_uid: xic wxh morbilliviruses, which include measles virus (mev), canine distemper virus, and rinderpest virus, are among the most important pathogens in their respective hosts and cause severe syndromes. morbilliviruses are enveloped viruses with two envelope proteins, one of which is hemagglutinin (h) protein, which plays a role in binding to cellular receptors. during morbillivirus infection, the virus initially targets lymphoid cells and replicates efficiently in the lymph nodes. the principal cellular receptor for morbillivirus is signaling lymphocyte activation molecule (slam, also called cd ), which is exclusively expressed on immune cells. this feature reflects the strong lymphoid cell tropism and viral spread in the infected body. morbillivirus infection, however, affects various tissues in the body, including the lung, kidney, gastrointestinal tract, vascular endothelium, and brain. thus, other receptors for morbilliviruses in addition to slam might exist. recently, nectin- has been identified as a novel epithelial cell receptor for mev. the expression of nectin- is localized to polarized epithelial cells, and this localization supports the notion of cell tropism since mev also grows well in the epithelial cells of the respiratory tract. although two major receptors for lymphoid and epithelial cells in natural infection have been identified, morbillivirus can still infect many other types of cells with low infectivity, suggesting the existence of inefficient but ubiquitously expressed receptors. we have identified other molecules that are implicated in morbillivirus infection of slam-negative cells by alternative mechanisms. these findings indicate that morbillivirus utilizes multiple pathways for establishment of infection. these studies will advance our understanding of morbillivirus tropism and pathogenesis. morbilliviruses belong to the order mononegavirales, family paramyxoviridae, and include measles virus (mev), rinderpest virus (rpv), and canine distemper virus (cdv). morbilliviruses are highly contagious for their respective hosts and mediate similar consequences of pathogenesis, such as fever, cough and coryza, and respiratory and gastrointestinal diseases. in particular, induction of severe transient immunosuppression along with the gain of life-long immunity are the most notable features of morbillivirus infection (griffin, ) . measles virus is a leading cause of mortality in children worldwide. in particular, strong immunosuppression causes secondary infection and leads to high childhood mortality in the developing world. rpv affects several species of wild and domestic clovenhoofed animals. the mortality rate can reach nearly % in highly susceptible cattle or buffalo herds; in fact, rinderpest had caused significant economic damage since records began. cdv is a cause of fatal disease in many species of carnivores. recently, fatal cdv infection has been reported in other species such as large felids (appel et al., ) , javelinas (appel et al., ) , and freshwater and marine seals (visser et al., ) . to limit these severe and fatal diseases of morbilliviruses, appropriate measures have been taken, including live attenuated and effective vaccines, which were developed more than years ago and control the viruses well. in particular, international campaigns have been conducted to eradicate both mev and rpv globally. as a result of vaccination efforts and culling of infected animals, eradication of rinderpest in all countries and territories was declared by oie ( ) and fao ( ) . rinderpest became the second viral disease, after smallpox, to be eradicated through human efforts. in the case of measles, vaccination has contributed to reducing the mortality rate in infants, and deaths due to measles were reduced by % worldwide between and (from , to , ) after a global campaign for vaccination (who, ) . morbilliviruses are enveloped virions that contain a nonsegmented, negative-stranded rna genome that encodes a single envelope-associated matrix protein (m), two glycoproteins (hemagglutinin h and fusion protein f), two rna-polymeraseassociated proteins (phosphoprotein p and large protein l), and a nucleocapsid protein (n) that encapsulates the viral rna (figure ) . the h gene encodes a key protein for morbillivirus and its animal hosts: the virus uses this protein to attach to cell receptors during the first step of infection (griffin, ) . the search for the receptor for morbillivirus began in vaccine strains of mev, and subsequently identified receptors for wild-type strains have revealed the closely related receptor usage and unique pathogenicity of the viruses. the viral particle contains the ribonucleoprotein complex consisting of the nucleocapsid (genomic rna and n proteins) and viral rna polymerase (p and l proteins), and the envelope consisting of the m, f, h proteins, and host cell-derived membrane. in this review, we introduce the identified receptors for morbilliviruses, mainly mev, and discuss cell tropism and pathogenicity in terms of receptor usage. measles virus was first isolated by enders and peebles ( ) from primary human kidney cells inoculated with the blood and throat washings of a child with measles. the virus strain (edmonston) was passaged multiple times in primary human kidney and amnion cells and then adapted to eggs and multiply passaged in chick embryo cells to produce the original edmonston b vaccine, which was licensed in (griffin, ) . administration of the live attenuated vaccine results in transient immunosuppression, but induces both expression of the neutralizing antibody and cellular immune responses sufficient for protection. vero cells derived from the african green monkey kidney had been utilized to isolate mev as a standard cell line because it is beneficial and safe. about years after mev was isolated, two groups reported in that cd acts as a cellular receptor for laboratory-adapted strains of mev. naniche et al. ( ) obtained a monoclonal antibody that inhibited cell fusion induced by recombinant vaccinia virus encoding the h and f proteins of the halle strain of mev. the antibody precipitated a cell-surface glycoprotein from human and simian cells but not from murine cells. n-terminal amino acid sequencing identified that the glycoprotein was human membrane cofactor protein (cd ), a member of the regulators of the complement activation gene cluster (naniche et al., ) . transfection of non-permissive murine cells with a cd expression vector confirmed that the human cd molecule serves as a mev receptor, allowing virus-cell binding, fusion, and viral replication. dorig et al. ( ) showed independently that hamster cell lines expressing cd produced syncytia and virus proteins after infection with the edmonston strain of mev and that polyclonal antisera against cd inhibited virus binding and infection. cd is a cell-surface, type i transmembrane - kd glycoprotein that belongs to the family of complement activation regulators and is ubiquitously expressed in all nucleated human cells. the most important function of cd is as an inhibitor of complement activation. it protects host cells from complement deposition by functioning as a cofactor for the factor-i-mediated proteolytic inactivation of c b and c b (liszewski et al., ) . in addition, cd has been implicated in the modulation of t-cell functions (marie et al., ) , generation of regulatory t-cells (kemper et al., ) , and control of interferon (ifn) production (katayama et al., ) . cd is also important during fertilization -it presumably promotes sperm-egg interaction (riley-vargas et al., harris et al., ) . cd exists in multiple isoforms, which are generated by alternative splicing of a single gene. it has four short consensus repeats (scr - ) comprising - aa each, an alternatively spliced serine/threonine/proline-rich region, a transmembrane region, and an alternatively spliced cytoplasmic tail. previous studies have located the mev binding site on cd to the scr and scr domains of the receptor (buchholz et al., ; hsu et al., ; casasnovas et al., ; christiansen et al., ; figure ). functional studies in vitro have suggested that signaling via cd is an important component of mev pathogenesis. for example, the high degree of interaction between mev-h and cd results in downregulation of cd from the surface of infected cells, rendering them more sensitive to c b-mediated complement lysis (schneider-schaulies et al., a,b; schnorr et al., ) . interestingly, cd -mediated immunosuppression in mev infection has been reported. one mechanism involves inhibiting activation-induced expression of interleukin (il)- , which is essential for the generation of successful effector t-cell responses, by cross-linking cd on the surface of monocytes by mev (karp et al., ; galbraith et al., ; karp, ; kurita-taniguchi et al., ) . interaction of mev-h and cd also induces il- , leading to inhibition of the contact hypersensitivity reaction (marie et al., ) . in contrast, mev binding to cd induces ifn production, which further triggers the early antiviral immune response naniche et al., ) . amino acid residues interacting with cd in the h protein have been identified (f , v , y , p , and i ; masse et al., ; santiago et al., ; vongpunsawad et al., ) . among them, two amino acid residues (v and y ) are crucial for determining the ability of mev strains to cause hemadsorption, cell fusion, and cd downregulation. although many studies clarified the interaction between mev-h and cd and the resultant cellular signaling events in vitro, it had also been revealed that these laboratory strains of mev do not induce any typical symptoms in non-human primate species, which are susceptible to wild-type mev. for example, rhesus and cynomolgus macaques have been described to cause frontiers in microbiology | virology figure | structure of cd . cd has four scrs at the amino terminus of its ectodomain. scr and scr interact with the laboratory strains of mev, whereas scr and scr interact with complement proteins c b and c b. outbreaks of measles in colonies; however laboratory strains of mev do not induce disease in these animals (kobune et al., ; van binnendijk et al., van binnendijk et al., , mcchesney et al., ) . furthermore, previous reports indicated that five strains of mev that were adapted for growth in vero cells showed little pathogenicity against experimentally infected macaques, whereas the bilthoven strain of mev, which grew in human cord blood cells, induced clinical symptoms of measles (auwaerter et al., ) . although vero cells had been used for mev isolation for a long time, the isolation was not highly efficient and usually required several blind passages. in contrast, kobune et al. found that an epstein-barr-virus-transformed marmoset b-cell line, b a, is , -fold more sensitive to the mev present in clinical specimens than vero cells. furthermore, mevs isolated and propagated in b a cells cause clinical signs in experimentally infected monkeys, which resemble those of human measles such as rashes and koplik's spots, leukopenia, and marked histological lesions in the lymphoid tissues (kobune et al., ) . subsequently, kobune et al. ( kobune et al. ( , reported that two strains of wild mev from the same patient, one isolated in b a cells and the other in vero cells, had different virulence in monkeys. the former induced acute signs of mev infection, whereas the latter did not induce any clinical signs of disease and caused milder histological lesions. these findings strongly indicated that mev isolated in b a cells maintains virulence similar to that in humans and that isolation in vero cells leads to loss of virulence. however, strains isolated in b a cells or human b-cell lines were shown to grow only in a limited number of lymphoid cell lines (kobune et al., ; schneider-schaulies et al., b; tatsuo et al., a) . furthermore, the h protein of mev isolated from b-cell lines neither induced downregulation of cd nor caused cell-cell fusion (upon coexpression of the f protein) in cd -positive cell lines (lecouturier et al., ; bartz et al., ; tanaka et al., ) . from these observations, it had been postulated that b-cell line-isolated strains do not use the ubiquitously expressed cd but utilize another molecule as a receptor (lecouturier et al., ; buckland and wild, ; bartz et al., ; hsu et al., ; tanaka et al., ; tatsuo et al., a) . tatsuo et al. ( b) performed a screening of a cdna library of b a cells, in which a non-susceptible human kidney cell line, t, was transfected with the cdna library and then screened with a vesicular stomatitis virus pseudotype bearing the h protein of mev isolated from b-cells and f protein from the edmonston strain. as a result, a single cdna clone capable of making transfected t cells susceptible to mev-h protein bearing pseudotype was identified. the sequence of the clone was a homolog of signaling lymphocyte activation molecule (slam), and consequently, human slam was identified as a lymphoid cell receptor for wildtype mev. importantly, the edmonston strain was found to utilize slam, in addition to cd , as a receptor, indicating that slam acts as a receptor not only for b-cell line-isolated mev strains but also for vaccine and laboratory-adapted strains (tatsuo et al., b) . subsequent studies have demonstrated that mev strains isolated and propagated by slam-positive cells show clinical signs of mev in infected animals (van binnendijk et al., ; mcchesney et al., ; zhu et al., ; auwaerter et al., ; el mubarak et al., ; bankamp et al., ) . therefore, it has been verified that slam acts as the principal cellular receptor for mev in vivo, and that use of cd may be the result of mev adaptation in vitro. furthermore, it has been demonstrated that all cdv and rpv strains use dog and cow slam as a receptor, respectively, and that slam is a common and principal receptor for morbillivirus (tatsuo et al., ) . signaling lymphocyte activation molecule is also known as cd and is expressed on thymocytes, activated lymphocytes, mature dendritic cells, macrophages, and platelets in humans and mice (sidorenko and clark, ; cocks et al., ; aversa et al., ) . in humans, cd + monocytes in tonsils and spleens express slam (farina et al., ) . slam is implicated in the regulation of t-cell activation by affecting t-cell antigen receptor signaling. in addition, slam has the ability to regulate the functions of several other immune cell types, including natural killer and dendritic cells. hence, slam has a broad involvement in the modulation of innate and acquired immune responses (veillette and latour, ; veillette et al., ; schwartzberg et al., ) . signaling lymphocyte activation molecule has two extracellular immunoglobulin superfamily domains, v and c , and is associated with the adaptor molecules, slam-associated protein (sap), or ews/flii-activated transcript (eat- ), in its cytoplasmic tail. the extracellular domain of slam associates with another slam molecule present on adjacent cells. in cd + t-cells, ligation of slam induces its binding to sap, and combined with t-cellreceptor (tcr)-mediated signals, triggers downstream signaling for the production of t helper (th ) cytokines such as il- and il- (veillette et al., ) . furthermore, slam controls production of il- , tumor necrosis factor α, and nitric oxide, presumably via eat- , by macrophages (veillette et al., ) . the v domain of slam is necessary and sufficient for mev receptor function and three amino acid residues, at positions , , and of human slam, are crucial for its function (ohno et al., ; figure ) . meanwhile, mutagenesis of the h protein based on its ability to induce slam-dependent cell-cell fusion has revealed that residues important for interaction with slam are i , d , d , y , d , t , r , h , y , and p (masse et al., ; vongpunsawad et al., ; navaratnarajah et al., ) . signaling lymphocyte activation molecule-isolated strains express typical clinical symptoms in experimental animal models. thus, the in vivo study of wild-type morbillivirus, in particular mev and cdv, has proceeded in conjunction with the establishment of a novel method for generating recombinant virus, known as reverse genetics (billeter et al., ) . to identify the host cells that support infection, a recombinant cdv that expressed green fluorescent protein (gfp) was produced by reverse genetics, based on a wild-type strain that is lethal to ferrets, and inoculated intranasally into animals (von messling et al., ) . cdv initially infected lymphocytes and massively replicated therein, thereby causing immunosuppression, systemic invasion, and host escape. in contrast, replication in epithelial cells was initially not detectable but substantial before host death. in a similar manner, gfp-expressing mev was also generated and inoculated into macaques via the aerosol route, and the time course of propagation was monitored . mev entered the host at the alveolar level by infecting macrophages or dendritic cells, which carried the virus to bronchus-associated lymphoid tissue, followed by regional dissemination by viremia. to further clarify the importance of slam for morbillivirus pathogenesis, recombinant viruses possessing h, which are incapable of recognizing slam but can enter epithelial cells (slam-blind), have been generated. signaling lymphocyte activation molecule-blind cdv infected primary ferret epithelial cells as efficiently as the parental wildtype cdv but was incapable of entering ferret peripheral blood mononuclear cells in vitro. experimentally infected ferrets indicated that the slam-blind virus is completely avirulent in ferrets; infection with this virus caused only a small, short-lived decrease in the blood leukocyte count (von messling et al., ) . signaling lymphocyte activation molecule-blind mev was also generated and inoculated intranasally into rhesus monkeys. as a result, the virus showed attenuated pathogenicity, inefficient infection of lymphocytes, and induced no clinical symptoms in these animals (leonard et al., ) . recently, our group has generated slam-blind rpv using a lapinized strain (rpv-l). rpv-l is highly virulent in rabbits and exhibits similar pathogenicity as virulent rpv in cattle. thus, rpv-l-infected rabbits should represent a useful model for studying in vivo pathogenicity after rpv infection. slam-blind rpv-l induced few clinical signs, which is in agreement with studies with cdv and mev, demonstrating that slam recognition is necessary for virulence. the virus was not detected in any of the lymphoid tissues, but was detected in lungs, suggesting that the slamblind rpv in rabbits could infect epithelial but not lymphoid cells (unpublished data). these results strongly indicated that slam-mediated cell entry is crucial for expression of full pathogenicity of morbillivirus. distribution and functions of slam provide a good explanation for the lymphotropism and immunosuppressive nature of morbillivirus. however, morbillivirus, in autopsied patients and some experimentally infected animals, has also been shown to infect the epithelial cells of the trachea, bronchial tubes, lungs, oral cavity, pharynx, esophagus, intestines, liver, and bladder (griffin, ) . these epithelial cells do not express slam, but the infected cells do shed virus, suggesting that entry into these slam-negative cells is mediated by other cellular receptors. in vitro studies have shown that a number of slam-negative cell types of epithelial or neuronal origin result in cytopathic effects and virus release. in particular, several well-differentiated polarized epithelial cell lines showed high susceptibility to wild-type mev (takeda et al., ; tahara et al., ) . further in vitro studies indicated that wild-type mev enters human polarized airway epithelium basolaterally, whereas progeny viral particles are released exclusively from the apical surface of these cells (tahara et al., ; ludlow et al., ) . moreover, it was shown that loss of tight junction proteins induced by the transcription repressor snail blocked infection with mev (shirogane et al., ) . these data strongly implied that polarized epithelial cells possess a putative epithelial receptor, epr, and that the receptor appears to be expressed on the basolateral side of the cells that is associated with tight junctions. from these studies, before identification of the components of epr, the region of the h protein that interacts with the epr was mapped to the h protein (i , l , l , p , y , and y ; leonard et al., ; tahara et al., ) . based on these data, an epr-blind mev maintaining slamdependent cell entry was generated and inoculated intranasally into monkeys (leonard et al., ) . as a result, epr-blind mevinfected macaques developed signs of measles comparable to those of animals infected with wild-type virus, including skin rash and anorexia, indicating that the epr-blind mev remained virulent in the macaques. however, epr-blind mev could not be isolated from the tracheal aspirates of all of the monkeys, unlike wild-type mev. this strongly suggested that mev crosses the respiratory epithelium only when it leaves the host and that epr-blind mev does not shed in the airways. in , two independent groups reported identification of the epr. both groups utilized microarray data from susceptible versus non-susceptible cell lines and compared the membrane protein gene transcripts. noyce et al. ( ) described the susceptibility of many different tumor cell lines to mev infection and selected susceptible and non-susceptible cell lines. they filtered the microarray data for membrane protein genes, and produced a short list of candidate receptors. of these, only human pvrl (nectin- ), a tumor cell marker found on breast, lung, and ovarian carcinomas, rendered cells susceptible to mev infection. transient knockdown of nectin- using sirna abolished mev infection in these cell lines. furthermore, antibodies specific for human nectin- inhibited mev infection. mühlebach et al. ( ) performed microarray analysis of seven epithelial cell lines from human airways or bladder previously characterized as permissive (three lines) or non-permissive (four lines), and identified that nectin- renders cho cells susceptible to mev. it was demonstrated that the v domain of nectin- binds strongly to mev-h (mühlebach et al., ; figure ) . the nectin family is a cell adhesion molecule family comprising four members (nectin- - ), and only nectin- functions as the epr (mühlebach et al., ; noyce et al., ) . nectins contain immunoglobulin-like domains, similar to slam. the nectin family proteins have recently been shown to be essential contributors to the formation of cell-cell adhesions and are novel regulators of cellular activities, including cell polarization, differentiation, movement, proliferation, and survival (takai et al., ; ogita et al., ) . nectins are also involved in the establishment of apical-basal polarity at cell-cell adhesion sites and the formation of tight junctions in epithelial cells (takai et al., ; ogita et al., ) . to date, details of the interaction mechanism of the newly identified receptor, nectin- , with mev-h has not been elucidated. in it has been postulated that the primary targets of mev are slampositive alveolar macrophages, dendritic cells, and lymphocytes of the immune system in the respiratory tract, rather than epithelial cells. this contention is supported by the finding that almost all cd + monocytes in human tonsils express slam. mev subsequently grows in slam-expressing lymphatic cells and spreads to lymph nodes throughout the body. after systemic infection, it is considered that the virus is transmitted from infected lymphocytes and dendritic cells to epithelial cells using nectin- on the basolateral side of epithelial cells, and virus particles are subsequently shed from the apical surface of these cells (figure ). from the above studies, the major transmission mode of morbillivirus, especially mev, has been drawn. however, many histopathological studies have indicated that morbillivirus is also detected in endothelial and neuronal cells (griffin, ) , suggesting the existence of other routes for virus propagation to these cell types. in particular, mev and cdv show strong neuronal tropism, and cause acute and persistent encephalitis (griffin, ) , nevertheless neural cells neither express slam nor nectin- . these cells may have their own receptors or be infected by virus via an inefficient receptor. previous studies using recombinant morbilliviruses expressing gfp have demonstrated that cell entry independent of slam and cd (and probably nectin- ) occurs in a variety of cell lines with www.frontiersin.org frontiers in microbiology | virology low infectivity (hashimoto et al., ; fujita et al., ; terao-muto et al., ) . this suggests the existence of inefficient but ubiquitously expressed receptors. previously, we have found that infection with several slam (and presumably nectin- ) negative cell lines with morbillivirus was inhibited by soluble heparin, and that virus bound to immobilized heparin. these results suggest that ubiquitously expressed heparin-like glycosaminoglycans are involved in morbillivirus infection (fujita et al., ; terao-muto et al., ) . more recently, we have also demonstrated a unique infection mechanism of mev, in which viral particles incorporate cellular cyclophilin (cyp)b on their surface and bind to cellular cd , a receptor for cypa and b, independently of mev-h (watanabe et al., ) . it is known that cypa incorporated into hiv- particles translocates to the surfaces of virions (misumi et al., ) , and that the interaction between cypa and cd enables hiv- to infect target cells via cd , independently of the binding of gp and cd (pushkarsky et al., ) . additionally, severe acute respiratory syndrome coronavirus (sars-cov) is proposed to use cd as a receptor in the same manner as hiv- (chen et al., ) . unlike hiv- and sars-cov, mev uses cypb instead of cypa for binding to cd . this finding is the first among viruses belonging to the order mononegavirales and shows a new infection mode of mev, which is independent of h protein. investigations aimed at identifying the receptors for morbillivirus started in with cd for vaccine strains of mev, followed by the lymphoid cell receptor, slam, in , and the epithelial cell receptor, nectin- , in , for wild-type viruses. along with the receptors, the cell tropism, transmission modes in the body, and unique pathogenicities of morbillivirus are being explained. however, many problems associated with morbillivirus remain to be clarified. in particular, the mechanism by which mev spreads in the central nervous system during fatal subacute sclerosing panencephalitis is unknown. further studies will lead to a better understanding of morbillivirus pathogenesis and to novel strategies for treatment and prevention. canine distemper virus infection and encephalitis in javelinas (collared peccaries) canine distemper epizootic in lions, tigers, and leopards in north america measles virus infection in rhesus macaques: altered immune responses and comparison of the virulence of six different virus strains engagement of the signaling lymphocytic activation molecule (slam) on activated t cells results in il- -independent, cyclosporin a-sensitive t cell proliferation and ifn-gamma production genetic changes that affect the virulence of measles virus in a rhesus macaque model differential receptor usage by measles virus strains reverse genetics of measles virus and resulting multivalent recombinant vaccines: applications of recombinant measles viruses mapping of the primary binding site of measles virus to its receptor cd is cd the cellular receptor for measles virus? crystal structure of two cd domains reveals an extended measles virusbinding surface function of hab g/cd in invasion of host cells by severe acute respiratory syndrome coronavirus evidence for distinct complement regulatory and measles virus binding sites on cd scr a novel receptor involved in t-cell activation predominant infection of cd + lymphocytes and dendritic cells during measles virus infection of macaques the human cd molecule is a receptor for measles virus (edmonston strain) infection of cynomolgus macaques (macaca fascicularis) and rhesus macaques (macaca mulatta) with different wild-type measles viruses propagation in tissue cultures of cytopathogenic agents from patients with measles global rinderpest eradication programme (grep) distinct responses of monocytes to toll-like receptor ligands and inflammatory cytokines host range and receptor utilization of canine distemper virus analyzed by recombinant viruses: involvement of heparin-like molecule in cdv infection morbillivirus downregulation of cd measles virus complement and complement regulators in the male reproductive system slam (cd )-independent measles virus entry as revealed by recombinant virus expressing green fluorescent protein artificial mutations and natural variations in the cd molecules from human and monkey cells define regions important for measles virus binding a single amino acid change in the hemagglutinin protein of measles virus determines its ability to bind cd and reveals another receptor on marmoset b cells measles: immunosuppression, interleukin- , and complement receptors mechanism of suppression of cell-mediated immunity by measles virus human receptor for measles virus (cd ) enhances nitric oxide production and restricts virus replication in mouse macrophages by modulating production of alpha/beta interferon activation of human cd + cells with cd and cd induces a t-regulatory cell phenotype marmoset lymphoblastoid cells as a sensitive host for isolation of measles virus nonhuman primate models of measles functional modulation of human macrophages through cd (measles virus receptor): production of il- p and nitric oxide in association with recruitment of protein-tyrosine phosphatase shp- to cd identification of two amino acids in the hemagglutinin glycoprotein of measles virus (mv) that govern hemadsorption, hela cell fusion, and cd downregulation: phenotypic markers that differentiate vaccine and wild-type mv strains measles virus selectively blind to signaling lymphocytic activation molecule (slam; cd ) is attenuated and induces strong adaptive immune responses in rhesus monkeys measles virus blind to its epithelial cell receptor remains virulent in rhesus monkeys but cannot cross the airway epithelium and is not shed membrane cofactor protein (mcp or cd ): newest member of the regulators of complement activation gene cluster wild-type measles virus infection of primary epithelial cells occurs via the basolateral surface without syncytium formation or release of infectious virus clinical isolates of measles virus use cd as a cellular receptor linking innate and acquired immunity: divergent role of cd cytoplasmic domains in t cell induced inflammation measles virus (mv) hemagglutinin: evidence that attachment sites for mv receptors slam and cd overlap on the globular head identification of a second major site for cd binding in the hemagglutinin protein from a laboratory strain of measles virus (mv): potential consequences for wild-type mv infection experimental measles. i. pathogenesis in the normal and the immunized host three isoforms of cyclophilin a associated with human immunodeficiency virus type were found by proteomics by using two-dimensional gel electrophoresis and matrix-assisted laser desorption ionization-time of flight mass spectrometry adherens junction protein nectin- is the epithelial receptor for measles virus human membrane cofactor protein (cd ) acts as a cellular receptor for measles virus a monoclonal antibody recognizes a human cell surface glycoprotein involved in measles virus binding evasion of host defenses by measles virus: wildtype measles virus infection interferes with induction of alpha/beta interferon production dynamic interaction of the measles virus hemagglutinin with its receptor signaling lymphocytic activation molecule (slam, cd ) tumor cell marker pvrl (nectin ) is an epithelial cell receptor for measles virus cell adhesion molecules nectins and associating proteins: implications for physiology and pathology histidine at position and its adjacent amino acid residues are critical for the ability of slam (cd ) to act as a cellular receptor for measles virus no more deaths from rinderpest cd facilitates hiv- infection by interacting with virusassociated cyclophilin a cd : expanding beyond complement regulation targeted and restricted complement activation on acrosome-reacted spermatozoa distinct kinetics for binding of the cd and slam receptors to overlapping sites in the measles virus hemagglutinin protein physical association of moesin and cd as a receptor complex for measles virus receptor usage and differential downregulation of cd by measles virus wild-type and vaccine strains measles virus-induced down-regulation of cd is associated with enhanced sensitivity to complement-mediated lysis of infected cells slam receptors and sap influence lymphocyte interactions, development and function epithelialmesenchymal transition abolishes the susceptibility of polarized epithelial cell lines to measles virus characterization of a cell surface glycoprotein ipo- , expressed on activated human b and t lymphocytes measles virus infects both polarized epithelial and immune cells by using distinctive receptorbinding sites on its hemagglutinin nectins and nectin-like molecules: roles in contact inhibition of cell movement and proliferation a human lung carcinoma cell line supports efficient measles virus growth and syncytium formation via a slam-and cd -independent mechanism the hemagglutinin of recent measles virus isolates induces cell fusion in a marmoset cell line, but not in other cd -positive human and monkey cell lines, when expressed together with the f protein virus entry is a major determinant of cell tropism of edmonston and wild-type strains of measles virus as revealed by vesicular stomatitis virus pseudotypes bearing their envelope proteins slam (cdw ) is a cellular receptor for measles virus morbilliviruses use signaling lymphocyte activation molecules (cd ) as cellular receptors heparin-like glycosaminoglycans prevent the infection of measles virus in slam-negative cell lines monkeys in measles research viral replication and development of specific immunity in macaques after infection with different measles virus strains consequence of the slam-sap signaling pathway in innatelike and conventional lymphocytes the slam family of immune-cell receptors comparison of two morbilliviruses isolated from seals during outbreaks of distemper in north west europe and siberia tropism illuminated: lymphocyte-based pathways blazed by lethal morbillivirus through the host immune system receptor (slam [cd ]) recognition and the v protein sustain swift lymphocyte-based invasion of mucosal tissue and lymphatic organs by a morbillivirus selectively receptor-blind measles viruses: identification of residues necessary for slam-or cd -induced fusion and their localization on a new hemagglutinin structural model cd /emmprin acts as a functional entry receptor for measles virus on epithelial cells measles mortality reduction: a successful initiative experimental measles. ii. infection and immunity in the rhesus macaque the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. key: cord- -thywse g authors: hwang, yoon jung; myung, heejoon title: engineered bacteriophage t as a potent anticancer agent in vivo date: - - journal: front microbiol doi: . /fmicb. . sha: doc_id: cord_uid: thywse g oncolytic viruses (ovs) induce antitumor effect by both direct lysis of target cells and eliciting immunogenic response to the virus and ultimately to the target cells. these viruses are usually natural human pathogens. bacteriophages are natural pathogens of bacteria that do not infect human and have greater advantages in safety, manipulation, and production over human viruses. we constructed an engineered bacteriophage t displaying a peptide, which targets murine melanoma cells and harbors a mammalian expression cassette of the cytokine granulocyte macrophage-colony stimulating factor (gm-csf) in viral genomic dna. the engineered phage was successfully transduced to b f melanoma cells both in vitro and in vivo. gm-csf was expressed from the transduced phage dna. all mice treated with the phage intravenously survived for days until the end of experiment, while only % of those not treated survived. during the days of phage treatment, phage t displaying homing peptide and expressing gm-csf inhibited tumor growth by % compared to the untreated control. serum cytokine levels of il- α, tnf-α, and gm-csf were seen to increase during the treatment. immunohistochemical analysis of tumor tissue revealed infiltration by macrophages, dendritic cells (dcs), and cd (+) t cells. migration of murine macrophages to bacteriophages was also observed in in vitro transwell assays in both time- and dose-dependent manners. taken together, the recombinant bacteriophage t efficiently inhibited tumor growth by changing the tumor microenvironment and recruiting anti-tumor immune cells. oncolytic viruses (ov) are viruses, which are known to attack and lyse cancer cells (raja et al., ; reale et al., ) . they destroy tumor mass by infecting and multiplying within the mass. in addition to viral lysis, the tumor mass, due to the presence of immunogenic viruses, is subject to attack by the immune system. partial remission of tumor mass when patients contracted viral diseases was observed by physicians as early as the beginning of twentieth century (kelly and russell, ). an hsv- based oncolytic virus (t-vec) has already been approved by the fda and is currently in clinical use (puzanov et al., ) . attenuated human pathogens, which have been tested as potential ovs, include the adenovirus (shaw and suzuki, ) , the vaccinia virus (haddad, ) , the measles virus (aref et al., ) , the mumps virus (ammayappan et al., ) , and the influenza virus (pizzuto et al., ) . other viruses known to be poor human pathogens, which have been tested as ovs, include the newcastle disease virus (tayeb et al., ) and the vesicular stomatitis virus (bishnoi et al., ) . potential ovs are often not powerful enough for solid tumors and safety has not always been matched with efficacy, as ovs are known to exhibit a certain range of toxic effects (raja et al., ; reale et al., ) . in addition, live viruses have been shown to be transferable from the primary treatment patient to healthcare workers and people inhabiting the same household (robilotti et al., ) . bacteriophages are viruses naturally infecting bacteria. since their discovery independently by frederick twort and felix d'herelle in early twentieth century, phages have mainly served as antibacterial agents and for the exploration of the basic mechanisms of life at the molecular level. in , accumulation of phages in cancer tissue and the inhibition of tumor growth were observed, and in , the binding of phages to cancer cells was seen both in vitro and in vivo (budynek et al., ) . phage t and its substrain hap were shown to bind melanoma cells and inhibit lung metastasis in a murine model (dabrowska et al., ) . interaction between lysine-glycine-aspartic acid (kgd) motif on phage capsid protein and the β integrin receptor on cell surfaces was hypothesized as being responsible for the activity. the same group of researchers showed that dendritic cells (dcs) primed with phage t and tumor antigens initiated differentiation accompanied by exhibiting an enhanced ability to prime t cells (pajtasz-piasecka et al., ) . application peritumorally of primed dcs retarded tumor growth in a mouse colon cancer model. eriksson et al. reported that peritumoral injection of m phages displaying peptide receptors or fab fragments in mouse melanoma cells led to delayed tumor growth and increased survival of tumor bearing mice (eriksson et al., ) . later, by investigating the tumor microenvironment, the same group of researchers showed that tumor destruction was caused by the activation of tumorassociated macrophages (eriksson et al., ) . hybrid adenoassociated virus/phage (aavp), a filamentous phage capsid packaging the cis-acting aav genomic element, was developed to facilitate targeted gene delivery without natural cell tropism (hajitou et al., ) . arginine-glycine-aspartic acid (rgd) peptide-displaying aavp expressing tnf-α with radiation therapy inhibited melanoma by modifying the tumor microenvironment in a mice model (quinn et al., ) . in this study, we developed phage t as an oncolytic phage (op). it displayed peptides targeting mouse melanoma cells and at the same time harbored a mammalian expression cassette for the cytokine granulocyte macrophage-colony stimulating factor (gm-csf). we tested its anti-tumor efficacy in vivo. the homing peptide used was pep (ctvalpggyvrvc) targeting grp on cancer cells (kim et al., ) . both strands of oligonucleotides encoding the peptide were synthesized (bioneer, korea) and annealed and ligated between ecori and hindiii sites in multiple cloning sites of t select - cloning kits (novagen, canada). the resulting t select vector was used for electroporation into escherichia coli blt (novagen, canada) to produce peptide-displaying phages. additionally, a cassette expressing gm-csf under cmv promoter was synthesized (bioneer, korea) and used to clone into the above described t genomic dna (genbank accession number v . ), which was cut with the restriction enzyme paci at , ~ , basepair. the synthesized cassette consisted of cmv promoter, kozak sequence, the orf encoding murine gm-csf (gene id ), and bgh polya signal. the recombinant phage was used to infect freshly cultured e. coli blt in a ml culture media at the multiplicity of infection (moi) of . . the mixture was incubated at room temperature for phage adsorption for h followed by shaking incubation at °c for h. chloroform was added to the culture at a final concentration of % (v/v) for complete lysis of bacteria and the culture was then incubated with shaking for more min. nacl was subsequently added at a final concentration of % (weight/volume) and the culture was incubated at °c for h. to remove any remaining bacterial cells or debris, the mixture was subjected to centrifugation at , × g for min. the supernatant was recovered and peg was added at a final concentration of % (weight/volume). the mixture was again subjected to centrifugation at , × g for min. supernatant was discarded and the pellet was resuspended in ml of sm buffer ( mm tris-hcl ph . , mm nacl, and mm mgso ). one milliliter of chloroform was added and the mixture was rigorously vortexed and subjected to a centrifugation at , × g for min. the upper phase was recovered and subjected to an ultracentrifugation. three milliliter of % glycerol was poured into an empty tube followed by the slow addition of % glycerol. the upper phase containing phages was then added to the tube and the remaining space was filled with sm buffer. the tube was centrifuged at , × g for hour. supernatant was discarded and the pellet containing phages was recovered by resuspension in ml of sm buffer. the method was described previously (branston et al., ) . triton x- was added to the phage sample at a final concentration of % (v/v) and the mixture was rigorously vortexed. after incubation on ice for min, the mixture was rigorously vortexed and subjected to centrifugation at , × g, °c for min. the upper phase was recovered and used for phage experiments in vitro and in vivo. the b f mouse melanoma cell line (kclb ) was obtained from the korean cell line bank at seoul national university. cells were grown in dulbecco's modified eagle's medium (dmem, thermo fisher scientific, usa) supplemented with fetal bovine serum (fbs, cellect, usa) at a final concentration of % (v/v) and penicillin/streptomycin (sigma aldrich, usa) at a final concentration of % (v/v). for staining bacteriophage t after transduction, × b f cells were seeded in a six-well plate with coverslip and grown in a co incubator. media was discarded after h and × pfu of phages in sm buffer were added to each well and incubated for min. unbound phages were washed out and the cells were fixed with cold acetone. blocking solution ( % bovine serum albumin in pbs) was added and the mixture was incubated for min. cells were then treated with : diluted anti-t tag antibody (ab , abcam, usa) for h followed by washing with pbs three times. secondary antibody ( : diluted anti-goat antibody, ab abcam, usa) was added and the mixture was incubated for h followed by washing with pbs three times. the nucleus was stained with ', -diamidino- -phenylindole (dapi) for min. a laser confocal microscope (lsm , carl zeiss, germany) was used for observations. for masking the receptor grp prior to phage transduction, : diluted anti-grp antibody (ab , abcam) was added to the cell culture and incubated for h followed by washing with pbs. visualization was performed by adding an alexa -labeled secondary antibody (anti-grp rabbit antibody, ab , abcam). for the labeling of phage dna, brdu (thermo fisher scientific, usa) was added at a final concentration of μm at the time of phage infection to the bacterial culture and the resulting progeny phages were collected. a total of × b f cells were seeded in a -well plate and incubated overnight. bacteriophages were added to the well at multiplicities of infection (moi) of or and the mixture was incubated for h followed by -( , -dimethylthiazol- -yl)- , -diphenyltetrazolium bromide (mtt) assay (cell viability assay kit, dong-in ls, korea) in accordance with the manufacturer's instructions. a total of × b f cells were seeded in a six-well plate and incubated overnight. engineered bacteriophage t displaying homing peptide (pep ) and expressing gm-csf was added to the culture at an moi of and cells were incubated for days. cells were harvested and lysed with cell extraction buffer ( mm tris-hcl, ph . , mm nacl, . % triton x- , . % sodium dodecyl sulfate, mm sodium orthovanadate, and mm naf) and the lysate was subjected to an sds-page analysis. expression was confirmed using the anti-gm-csf antibody (ab , abcam, usa) and the anti-rabbit secondary antibody (ab , abcam, usa) in a western blot analysis. the same lysate was used for the extraction of total rna using the trleasy total rna ultrapurification kit (rbc, taiwan). fifty milliliter of extracted rna was treated with unit of dnase i (solgent, korea) at room temperature for min for complete removal of dna as described (rio et al., ) . the mixture was cleaned up through a column included in the above rna isolation kit. five microgram of total rna was mixed with oligo (dt) primer and , u of reverse transcriptase (dynebio, korea). the mixture was annealed at °c for min followed by incubation at °c for min to allow for enzyme reaction. the reaction was then stopped by incubation at °c for min. real-time pcr (rt-pcr) was performed with the resulting cdna, primer, and sybr green qpcr x premix (dynebio, korea). the primer sequence used was forward: 'ggccttggaagcatgtagag ' and reverse: 'ccgtagaccctgctcgaata '. as a control, the extracted rna with dnase i treatment was subjected to a pcr reaction to detect any remaining dna. all animal studies were approved by, and complied with, the regulations and guidelines of the ethical committee for animal experiments of hankuk university of foreign studies (approval number hufs- - ). six-week-old female balb/c mice were obtained for the experiments (young bio, korea). for tumor size measurement, mice were divided into six groups. in vitro cultured × b f cells were subcutaneously injected into the right flank of each mouse. tumor mass was allowed to grow for week until its diameter reached ca. mm. treatment started week post melanoma cell graft. group contained control mice with sm buffer treatment. group mice were treated with × pfu of wild type bacteriophage t every day for days. group mice were treated with × pfu of pep -displaying bacteriophage t every day for days. group mice were treated with × pfu of pep -displaying bacteriophage t harboring expression cassette of gm-csf every day for days. group mice were treated with × pfu of pep -displaying bacteriophage t and ng of gm-csf (catalogue number z , genscript, usa) every day for days (sun et al., ) . group mice were treated with ng of gm-csf every day for days. all treatments were injected intravenously (iv) in the tail vein. tumor volume was measured during the treatment period. after days of treatment, the mice were sacrificed and tumor mass was removed for immunohistological analysis. for serum cytokine analysis, μl of orbital blood collection was performed for each mouse. for survival observations, mice were divided into six groups as above and survival was monitored for days. when tumor mass exceeded % of total body weight, the mouse was euthanized. the survival graph was plotted in accordance with kaplan-meier plot and drawn with prism, graphpad software. in vivo imaging was described previously (kelly et al., ) . briefly, × pfu of pep -displaying bacteriophage t or wild type bacteriophage t was fluorescently labeled with . mg/ ml of fluorochrome-hydroxyl-succinate ester (cy . ) in a dark room at room temperature for h. cy . -labeled phages were injected in tail veins of balb/c mice bearing b f grafted tumor mass and in vivo live imaging was performed using an fmt -lx imager (institute pasteur korea) after h. cytokine elisa serum was obtained from mouse blood by centrifugation at × g for min. mouse cytokines il- α, tnf-α, and gm-csf were measured using multi-analite elisarray kits (qiagen, germany) in accordance with the manufacturer's instruction. the assay was performed in triplicate. tumor-bearing mice treated with various phages and/or cytokine were sacrificed and tumor masses were removed. these were then fixed in % formalin and hematoxylin-eosin (he) staining and immunohistochemistry (ihc) were performed (logone bio convergence research foundation, seoul, korea). all experiments were performed in triplicate and statistical significance was obtained using one way anova followed by tukey's test (prism, graphpad software). p < . was considered as statistically significant. kaplan-meier analyses were used and the log-rank mantel-cox test was employed to determine any difference between the survival curves of the groups. p < . was considered as statistically significant. twelve millimeter transwell with . μm pore (corning transwell polyester membrane cell culture inserts, cls ) was used. a total of × b f cells were seeded in the lower chamber cell viability assay of b f cells after exposure to bacteriophages. two different concentrations (multiplicity of infection of either or ) of native t or engineered t were added to the culture and incubated for h before mtt assay was performed. control was treated with sm buffer. (c) homing and internalization of phage particle, and nuclear localization of phage dna. first and second row: wild type phage t (wt t ) or t displaying the homing peptide (t -pep ) was added to in vitro cultured b f melanoma cells and binding was observed under a fluorescent laser scanning confocal microscope. the nucleus was stained with dapi (blue) and the phage particle was stained with anti-t antibody (green). third row: b f cells were first treated with anti-grp antibody (red) to mask the receptor for pep . then t -pep (green) was added and binding was observed. fourth row: t -pep was produced in the presence of brdu (green) to label the genomic dna and added to cultured b f cells. internalized phage dna to dapi (blue) stained nucleus is shown. (d) real-time pcr (rt-pcr) analysis of mrna encoding gm-csf from t -pep or t -pep _g transduced b f cells. relative amounts of mrna encoding gm-csf from cells treated with t -pep or t -pep _g are shown in black or white bars, respectively. control was treated with sm buffer. t -pep , phage t displaying pep and t -pep _g, phage t displaying pep and expressing gm-csf. (e) western blot analysis of gm-csf from t -pep _g transduced b f cells (lane ) and empty cell (lane ). gapdh was used as an internal control. for statistical analysis, one way anova was performed and then tukey's test was conducted. *above each vertical bar indicates statistical significance of each test to the control. *above each horizontal bar indicates statistical significance of each test between corresponding pairs. and incubated at °c for h. a total of × pfu of phage t displaying pep harboring expression cassette of gm-csf was added to the confluently grown cells and incubated for h. then, × raw . cells were loaded in the upper chamber and incubated at °c for , , or h. media were discarded and migrated cells on membrane surfaces were fixed with ml of % ethanol at room temperature for min followed by drying for min. fixed cells were stained with . % crystal violet at room temperature for min followed by washing with distilled water three times. cells were then observed under a light microscope. the engineered bacteriophage t displaying homing peptide (pep ) and harboring a mammalian expression cassette of murine gm-csf was produced and the phage solution was nearly completely cleared ( %) of endotoxins ( figure a) . we first tested if this preparation exerted any toxicity in vitro. an mtt assay was performed with the results indicating that neither wild type t nor its engineered version had a significant effect on the viability of murine melanoma cells ( figure b) . next, we verified whether the engineered phage homed into b f cells in vitro. both wild type t and its engineered version were added to cultures of murine melanoma cells and stained with anti-t antibody followed by observation under laser scanning confocal fluorescent microscope ( figure c) . as anticipated, wild type t was washed out while engineered t displaying pep remained attached to melanoma cells. grp is known to be the receptor for pep (kim et al., ) . when the melanoma cells were treated with anti-grp antibody prior to the addition of phages, phage t displaying pep could not bind to the cells. we then grew phage t in the presence of brdu to label the phage genomic dna and used the phage for transduction. we observed localization of brdu labeled dna to the nucleus, suggesting that phage genomic dna enters the nuclei of melanoma cells. since the phage genomic dna harboring the expression cassette of gm-csf was localized to nuclei, we expected production of cytokine gm-csf from the transduced culture of melanoma cells. we could observe the transcription of cassette in a time-dependent manner using rt-pcr analysis ( figure d) . expression of gm-csf was also observed by western blot ( figure e ). as homing of the engineered phage was seen in vitro, we next investigated whether it homed into targets in vivo. after grafting of in vitro grown b f cells in the mice and after their tumor masses had built up, either wild type phage t or the engineered version was given to the mice intravenously and in vivo live imaging was performed (figure ) . fluorescently labeled phage t displaying pep localized to tumor masses four times more than wild type t . the location of wild type t looked peritumoral, rather than intratumoral, while the location of the majority of the engineered t looked intratumoral. since the engineered phage t was seen to home in tumor mass in vivo and to express gm-csf in vitro, we next explored whether this phage could inhibit tumor growth in vivo. in vitro cultured b f murine melanoma cells were implanted into mice and allowed to grow for days before the start of phage treatment. phages were intravenously injected into mice once every day for days and the survival, and changes in, tumor masses were observed (figures a,b) . tumor mass was measured , , , and days post implantation with various treatments (figures c-e) . phage t displaying homing peptide and expressing gm-csf (t -pep _g) inhibited tumor growth by % compared to the untreated control. treatment with phage t displaying pep (t -pep ) or in combination with externally added protein gm-csf led to a decrease in tumor volume by %. no additional effects were observed at the given concentration of gm-csf. the amount of total gm-csf available in the body could be one determining factor for the efficacy of this treatment (see figure ) . alternatively, the availability of gm-csf in the tumors' microenvironment could be another determinant. isolated tumor mass after sacrifice is shown for each treatment (figure c ). forty percent of untreated mice survived, while % of mice treated with phage t displaying the homing peptide and expressing gm-csf (t -pep _g) survived at the end of the experiment (figure f) . untreated mice began to die from day and % died by day . sixty percent of mice treated with wild type t displaying homing peptide (t -pep ) or with protein gm-csf survived, but protein gm-csf-treated mice had earlier deaths. eighty percent of wild type t or t displaying pep plus added gm-csf survived, but wild type t -treated mice had earlier deaths. one can note the following, in figure , the live imaging was performed at days post graft and hours after iv injection of phages. thus, the tumor sizes were identical at that particular time point. in figure , tumor mass was measured from to days post graft, where effects of injected phages were observed. thus the initial tumor sizes between the two experiments were different. animal cells are not natural hosts for bacteriophages and we cannot expect cell lysis from phage multiplication. one possibility for the lysis of tumor cells is immunological attack. since the recruitment of immune cells to the tumor could be mediated by various cytokines, we next checked the increase in serum cytokine after the administration of the phages (figure ) . three inflammatory cytokines, il- α, tnf-α, and gm-csf, were measured in mice serum after each treatment. eight-or three-fold increases in serum il- α were observed when the mice were administered with phage t displaying pep and expressing gm-csf (t -pep _g) or phage t displaying pep plus the externally added protein gm-csf (t -pep + g), respectively. neither the phage alone nor the protein gm-csf alone lead to any increases ( figure a) . however, homing phage t displaying pep (t -pep ) alone could strongly increase serum tnf-α ( figure b) . the latter effect decreased in the presence of gm-csf. t -pep alone induced tnf-α, but not other cytokines. in case of viral infection, inflammatory cytokines are induced at different levels. for example, epstein-barr virus infection in b or t cells induced tnf-α, but not il- α (lay et al., ) . in another example, sars-cov infection led to a preferential production of tnf-α, resulting in loss of germinal center cells (kaneko et al., ) . phage t may preferentially induce tnf-α rather than other cytokines. phage t displaying pep and expressing gm-csf (t -pep _g) induced the highest level of the cytokines (figure c) . t displaying the homing peptide (t -pep ) alone did not induce the production of gm-csf. taken together, for statistical analysis, one way anova was performed and tukey's test was conducted; * or **above each vertical bar indicates statistical significance of each test to the control; * or **above each horizontal bar indicates statistical significance of each test between corresponding pairs. (f) survival of mice bearing tumor mass was monitored for each treatment for days post implantation (n = /group). log rank test was used to test the significance. both t displaying the homing peptide and gm-csf were the determinants for the induction of il -α, while the presence of t displaying the homing peptide is the major determinant for the induction of tnf-α. t displaying pep (t -pep ) was not an inducer of gm-csf in these mice. as the expression of cytokines could lead to the activation and recruitment of immune cells to tumor mass, mice bearing tumor mass were treated with phages and/or cytokine gm-csf, and immunohistochemical observation was performed (figure ) . massive necrotic or damaged tumor cells were seen after treatment with the recombinant phage ( figure a) . tumor destruction and the shrinkage of cells were most prominent in mice treated with phage t displaying pep and gm-csf, which was either expressed from the phage or externally added. wild type t , t displaying pep , or externally added protein gm-csf alone, induced a limited destruction of tumor mass and shrinkage of cells. the highest degree of macrophage infiltration was observed when both t displaying pep and gm-csf were present ( figure b ). lesser infiltration was seen when t displaying pep or gm-csf alone was treated. for dc or cytotoxic t cells, t displaying pep and expressing gm-csf strongly recruited the immune cells (figures c,d) . considering the amount of total gm-csf detected (figure c) , gm-csf seems to play a significant role in recruiting the immune cells. for natural killer (nk) cells, little recruitment was seen with any treatment (figure e ). multiple sections of tumor tissue were stained immunohistochemically and observed for each immune cell. to observe phage-induced macrophage infiltration quantitatively in vitro, we performed a transwell migration assay. various doses of either wild type t or t displaying pep and expressing gm-csf (t -pep _g) were added and macrophage migration was detected at various points in time (figure ) . it was seen that wild type t hardly induced any migration of macrophages, while t expressing gm-csf induced a massive recruitment of macrophages in both dose-and time-dependent manners. in this experiment, a very efficient homing of phage t displaying pep , targeting cell surface receptor grp , was verified both in vitro and in vivo. in addition, in vivo imaging showed that accumulation of wild type t (without tropism for b f cells) occurred at the tumor, although at a much lesser degree. earlier literature reported the accumulation of phages in cancer tissue and the inhibition of tumor growth in (budynek et al., ) . this observed accumulation was both intratumoral and peritumoral. we speculate that the peritumoral accumulation of wild type phages in the latter experiment was in the tumor vasculature (forbes et al., ) . for example, bacteria were also shown to accumulate in the chaotic vasculature of tumors. bacteria are better at infiltrating inside tumors thanks to their motility, which phages do not have (forbes, ) . nonetheless, a small portion of phages were seen inside tumor masses in vivo in this experiment. an earlier experiment using the continued) bacteriophage lambda also reported that unmodified phage particles were capable of transducing mammalian cells in vivo, although much less efficiently than the surface modified phage targeting mammalian cells (lankes et al., ) . once attached to the cell surface, phage t particle was efficiently internalized and phage genomic dna was delivered to nuclei followed by the expression of gene encoding gm-csf from the cassette. it is coherent with previously reported gene deliveries using native phage lambda (lankes et al., ) , phage lambda displaying cyclizable rgd peptide to cos- cells (dunn, ) , phage m displaying epidermal growth factor (egf) to cos- cells (kassner et al., ) , m targeting her- receptor (urbanelli et al., ) , or aavp displaying rgd peptide (hajitou et al., ; trepel et al., ; kia et al., ; stoneham et al., ; smith et al., ; przystal et al., ) . trafficking of internalized phage particles was shown to be mediated by clathrin-mediated endocytosis followed by endo-lysosomal delivery (stoneham et al., ) . the latter is a common pathway for internalized materials and escaping from the endo-lysosome seems to be critical for more efficient expression of phage transgenes. increases in serum cytokines may be a factor leading to tumor regression in this experiment. recently approved oncolytic virus t-vec harbors two copies of gene encoding gm-csf (conry et al., ) . gm-csf promotes dc accumulation at sites of inflammation and enhances antigen presenting cell functions (kaufman et al., ) . since gm-csf was expressed from transduced tumor cells, the local concentration is expected to be higher than in total serum. two other proinflammatory cytokines, il- α and tnf-α, were also increased in the presence of t displaying homing peptide and harboring expression a b figure | transwell migration assay of macrophages. cultured b f cells in the lower chamber were treated with three different titers (moi , , or ) of wild type t (wt t ) or t displaying the homing peptide and harboring an mammalian expression cassette of gm-csf (t -pep _g). macrophages (raw . ) in the upper chamber were allowed to migrate for three different time periods. (a) staining and visualization of membrane after migration. white pores are seen from the membrane and macrophages are stained with crystal violet. (b) three random fields were chosen and the migrated cells were quantitated. for statistical analysis tukey's test was performed. *above each vertical bar indicates statistical significance of each test to the control. *above each horizontal bar indicates statistical significance of each test between corresponding pairs. cassette of gm-csf. in addition, tnf-α increased in the presence of t displaying the homing peptide without expression of gm-csf, suggesting that locally accumulated, high titer of the phages themselves could stimulate cytokine production. interestingly, less increases of tnf-α were observed, where gm-csf was expressed. therefore, there may be interference between the productions of the two cytokines in this experiment. changes in the tumor microenvironment, including locally accumulated phages and increases in proinflammatory cytokines accompanied by the recruitment of various immune cells, seems responsible for tumor regression. in fact, macrophages were found in tumor tissue when various phages and/or gm-csf were administered. on the other hand, dc and cd + t cells were predominantly found in tumor tissue when the t displaying the homing peptide and harboring expression cassette of gm-csf was administered. local expression of gm-csf might lead to enhanced t-cell priming by dc. antigen priming by dcs and the direct killing of tumor cells by t cells are the most powerful way of bringing about immune-mediated tumor regression. we could observe the same set of immune cells clearly infiltrating tumor tissue, where homing phages accumulated and gm-csf was produced by the tumor cells themselves, which could lead to locally increased concentrations. in case of nk cells, we could not observe any strong recruitment. one study reported a dual role of gm-csf in recruiting nk cells (nandagopal et al., ) . at high concentrations, gm-csf strongly induced migration of nk cells, while at low concentrations, it induced hyper-polarization, immediate arrest of nk cells, and little/or no nk-cell migrations. the observation that macrophage, one of the immune cells recruited in vivo, also migrated to tumor cells treated with phage t displaying the homing peptide and harboring expression cassette of gm-csf in time-and dose-dependent manners in an in vitro transwell assay is coherent with in vivo results. the immuno-modulatory nature present in the mucosal immunity of bacteriophages was recently reported (gogokhia et al., ) . the authors described that bacteriophages or phage dna could stimulate ifn-γ production via tlr- sensing, resulting in exacerbated colitis. a greater and greater body of evidence suggests relationships between phages and mammalian immunity. there have been several reports describing tumor regression in the presence of phages. in this experiment, we have demonstrated advancements in the utilizing of phages for this purpose. first, by displaying that the homing peptide (pep ) is more highly selective for target cells when compared to kgd or rgd peptides previously described (dabrowska et al., ; quinn et al., ) . although a report had already described the peritumoral injection of phage m displaying a homing peptide (eriksson et al., ) , we utilized intravenous injections and obtained greater tumor regression, suggesting better targeting methods and the increased stability of our system. t has much more packaging capacity when compared to m , and the double stranded nature of genomic dna has a better expression and prolonged stability inside mammalian cells. unlike aavp (hajitou et al., ) , t has no dna of animal virus origin, leading to less possible side effects. taken together, the combination of tumor-targeting bacteriophages and intratumoral expression of gm-csf from transduced phage dna can efficiently stimulate the immune system, leading to tumor regression. the datasets generated for this study are available on request to the corresponding author. the animal study was reviewed and approved by 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-thazunob authors: smith, matthew l.; gandolfi, stefano; coshall, philippa m.; rahman, pattanathu k. s. m. title: biosurfactants: a covid- perspective date: - - journal: front microbiol doi: . /fmicb. . sha: doc_id: cord_uid: thazunob the recent outbreak in severe acute respiratory syndrome – coronavirus- (sars-cov- ) has demonstrated the complete inability of nations across the world to cope with the pressures of a global pandemic, especially one in which the only current feasible treatments are those which deal with the symptoms alone and not the viral cause. as the death toll rises, scientists begin to fall toward new avenues of research, with novelty showing itself to be an incredible and so far, underrated resource. in this case, the use of biosurfactants in dealing with this pandemic justifies extensive study with their potential applications being in the prevention of viral spread; dealing with the symptoms that develop after the incubation period; directly targeting viral infected cells and preventing the spread of the virus throughout the host, all in addition to also acting as potential drug delivery systems and cleaning agents. this extensive avenue of biosurfactants owes to the simplicity in their amphiphilic structure which permits them to interact directly with the lipid membrane of the coronavirus, in a way which wouldn't be of significant threat to the host. although it could possibly interact and affect the virus, it could also affect human internal organs/cells by interacting with lipid membrane, if (biosurfactant is) ingested, and it still needs further studies in human models. the structure of the coronavirus, in this case sars-cov- , is detrimentally dependent on the integrity of its lipid membrane which encloses its vital proteins and rna. biosurfactants possess the innate ability to threaten this membrane, a result of their own hydrophobic domains across their amphiphilic structure. with biosurfactants additionally being both natural and sustainable, while also possessing a remarkably low cytotoxicity, it is of no doubt that they are going to be of increasing significance in dealing with the current pandemic. introduction sars-cov- poses a serious and escalating threat to public health, after having triggered a pandemic that stems from its first recorded infection in wuhan, china during early december (hong et al., ) . in many cases, infection will induce flu-like symptoms in the host who will additionally become highly contagious, having an averaged r of roughly . , even throughout the initial incubation period which has been suggested to last up to - days (backer et al., ; liu et al., ) . complications in the immune response or infection occurring in those who already have underlying health conditions, may result in more serious symptoms such as pneumonia or acute respiratory distress syndrome (ards) which can often be fatal. this incidence of fatality paired with the ease of viral transfer which often occurs through droplet and aerosol transmission is what makes this particular strain of coronavirus so deadly (rothan and byrareddy, ) . there is current debate into the significance of the political action of many countries which could have prevented the spread from having such a global impact and whether more could have been done to better prepare healthcare services to deal with the virus. having been better equipped, hospitals may have likely been able to decrease the overall rates of death from the virus. regardless of where the responsibility for this pandemic lies, it is clear that we currently rely on international collaboration and extensive scientific prowess to deal with the issue and recover from it in a way which will best reduce the impact it can cause in the future. one such avenue of investigation is the use of biosurfactants which have proven themselves to be significant in a variety of processes, all of which being crucial in managing the pandemic by dealing with both the virus itself and the symptoms in which it can cause. the structure of the coronavirus, similarly to others of this type, consists of a lipid membrane which encloses its vital proteins and positive sense rna (vellingiri et al., ) . this is an incredibly simple structure but can cause tremendous harm when its membrane fuses with that of an animal cell, allowing its rna to be synthesized by the host. this will result in the replication of the virus with the use of the host's cellular mechanisms spreading to other cells in the body, causing exponential damage in the process. the lipid membrane, along with embedded spike proteins is crucial in the virus's ability to both maintain its integrity and also pass our cells' phospholipid bilayer which allows it to initiate its mechanism of infection (das, ) . the amphiphilic nature of biosurfactants allows them to interact with the hydrophobic domain of the viral membrane with a significant enough affinity to disrupt it, resulting in a breakdown of its structure and therefore disabling it. biosurfactants are currently used across a large range of industrial and medical processes and their innate versatility open up their use for a large variety of coronavirus related applications (randhawa and rahman, ) . in dealing with this pandemic, it is crucial that we target the virus at every stage of its transmission and incubation. the use of biosurfactants will therefore be considered in handwashes and cleaning agents to prevent the spread of the virus; targeting and relieving the symptoms after infection; acting as drug delivery systems and additionally their use in other important areas with a key example being the production reliable antiviral facemasks. biosurfactants are defined as being amphiphilic moieties which possess the ability to reduce surface tensions across the interface of typically polar substances such as oil and water, therefore exhibiting emulsification properties. biosurfactants stand out from synthetic surfactants mainly because of their biological and therefore renewable origins, being predominantly made by microbial species and some plants. compared to their synthetic counterparts biosurfactants have greater emulsification activities, work across a broader range of temperature conditions and most importantly, they have been proven to exhibit a significantly low degree of cytotoxicity (abdel-mawgoud et al., ) . the amphiphilic nature of biosurfactants means that their hydrophobic domain is able to interact with the lipid membrane of the virus, while simultaneously interacting with other hydrophilic substances such as water. this property is what allows them to disrupt the virus structure and therefore deactivate it (sandeep and rajasree, ) . biosurfactants additionally have an interesting trait in which they are able to form micellar structures around their critical micelle concentration (cmc), a value that differs greatly between the different biosurfactant types. this structure will be significant in directly targeting the virus, impacting its overall emulsification activity, while also being crucial in our application of biosurfactants in drug delivery. the micelles have the potential to work as liposomes which could directly deliver a drug to the site of infection while also protecting it from the harsh conditions in the body which would otherwise impact its function (nakanishi et al., ) . the versatility in the use of biosurfactants alongside their already large presence in both the pharmacological and food industries prove them to be a significant route in finding novel solutions to the covid- pandemic, therefore justifying their extensive research moving forward (campos et al., ; fracchia et al., ; nitschke and silva, ; ribeiro et al., ) . due to biosurfactant unique chemical structures, understanding the functional mechanisms of action as well as their toxicity to human body is crucial for their exploitation in medical field. nowadays, biosurfactant find applications as antimicrobial, antiadhesive, in immunomodulation and as antitumor. lipopeptides and glycolipids are the most effective as antimicrobial and represents an important source for the discovery of new antibiotics. biosurfactant have been shown in mammalian cells, to participate in several intercellular molecular recognition steps such as signal transduction, cell differentiation and cell immune response acting as antitumor agents by interfering with cancer progression processes (gudiña et al., ; fracchia et al., ; sajid et al., ) . the antimicrobial and anti-adhesive properties of biosurfactant relays on membrane damage/disruption, causing metabolite leakage by modification of membrane protein morphology; by affecting energy generation and metabolites transport as well as by altering the bacterial lipopolysaccharide system (lps), thus reducing cell adhesion and biofilm formation (van hamme et al., ; fracchia et al., ) . different lipopeptides have reached a commercial antibiotic status, like echinocandins, micafungin, anidulafungin, and daptomycin (fracchia et al., ) . moreover, some biosurfactant have shown immunomodulation activities (sajid et al., ) . as examples surfactin is an interesting molecule, believed to reduce the activity of macrophage by down regulating the expression of several cell surface molecule (i.e., cd ), thus being a potential candidate in the treatment of hypersensitivity related immune disorders (paine et al., ) . surfactin is also known to have anti-inflammatory activities because of its inhibitory properties on phospholipase a , on the release of interleukin and nitric oxide (kim et al., ; byeon et al., ; backhaus et al., ) . in a similar way sophorolipid injection in animals showed inhibition of pro-inflammatory cytokine and nitric oxide in the treatment of sepsis (fu et al., ) . biosurfactant have been also proposed as new molecule for the treatment of autoimmune diseases as well as potent immuno-modulator and anticancer agent (sajid et al., ) . despite their versatility some biosurfactant are produced by opportunistic bacteria and it is essential to consider their in vivo toxicity and safety. scarcity of clinical data on the use and validation of such molecules in animal models and human volunteers pose a major challenge. nonetheless, some biosurfactants have proven their efficacy in different sectors, fulfilling drug regulatory bodies requirements as biocompatible and non-toxic molecules. although reasons behind microbial biosurfactant production are currently unresolved, a likely explanation has been proposed through evolutionary analyses. this view recognizes competitive advantages generated through biosurfactant production, aiding in areas such as resource acquisition and defense, therefore increasing survivability when compared to other organisms who may be disadvantaged as a result (cameotra et al., ; kiran et al., ) . biosurfactant production has often been found to occur where species have experienced depleted resources, as well as during times where they may benefit from their antimicrobial nature. previous studies have explored the defensive nature of surfactants by expanding the application of bioactive peptides to inactivate enveloped viruses. cyclosporin a (csa) is a biopeptide produced from the fungus tolypocladium inflatum, already known to inhibit the propagation of the influenza virus by interfering with the viral cycle (garoff et al., ; khan, ) . csa does not affect adsorption or rna replication but instead inhibits steps after protein synthesis, such as assembly or budding (garoff et al., ) . this is extremely important as budding enables viruses to exit host cells and attach to derived membranes enriched in viral proteins encouraging spread and infection (hamamoto et al., ) . by targeting later events of the virus life cycle the problem of resistance to available drugs will be overcome as well as limiting spread. lipopeptide used as adjuvant or linked to low mass antigenic molecules have also been used to stimulate the immune system to produce antibodies. synthetic lipopeptide vaccines have been shown to be able to induce virus specific cytotoxic t-lymphocytes against the influenza nucleoprotein epitope (deres et al., ) . similar results have been observed against foot-and-mouth disease in vivo b-and t hcell response to hiv- loleit et al., ) . this might have a very interesting application in novel vaccines discovery and production. sophorolipids (sl), a group of microbial glycolipids produced by yeasts have shown properties as immunomodulators, anti-inflammatory and improved sepsis survival in experimental animal models (borsanyiova et al., ) . by acetylation of the sophorose head groups, sl have been active against herpes virus and hiv virus. such modification is considered to improve hydrophilicity of sl thus promoting its antiviral and cytokine-stimulating property (shah et al., ; gross and shah, ) . these are potential issues that may arise with sars-cov- . this necessitates for the screening of potential agents with novel modes of action to eliminate harmful life-threatening effects. timely antiviral administration is key throughout global pandemics and the prompt treatment of ill individuals is key to managing future cases. extreme measures such as closing public areas and social distancing may reduce the infection rate of a disease. however, only antiviral drugs or vaccines are effective in curing and preventing infection when directly exposed to sars-cov- . when facing a new and previously uncategorized virus, vaccine production is likely to be a slow process-unless the viral strain closely resembles a previously identified virus in which we have pre-established treatments. therefore, during such unprecedented times, before a cure is available it is vital to encourage safe and efficient cleaning procedures that effectively eliminate dormant forms of the virus that may lay on surfaces in public areas, clothes or homes. anionic surfactant types are commonly used in cleaning products and detergents. when applied to a surface the fatty acid chains of the surfactant bind to the hydrophobic components of microbes, while the surfactants hydrophilic domain will simultaneously bind to water with a significant affinity, resulting in the solubilization of that microbe. this emulsification reaction therefore enables the surface to be cleaned whilst depositing an effective surfactant layer. once attached the anionic detergent particles electric charge removes harmful substances from the surface and solubilize them into smaller droplets which results in an emulsification of dirt and detergent. as the surfactant molecules are continuously attached to the dirt/harmful substance the process of repulsion continues effectively preventing the same particle from being reintroduced to the surface. a visual is shown in figure . surfactants are the single most important ingredients in laundry and household cleaning products and typically account for - % of total detergent composition (yangxin et al., ) . through increased research, various combinations of surfactant types as well as alterations to surfactant volume within existing and new products have proven surfactants to be effective over traditional products such as bleach. there is no doubt that bleach has proven itself to be an incredible antimicrobial agent with its active ingredient sodium hypochlorite being effective in destroying bacteria, fungi and viruses. however, bleach can irritate skin, airways and mucus membranes, which suggests prolonged exposure to be harmful. bleach also decomposes under heat and light and reacts easily with other chemicals decreasing its effectiveness. improper use of bleach such as deviations from recommended dilutions may also affect performance and its overall use is likely to carry with it its own environmental implications which increase in significance when we consider the large-scale washing of public areas to prevent viral spread. all the issues stated above highlight how using bleach alone for disinfection in public areas or areas of high risk such as hospitals/surgeries can involve injury to health-care workers and increase immunosuppression which may lead to increased susceptibility to sars-cov- especially in areas where exposure to the virus is more likely [world health organization (who), ]. therefore, using products that contain biosurfactants in conjunction with or as an alternative to heavily chemical cleaners may be more effective in efficient disinfection. biosurfactants have great advantages as eco-friendly and lesstoxic alternative to synthetic surfactant and to date, glycolipids (sophorolipids, rhamnolipids, and mannosylerythritol lipids) are the most commercialized in cleaning applications by different companies world-wide. companies like saraya, ecover, and henkel apply sl in their laundry, dishwashing and cleaning products whereas basf, evonik, teegene and unilever are commercializing rhamnolipids and lipopeptide biosurfactants based products (klosowska-chomiczewska et al., ; fracchia et al., ; randhawa and rahman, ; singh et al., ) . since the start of the pandemic in december , demand for various products -especially gloves, soaps, disinfectants and hand sanitizers has increased dramatically. a huge emphasis has arisen on the necessity of cleanliness, specifically the efficient cleaning of hands after coming into contact with potentially contaminated surfaces both indoors and outdoors. due to this, governments globally have had to update and define technical and regulatory standards for such products to ensure safe practice and manufacture whilst dealing with the pandemic. information regarding proper hand washing techniques as well as products content has been published. specifically, the uk government has released technical specifications for the production of hand wash and associated legislation of personal protective equipment (ppe) as well as prerequisites before items are sold. stricter regulations ensure consistency and guaranteed effectiveness of hand washing products which are vital during times of a pandemic (cabinet office gov.uk, ) the effectiveness of hand sanitizers compared to washing hands with soap and water has been of constant debate as of recently. the c.d.c (center for disease control and prevention) has stated that using soap when washing hands is more effective than hand sanitizer or water alone [centers for disease control and prevention (cdc), ]. this is because surfactants present in soaps lift and remove microbes, harmful substances and dirt from skin. furthermore, the lather produced from soap as well as effective scrubbing work well to remove contaminants. the use of alcohol-based hand sanitizer that contain at least % alcohol is still recommended if washing hands with soap is not available. hand sanitizers do not get rid of all types of germs and may not be as effective where hands are excessively greasy or dirty [centers for disease control and prevention (cdc), ]. alcohol is extremely effective, but efficacy differs among different types. for example, ethyl alcohol ( %) is a powerful germicide and is considered generally superior to isopropyl alcohol. it is also important to consider the prolonged and repeated use of alcohol-based products such as a disinfectant which can cause discoloration and damage to the skin [world health organization (who), ]. whereas, teegene has reported about lipopeptide and rhamnolipid biosurfactant based cosmetics (randhawa and rahman, ; focus on surfactants, ) and evonik has reported their sophorolipid biosurfactant for skin conditioning, refatting and moisturizers properties to be used in shampoo, shower gel and household cleaners (focus on surfactants, ) and it also has a potential use in handwash applications. acute respiratory distress syndrome (ards) is a progressive medical syndrome, characterized by a build-up of fluid in the patient's alveoli which result in inefficient oxygen transfer across these alveolar membranes into the blood (matthay et al., ) . ards is often a consequence of an already serious medical concern, in this case covid- , with the resulting lack of oxygen to organs contributing to the high fatality rates seen in those who begin to develop symptoms (ware and matthay, ) . one leading cause for this alveolar fluid build-up, in response to infection by sars-cov- , is surfactant dysfunction which has negative consequences on the emulsification and thereby clearance of liquid from this particular region. current treatments for this rely on ventilators to supplement the body with the oxygen which otherwise would not be able to successfully transfer into the blood, relieving the immediate symptoms which would otherwise lead to issues such as hypoxia (luks et al., ) . socioeconomic factors have played a large role in the effectiveness of such a treatment, with providing enough ventilators and facilities for each patient affected proving to be difficult and, in some cases, impossible considering the cost, space and training required to use them. with this in mind, biosurfactants present themselves to be a promising area of study in identifying novel treatments for ards which could overcome the socioeconomic barriers that have so far limited the effectiveness of ventilators. it is for this reason that future study for their use as a direct treatment for ards, through solubilizing the alveolar substrate, is likely to generate positive results and is therefore crucial in combating covid- in the future. when considering possible treatment opportunities for the covid- pandemic, it is crucial to decide upon a mode of drug delivery which doesn't compromise the molecular nature of the product while also being able to deliver it successfully to the area of interest. with the sars-cov- predominantly impacting the respiratory system and upper gastrointestinal tract, a likely mode of delivery would be via aerosol or lozenge. the micellar nature of biosurfactants result in them being the ideal candidates for either system of drug delivery, allowing them to form a stable liposome which will encase the drug, protecting it from damage which may otherwise cause dysfunction (sosnowski and gradon, ). the physicochemical characteristics of biosurfactants allow them to maintain their integrity while used in an aerosol, this would be the likely mode of drug delivery considering the main area of virulence to be within the lungs. the solubility of biosurfactants will work to their advantage throughout this process as they will evidently increase the bioavailability of the drug, once it has been administered. this self-solubilising nature of biosurfactants therefore advances the drugs dose proportionality, resulting in more consistent impacts across patients (omkar et al., ) . the ability for biosurfactants to mediate drug delivery is apparent, however the benefits of their use in this way are -fold. in addition to providing safe passage for the drug to the target, the biosurfactants will also exhibit natural antiviral properties at the site of infection, in addition to also relieving surfactant dysfunction in the alveoli, another consequence of sars-cov- infection. in this way, they will be able to inhibit a number of viruses present around that given area while also directly relieving symptoms, a significant factor reducing its virulence and transmission between hosts. this characteristic has furthermore been expanded upon as researchers ponder the ability for biosurfactants to themselves behave as a treatment in this way. one such example includes the addition of clinically approved biosurfactants in gummies or lozenges, as they are consumed the biosurfactants will directly reach parts of the mouth and esophagus which may be impacted and therefore provide symptomatic relief (vellingiri et al., ) . in addition to this, the biosurfactant will likely form a vapor in the mouth which can be inhaled through the process of ingestion, allowing it to reach areas of the respiratory tract to potentially provide relief in that area as well. the covid- pandemic is one which has had a detrimental impact on public health, seriously hindering the normal functionality of our society and therefore resulting in massive hardship across both our economy and public well-being. it is for this reason that scientists have been at a bid to find novel ways in which this pandemic can be combatted, across all of its planes of influence throughout our society. biosurfactants have not only proven themselves to be the ideal candidates to behave as this novel solution but have done so in a way which targets many of the avenues that are crucial in resolving a pandemic of this scale. their potential in behaving as safe and effective cleaning solutions have been discussed and they have been recognized as being a valid alternative to current procedure. in addition to this, biosurfactants have been found to act as significant drug delivery systems, housing multiple benefits to their use, most importantly showing a great potential in being able to successfully deliver drugs, maintaining dose proportionality in the process. finally, the potential for biosurfactants to exhibit medicinal qualities in the treatment of sars-cov- infection, particularly in the relief of symptoms associated with ards, have shown exceptional promise. the lack of knowledge shrouding this phenomenon therefore justify extensive future research. a prominent barrier remains in the large production costs associated with biosurfactant bioprocessing, a significant factor which must not be overlooked and should remain a focal point for future study. reducing the cost of bioprocessing, alongside the continued research of biosurfactant applications in this area are key to success within this field. the incredible versatility found across the structure and functions of biosurfactants mean that their reach in effectiveness is by no means limited to the applications which have been so far highlighted. as research progresses in this area, the feasibility of both their use and their socioeconomic development increase. the results of this pandemic have been so far detrimental however, it is crucial that we push forward and adapt such ideas in finding the very solutions that we need to combat the consequences of this virus and pathogens in the future. in this way, biosurfactants bring promise to a scenario that is often shrouded in despair, with research and scientific prowess we will not only overcome the issues of this pandemic, but we will additionally better equip ourselves for the future. all datasets presented in this study are included in the article/ supplementary material. ms, sg, pc, and pr have equally contributed in the preparation of this manuscript. this study was funded by the expanding excellence in england (e ) scheme, research england, uk. rhamnolipids: diversity of structures, microbial origins and roles incubation period of novel coronavirus ( -ncov) infections among travellers from wuhan, china surfactant inhibits atp-induced release of interleukin- via nicotinic acetylcholine receptors biological activity of sophorolipids and their possible use as antiviral agents surfactin blocks no production in lipopolysaccharide-activated macrophages by inhibiting nf-κb activation technical specifications for personal protective equipment (ppe) synthesis of biosurfactants and their advantages to microorganisms and mankind microbial biosurfactants as additives for food industries keeping hands clean can bioactive lipids inactivate coronavirus (covid- )? in vivo priming of virus-specific cytotoxic t lymphocytes with synthetic lipopeptide vaccine teegene biotech develops method to produce biosurfactants using unique strains of bacteria evonik commercializes biosurfactants potential therapeutic applications of microbial surface-active compounds 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biosurfactant production: emerging trends and promising strategies influence of a biosurfactant on entrainment and de-aggregation of powders physiological aspects: part in a series of papers devoted to surfactants in microbiology and biotechnology covid- : a promising cure for the global panic the acute respiratory distress syndrome novel lowmolecular-weight synthetic vaccine against foot-and-mouth disease containing a potent b-cell and macrophage activator annex g -use of disinfectants: alcohol and bleach, " in infection prevention and control of epidemic-and pandemic-prone acute respiratory infections in health care development of surfactants and builders in detergent formulations authors thank research england for funding support through the expanding excellence in england (e ) scheme. the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © smith, gandolfi, coshall and rahman. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- -j r nq authors: hernando-amado, sara; coque, teresa m.; baquero, fernando; martínez, josé l. title: antibiotic resistance: moving from individual health norms to social norms in one health and global health date: - - journal: front microbiol doi: . /fmicb. . sha: doc_id: cord_uid: j r nq antibiotic resistance is a problem for human health, and consequently, its study had been traditionally focused toward its impact for the success of treating human infections in individual patients (individual health). nevertheless, antibiotic-resistant bacteria and antibiotic resistance genes are not confined only to the infected patients. it is now generally accepted that the problem goes beyond humans, hospitals, or long-term facility settings and that it should be considered simultaneously in human-connected animals, farms, food, water, and natural ecosystems. in this regard, the health of humans, animals, and local antibiotic-resistance–polluted environments should influence the health of the whole interconnected local ecosystem (one health). in addition, antibiotic resistance is also a global problem; any resistant microorganism (and its antibiotic resistance genes) could be distributed worldwide. consequently, antibiotic resistance is a pandemic that requires global health solutions. social norms, imposing individual and group behavior that favor global human health and in accordance with the increasingly collective awareness of the lack of human alienation from nature, will positively influence these solutions. in this regard, the problem of antibiotic resistance should be understood within the framework of socioeconomic and ecological efforts to ensure the sustainability of human development and the associated human–natural ecosystem interactions. the problem of antibiotic resistance (ar) has been traditionally addressed by focusing on humanlinked environments, typically health care facilities. nevertheless, it is now generally accepted that most ecosystems may contribute to the selection and spread of ar (aminov, ; martinez et al., ; davies and davies, ; martinez, ; berendonk et al., ; larsson et al., ) . a key conceptual point is that, based on cultural, humanitarian, and economic reasons, we have historically preserved the health of individual humans and farming animals. to that purpose, the same families of antimicrobial agents have been used. as a consequence, their positive (healing) and negative (selection of ar, therapeutic failure) effects have influenced the common health of humans and animals in particular locations (one health). the concept one health, first used in early twentieth century, expands the integrative thinking about human and animal medicine, including for the first time ecology, public health, and societal aspects (zinsstag et al., ) . in the case of ar, the one health perspective focuses on the risk assessment of emergence, transmission, and maintenance of ar at the interface between humans, animals, and any other linked (local) environment (robinson et al., ; jean, ) . consequently, the application of one health approaches demands integrative surveillance tools and interventions based on multidisciplinary approaches that include ecological and sociodemographic factors, besides more classic epidemiological models. global health is based on a broad collaborative and transnational approach to establish "health for all humans." in this case, it focuses ar at a general (global) scale, considering that the selection and global spread of antibiotic-resistant bacteria (arbs) and antibiotic resistance genes (args) are a problem that influences the health of human societies with disparate social and economic structures and is linked to many societal and ecological factors (chokshi et al., ) . interventions to reduce ar burden in a global world certainly require common and integrated policy responses of countries, international organizations, and other actors (stakeholders included). its goal is the equitable access to health and minimizing health risks all over the globe. besides its objective aspects (i.e., how travelers, migrating birds, or international commerce may contribute to ar spread), it has important international political aspects. it focuses in how countries and international organizations address the elements connecting and potentially spreading ar among humans, animals, and natural ecosystems at the earth scale (wernli et al., ) . in summary, the problems and the potential solutions concerning ar are not confined to particular regions, but have a global dimension: a problem for all humans, animals, and natural ecosystems, which should be solved with interventions aiming to improve health for all of them koplan et al., ; laxminarayan et al., ) . in the context of ar, a healthy environment would be an environment where ar is low or can be controlled by human interventions (hernando-amado et al., ; andersson et al., ) . of course, the global health concept of "health of an environment" (iavarone and pasetto, ; pérez and pierce wise, ; bind, ; van bruggen et al., ) or, in general, planetary health (lerner and berg, ) , has an unavoidable anthropogenic flavor. in practice, we consider "healthy environments" or "healthy ecosystems" those that minimize their current or their potential harm for the human individual or the society, in our case for ar. in other words, we adopt a selfish strategy, which should be necessarily implemented by the international (global) institutions. selfishness (kangas, ) applies mainly to individuals, but also to societal groups. however, these groups have not enough possibilities to act alone in the case of infectious diseases in general and ar in particular, which may expand worldwide. therefore, individual selfishness for health should be integrated in local one health and also in global health actions. the goal of controlling ar is a highly complex one, and its dimension has been compared to climate change or biodiversity loss, problems where individual actions are not enough for providing a solution, and consequently, individual freedom is confronted with collective responsibility (looker and hallett, ) . the construction of human societies reflects the tension between individual freedom and social rules/laws. the implementation of different social rules/laws for regulating human activities within a society is mainly based on moral (as kant's categorical imperative (kant, ) or religious-based brotherhood (matthew : - ) statements), social stability (as anticrime laws; schiavone, ) , organizative (type of government and how it is formed, group identity), and efficacy (as antitrust laws; ricardo, ) arguments. however, these arguments mainly apply for establishing the socioeconomic organization as well as the individual welfare within a society. the situation concerning human health is somehow different. there are individual diseases, such as cancer or stroke, and social diseases, such as transmissible infections. for the firsts, social norms (as consciousness of the importance of the control of cholesterol, excess sugar uptake, or hypertension levels) are well established, and even laws (non-smoking regulations) had been implemented in occasions. however, the main impact of these regulations is at the individual health level (wikler, ) , because the associated diseases are not physically transmissible. a different situation happens in the case of infectious diseases in general and of ar in particular. for these diseases, everything that happens in a single person affects any one around. further, the fact that an arg emerging in a given geographic area can spread worldwide implies that neither individual norms nor country-based norms have been sufficient until now to counteract the worldwide spread of ar. one important aspect of laws in democratic societies is that they must be well accepted by the community, so that the acceptation of social norms usually comes first than their implementations as rules/laws. actually, the efficiency of democracy for responding to social crisis (as current ar or covid- crises), in opposition to other more autocratic regimens where decisions are implemented top-down, had been the subject of debate from the early beginning of democratic revolutions (tocqueville, ; hobbes, ; rousseau, ; spinoza, ) . in this regard, it is important to remark that one health aspects of ar can be tackled in the basis of countrylevel regulations that are linked to the socioeconomic and cultural aspects of each country (chandler, ; chokshi et al., ) . however, because global earth governance does not exist, global health control of ar is based on recommendations, rather than in rules/laws. consequently, the acceptance of social norms, starting within individuals or small organizations and expanding throughout the whole society (figure ) , is fundamental to provide global solutions to the ar problem (nyborg et al., ; chandler, ) . the acceptance by the community of these social norms, considering that the way of promoting these norms might differ in different parts of the world (cislaghi and heise, ; cislaghi and heise, ) , largely depends on the transfer to the society of the knowledge required to understand the mechanisms and the impact for human health of the emergence and transmission of ar, an information that is discussed below. figure | how the interactions among individual health, one health, global health, and social norms influences antibiotic resistance. the right panel shows the different levels of dissemination of antibiotic resistance. in the left panel, the different types of norms (from individual to global norms) that can impact antibiotic resistance at each level are shown. these norms influence all levels of transmission: the individual promotes (red arrows) his own individual health, but doing it also promotes the health of the group, and the health of the group promotes global health of the human society at large. at each level, there is a positive action (red broken lines) on antibiotic resistance. such dynamics largely depends on social norms (blue arrows) rewarding the individual or the groups whose behavior promotes health. below the left panel, the basic social norm, progress and development, has consequences on the whole ecobiology of the planet (lower panel with bullet points), influencing the undesirable open circulation of antimicrobial resistant bacteria (with their mobile genetic elements) and antibiotic resistance genes. the classic definition of ar is based only on the clinical outcome of the infected patient. an organism is considered resistant when the chances for the successful treatment of the infection it produces are low . this definition, which is the most relevant in clinical settings, presents some limitations for studies based on one health approaches that include the analysis of non-infective organisms, which lack a clinical definition of resistance, as well as analysis of the distribution of args, in several occasions, using non-culture-based methods . even in the case of animal medicine, antibiotic concentration breakpoints defining resistance are still absent for some veterinary-specific antimicrobials and poorly defined for different types of animals with disparate weights, which would influence the availability of the drug inside animal body (toutain et al., ; sweeney et al., ) . to analyze ar beyond clinical settings, the term resistome, understood as the set of genetic elements that can confer ar, irrespectively of the level of resistance achieved, in a given organism/microbiome was coined (d'costa et al., ; wright, ; perry et al., ) . ar acquisition is the consequence of either mutation (or recombination) or recruitment of args through horizontal gene transfer (hgt), transformation included. ar mutations are generally confined to their original genomes, propagating vertically and not spreading among bacterial populations, although some few exceptions of horizontal transfer of chromosomal regions containing ar mutations have been described (coffey et al., ; ferrandiz et al., ; novais et al., ; nichol et al., ) . the set of mutations that confer ar can be dubbed as the mutational resistome. current wholegenome-sequencing methods of analysis can allow defining the mutational resistome in an isolated microorganism (cabot et al., ; lopez-causape et al., ) . however, they are not robust enough yet for determining the mutational resistome in metagenomes. consequently, the impact of these analyses in one health studies is still limited and will not be further discussed in the present review. concerning their relevance for acquiring ar, args can be divided in two categories. the first one comprises the genes forming the intrinsic resistome (fajardo et al., ) , which includes those that are naturally present in the chromosomes of all (or most) members of a given bacterial species and have not been acquired recently as the consequence of antibiotic selective pressure. despite that these genes contribute to ar of bacterial pathogens, they are responsible just for the basal level of ar, which is taken into consideration when antibiotics are developed. in this regard, unless these genes, or the elements regulating their expression mutate, they are not a risk for acquiring resistance and have been considered as phylogenetic markers . further, it has been discussed that these genes may contribute to the resilience of microbiomes to antibiotic injury (ruppe et al., b) , hence constituting stabilizing element of microbial populations when confronted with antibiotics more than a risk for ar acquisition by pathogens. the second category, dubbed as the mobilome, is formed by args located in mobile genetic elements (mges) that can be transferred both vertically and horizontally, hence allowing ar dissemination among different bacteria (frost et al., ; siefert, ; jorgensen et al., ; lange et al., ; martinez et al., ) . while the analysis of the resistome of microbiota from different ecosystems has shown that args are ubiquitously present in any studied habitat (d'costa et al., ; walsh, ; jana et al., ; lanza et al., ; chen et al., b) , the impact of each one of these args for human health is different. indeed, it has been stated that the general resistome of a microbiome is linked to phylogeny and to biogeography, indicating that most args are intrinsic and do not move among bacteria (pehrsson et al., ) . however, some args escape to this rule and are shared by different ecosystems and organisms (forsberg et al., ; fondi et al., ) . these mobile args, frequently present in plasmids (tamminen et al., ; pehrsson et al., ) , are the ones that are of special concern for human health. although not belonging to the antibiotic resistome, genes frequently associated with resistance to other antimicrobials, such as heavy metals or biocides, as well as the genes of the mges backbones, eventually involved in the transmission and selection of args among microbial populations, the mobilome at large, are also relevant to track the emergence and dissemination of ar among different habitats martinez et al., ; baquero et al., ) . hgt processes are recognized as the main mechanisms for transmission of genetic information (baquero, ) . from the ecological point of view, hgt should be understood as a cooperative mechanism that allows the exploitation of common goods as args by different members within bacterial communities. in fact, some studies suggest that the ecological consequences of hgt events in ar evolution are contingent on the cooperation of complex bacterial communities, besides the acquisition of individual adaptive traits (smillie et al., ) . however, the understanding of the ecological causes and consequences of args transmission among organisms and microbiomes is still limited from the one health and global health perspectives. hgt-mediated ar is a hierarchical process (figure ) in which args are recruited by gene-capture systems as integrons and afterward integrated in mges as plasmids, insertion conjugative elements, or bacteriophages (frost et al., ; garcia-aljaro et al., ; gillings et al., ; botelho and schulenburg, ) , which afterward are acquired by specific bacterial clones. selection at each of these levels will also select for all the elements involved in ar spread. for instance, the acquisition of an arg by a clone may promote the expansion of the latter (and of all the genetic elements it contains, other args included) in antibiotic-rich environments, such as hospitals or farms schaufler et al., ) , and vice versa, the introduction of an arg in an already successful clone may increase the chances of this resistance gene for its dissemination even in environments without antibiotics, unless the associated fitness costs are high. in this sense, if arg acquisition reduces the fitness, and this implies a decreased capability for infecting humans (see below), the burden for human health might eventually be lower. nevertheless, it is relevant to highlight that ar transmission cannot be understood just by analyzing the genetic mechanisms involved and the consequences of such acquisition for the bacterial physiology. indeed, as discussed below, there are ecological and socioeconomic elements that strongly influence ar dissemination. the evolution of ar comprises the emergence, the transmission, and the persistence of arbs (martinez et al., ; baquero et al., ) . concerning human health, selection of arbs/args is particularly relevant at the individual health level, whereas transmission is a main element to be taken into consideration at the one health and global health levels (figure ) . indeed, unless ar is transmitted, it will be just an individual problem that would not affect the community at large. it is generally accepted that non-clinical ecosystems are often primary sources of args (davies, ) . as above stated, after their capture and integration in mges (figure ), args and their bacterial hosts can contaminate different ecosystems, which might then be involved in their global spread (martinez, ; fondi et al., ; gillings, ; gillings et al., ) . this means that nearly any ecosystem on earth, along with the humandriven changes produced in it, may modulate evolution of ar. importantly, the huge escalation and worldwide expansion of a limited set of animals, plants, and their derived products, including foods, due to the anthropogenic selection of a few breeds and cultivars for mass production in livestock and agricultural industries (okeke and edelman, ; zhu et al., ) of economic interest have collapsed the variability and biodiversity of animals and plants (seddon et al., ) . because these organisms harbor particular host-adapted bacteria, which are frequently under antibiotic challenge, this situation, together with the ecological similarities of human habitats, might favor ar spread (martiny et al., ; manyi-loh et al., ) . indeed, while in underdeveloped areas of the world food animals are very diverse, intensive farming, common in developed countries, ensures a "shared-stable" environment where only the most productive types prevail (kim et al., ) . the common genetic origin of these types and the process of microbiota acquisition from nearby animals in intensive farming should homogenize also their microbiomes with consequences for ar dissemination. actually, it has been shown that the loss of microbial diversity figure | genetic, ecological, and socioeconomic elements mediating the transmission of antibiotic resistance. args are ubiquitously present in any studied microbiome (a). however, only a few of them are transferred to human/animal pathogens, hence constituting a health problem. the genetics events implied include the acquisition of args by gene-recruiting genetic elements such as integrons (b); the integration of these elements in mges as plasmids, bacteriophages, or frontiers in microbiology | www.frontiersin.org figure | continued insertion conjugative elements (c); and the acquisition of these elements by specific bacterial clones (d). these arbs can share these elements among the members of gene-sharing communities (e) and also move among different ecosystems, including humans, animals (particularly relevant farm animals), and natural ecosystems (with a particular relevance for water bodies). the connection of these ecosystems, as well as the reduced diversity of animals, plants, and in general habitats as the consequence of human activities, allows the different microbiomes to be in contact, favoring args transmission among the microorganism they encompass (f). this transmission is facilitated at the global scale by travel, animal migration, trade of goods, and eventually by meteorological phenomena, climate change included (g), hence producing a global health problem (h). while most studies on the dissemination of args focus on mges (davies, ; muniesa et al., ; lanza et al., ; garcia-aljaro et al., ) , recent works suggest that the contribution of natural transformation (orange arrow), allowing the direct uptake of args by natural competent microorganisms, may have been underestimated (domingues et al., ; blokesch, ) . further, competence can occur due to interbacterial predation (veening and blokesch, ) , a biological interaction that may facilitate the acquisition of beneficial adaptive traits by predator bacterial species (cooper et al., ; veening and blokesch, ) . other hgt mechanisms, such as dna packing in extracellular vesicles (ecv) or transference of dna through intercellular nanotubes, also seem to be relevant in nature (dubey and ben-yehuda, ; fulsundar et al., ) . while the biotic conditions that may enhance hgt have been studied in detail, less is known concerning abiotic modulation of args transfer. under contemporary conditions, at least microorganisms are affected by a freeze-and-thaw cycle, at least are agitated by sand, and at least are subjected to conditions suitable for electrotransformation every year (kotnik and weaver, ) . may favor ar spread (chen et al., a) . note that, beyond the transmission of particular ar spreading clones, ar is expected to spread in farms by the modification (eventually homogenization) of animals' microbiota. notwithstanding, even farm workers are subject to microbiome acquisition from animals, leading to microbiome coalescence sun et al., ) . it is to be noticed, and the recent covid- crisis exemplifies it, that besides economic development, cultural habits are relevant in the use of animals for food, a feature that has not been analyzed in detail, particularly with respect to their role as vectors potentially involved in ar dissemination. despite that the homogenization of hosts may help in ar transmission, the spread of arbs has some constraints, because the differential capability of each bacterial clone for colonizing different hosts may modulate their dissemination. indeed, while some species and clones are able to colonize/infect different animal species, humankind included, several others present some degree of host specificity (price et al., ; sheppard et al., ) . further, it has been shown that the capacity to colonize a new host is frequently associated with a reduction in the capacity for colonizing the former one. the same happens for mobile args; they are encoded in mges that present different degrees of host specificity, which defines the formation of gene-exchange communities, where the interchange of genetic material among members is facilitated (skippington and ragan, ) . conversely, the incorporation of different replicons and modules within plasmid backbones, a feature increasingly reported (douarre et al., ) , would enable arg replication in different clonal/species background and thus modify the community network of args. actually, the risk for humans of animal-based ar seems to be linked in most cases to shuttle, generalist clones able to colonize humans and particular animals (price et al., ; sheppard et al., ) . the understanding of the elements driving the transfer of ar among animals, humans included (figure ) , requires the comprehensive survey of the clones and args that are moving among them (european food safety authority et al., ). tools to track the global epidemiology of antimicrobial-resistant microorganisms such as bigsdb (jolley et al., ) or comprehensive databases of args, ideally providing information of their mobility (zankari et al., ; alcock et al., ) , are fundamental for studying ar transmission at a global level. it is worth mentioning that, because humans constitute a single biological species, the human-associated organisms spread easily among all individuals. in fact, more prominent differences in humans' microbiome composition can be observed between individuals than among ethnic groups, even though, as expected, the resemblance in microbiotas is higher among those groups that are geographically clustered (deschasaux et al., ; gaulke and sharpton, ) . some groups of human population are, however, more prone to acquire arbs, due either to socioeconomic or to cultural factors. in lmics (low-to medium-income countries) and brics (brazil, russia, india, china, and south africa) countries, the combination of wide access to antibiotics, weak health care structures, and poor sanitation defines certainly a dangerous landscape. moreover, the progressive aging of the western population might favor the establishment and further expansion of an elderly reservoir of arbs and args, an issue that deserves further studies. the hypothesis that the microbiome of elder people might be a reservoir of ar is based not only on their cumulative history of antibiotic exposure and contacts with health care centers, but also on the rampant use of antibiotics of this population more prone to suffer from acute, chronic, or recurrent infections. significant worldwide advances in the organization of medical care of the elderly people lead to frequent hospitalizations, but health care centers may also facilitate the selection and further amplification of ar in the community. in addition, this may subsequently favor the entry of high-risk clones and of args in the hospital setting (hujer et al., ) . as stated above, there is a global increasing permeability of the natural biological barriers that have historically prevented bacterial dissemination through different ecosystems. besides local spread of ar in environments shared by animals and humans, which has to be addressed under a one health approach, ar can disseminate worldwide (figure ) by economic corridors that promote the global interchange of goods and trade or human travelers or by natural bridges, such as animal migration paths or natural phenomena such as air and water movements (okeke and edelman, ; baquero et al., ; allen et al., ; overdevest et al., ; kluytmans et al., ; fondi et al., ) . the result is the appearance of similar arbs and args in different geographic areas. as the consequence, ar is a global health problem in the sense that an arb that emerges in a given place can rapidly spread worldwide. indeed, multidrugresistant bacteria, similar to those encountered in clinical settings, have been detected in human isolated populations that were not previously in contact with antibiotic, as well as in wildlife (clemente et al., ) . this indicates that pollution with args is present even in places where antibiotic concentrations are low (kümmerer, ) and might involve mechanisms of transmission that do not require selection. for instance, migrating birds can carry enteropathogenic bacteria resistant to different antibiotics (middleton and ambrose, ; poeta et al., ) , and international travelers, even those not receiving antibiotic treatments, also contribute to ar transfer among different geographic regions (murray et al., ; reuland et al., ) . in the group of long travelers are refugee people, in which dissemination of multidrug-resistant strains is favored by the poor sanitary conditions and overcrowding camps that refugees confront (maltezou, ) . a final issue concerning ar is its stability in the absence of selection. it has been proposed that the acquisition of ar reduces bacterial competitiveness in the absence of antibiotics (fitness costs) (andersson and hughes, ; martinez et al., ) ; certainly, a wishful proposition such as, if true, the reduction in the use of drugs or eventually antibiotic-cycling strategies should decrease ar (beardmore et al., ) . nevertheless, eliminating the use of an antibiotic does not produce a full decline of ar (sundqvist et al., ) . in fact, different studies have shown that ar not always reduces fitness but also can even increase bacterial competitiveness (andersson and hughes, ; schaufler et al., ) . in addition, compensatory mutations or physiological changes that restore fitness can be selected in resistant bacteria (andersson, ; schulz zur wiesch et al., ; olivares et al., ) . it is a fact, however, that although arbs are found nearly everywhere, including wild animals, natural ecosystems, or people from isolated populations without contact with antibiotics, among others (durso et al., ; clemente et al., ; alonso et al., ; fitzpatrick and walsh, ; power et al., ) , ar prevalence is consistently lower when antibiotics are absent, which suggests that pollution may impact ar, a feature that is discussed below. pollution of natural ecosystems is associated with activities that have driven relevant economic transition, in principle favoring human welfare, such as mining, industry, intensive land use, or intensive farming, among others. notwithstanding, globalization of health services, as well as the shift toward intensive farming, besides their positive contribution to human wellbeing, has rendered an increasing pollution by compounds with pharmacological properties of natural ecosystems, particularly water bodies, which may disrupt the stability of these ecosystems (oldenkamp et al., ) . among them, antibiotics are considered the most relevant cause of ar selection. despite regulations for reducing their use (van boeckel et al., ) , a substantial increase in global antibiotic consumption has occurred in the last years, and an even greater increase is forecasted in the next years (klein et al., ) . however, antibiotics are not the unique pollutants that can prime the selection and spread of ar. in this regard, it is important to highlight that heavy metals are one of the most abundant pollutants worldwide (panagos et al., ) . their abundance results from anthropogenic-related activities, such as mining, industry, agriculture, farming, or aquaculture and even for therapeutic use in ancient times. importantly, they may persist in nature for long periods of time. further, likely because metal pollution occurred before the use of antibiotics, heavy metal resistance genes were incorporated to mge backbones before args (mindlin et al., ; staehlin et al., ) . this means that heavy metals may coselect for mges and the args they harbor (partridge and hall, ; staehlin et al., ; zhao et al., a) . even more, the presence of heavy metals, as well as of biocides or sublethal antibiotic concentrations (jutkina et al., ; zhang et al., ) , may stimulate hgt, as well as modify the dynamics of antibiotics, such as tetracyclines, in natural ecosystems (hsu et al., ) . coselection may also occur when a single resistance mechanism, such as an efflux pump, confers resistance to both heavy metals and antibiotics (cross-resistance) (pal et al., ) . although most published works analyze the effect of different pollutants on their capacity to select arbs or args, it is important to highlight that args should also be considered pollutants themselves. actually, a recent work indicates a close relationship between the abundance of args and fecal pollution (karkman et al., ) . in this respect, it is worth mentioning that, differing to classic pollutants, args/arbs are not expected to disappear along time and space, but rather, their abundance may even increase as the consequence of selection and transmission (martinez, ) . while the direct selection of ar by antibiotics or the coselection mediated by other pollutants, as the aforementioned heavy metals, has been discussed (wales and davies, ) , the effect of other types of human interventions on the dissemination of args and arbs through natural ecosystems has been analyzed in less detail. as an example, it has been proposed that wastewater treatment plants, where commensals, arbs, args, and antibiotics coexist, could act as bioreactors favoring the selection and transmission of args between different organisms (rizzo et al., ; su et al., ; manaia et al., ) , although evidences supporting this statement are scarce (munck et al., ; azuma et al., ) . in addition to the aforementioned pollutants with a direct effect in ar selection, it is worth noting that there are other abundant contaminants, such as sepiolite (present in cat litters or used as a dietary coadjuvant in animal feed) or microplastics, present in almost all aquatic ecosystems, which can favor the transmission of args or mges between bacterial species (rodriguez-beltran et al., ; kotnik and weaver, ; arias-andres et al., ) , hence amplifying the ar problem at a global scale. finally, the possible effect of climate change on the spread of ar is worth mentioning. indeed, it modifies the biogeography of vectors (such as flies, fleas or birds) involved in the spread of infectious diseases (fuller et al., ; beugnet and chalvet-monfray, ) . in addition, the increase of local temperatures seems to correlate with an increased ar abundance in common pathogens (macfadden et al., ) . besides, climate change is affecting ocean currents (martinez-urtaza et al., ) , which may allow the intercontinental distribution of arbs and args (martinez, a,b) . although this phenomenon might contribute to the globalization of ar, further research is needed to clearly demonstrate a cause-effect relationship. it is relevant to mention that increased pollution and climate change are the unwanted consequences of human development. it would then be worth discussing how human development in general may impact (positively and negatively) ar, a feature that is analyzed below. human development is a necessity of our human behavior, although different models of development have been and are proposed, each one producing different impacts in the structure of human societies and on the preservation and stability of natural ecosystems (fenech et al., ; farley and voinov, ; seddon et al., ) . nevertheless, even for different socioeconomic models, there are some social norms that tend to be widely accepted, in particular those aiming to improve individual well-being. this implies the establishment of a society of welfare, understood as a right of any human on earth, a feature that depends on the economic development, and can be particularly relevant in the case of transmissible infectious diseases in general and of ar in particular. a continuously repeated mantra in worldwide ar policies is that the abusive consumption of antibiotics for the treatment or prevention of infections in humans and animals constitutes the major driver of ar. however, we should keep in mind that antibiotics constitute an important example of human progress supporting individual and global human health. in fact, the origin of the massive production of antimicrobials was a consequence of the needs resulting from world war ii in the s. this was followed by many decades of human progress, most importantly by the common understanding of equal human rights, which was followed by the economic and social development (including medicine and food industry) of densely populated regions in the planet, including india and china. these countries are currently among the leaders in the production and consumption of antimicrobial agents. notwithstanding, as in any area of economy, progress bears a cost that, in this case, is antibiotic pollution of the environment, globally accelerating the process of the emergence, the transmission, and the persistence of arbs (martinez et al., ; baquero et al., ) . the non-controlled use of antibiotics is facilitated in lmics with disparate economic growth by different factors. heterogeneous regulation of antibiotic sales and prescriptions (often weak or missing) and the increase of online on-bulk sales in recent years contribute to their overuse (mainous et al., ). most of live-saving medicines represent out-of-pocket costs in most lmics, which led to an exacerbated use of cheap (usually old and less effective) antibiotics, phasing out their efficacy and increasing the demands and prices for the most expensive ones, eventually resulting in treatment unavailability (newton et al., ) . further, the cost of treating ar infections is much higher than that of treating susceptible ones, which is increasing the cost of health services (wozniak et al., ) . conversely, the growing economic capability of lmics in the brics category triggers the access of the population to health services and last-resort antibiotics. these countries also face a sudden high demand for meat and thus a prompt industrialization of animal production that has favored the misuse of antibiotics for growth promotion facilitated by their online availability (mainous et al., ). in addition, counterfeit or substandard antibiotics recently become a serious global problem (gostin et al., ) , which is exacerbated in lmics, where they represent up to a third of the available drugs. noteworthy, % of all reports received by the who global surveillance and monitoring system on substandard and falsified medicines worldwide come from africa, and most of them correspond to antimalarials and antibiotics (newton et al., ; gostin et al., ; hamilton et al., ; petersen et al., ) . despite this situation, it is important to highlight that human consumption of antibiotics is an unavoidable need to preserve human health. in fact, most health problems dealing with infections in lmics are still caused by a poor access to antibiotics, not by an excessive use of them. proof of this is the fact that the distribution of antibiotics has reduced endemic illnesses and children mortality in sub-saharan africa (keenan et al., ) . this means that, while a global decline in the use of antibiotics would be desirable to diminish the problem of ar, there are still several parts in the globe where antibiotic use should still increase to correctly fight infections. in fact, our primary goal should not be to reduce the use of antibiotics, but to ensure the effective therapy of infectious diseases for the long term. this does not mean that ar is not a relevant problem in lmics; it means that reducing antibiotic use is not enough to solve the problem. indeed, the current high morbidity and mortality due to infectious diseases (malaria, tuberculosis, low respiratory infections, sepsis, and diarrhea) in lmics will be worsened in the absence or low efficiency of therapeutic treatments. further, ar has economic consequences. according to world bank, . million people could fall into extreme poverty by because of ar, most of them from lmics (jonas and world bank group team, ) . consequently, besides a global health problem, ar has an important economic impact (rudholm, ) , hence constituting a global development problem, endangering not only the achievements toward the millennium development goals but also the sustainable development goals (van der heijden et al., ). world bank estimates that ar could impact the gross domestic product from to . %, which is even higher than what is estimated for the climate change (jonas and world bank group team, ) . these economic foresights are linked to the threads of increased poverty, food sustainability, global health deterioration (associated with both food safety and affordability to health care), and environment protection. all these issues are also impacted by the overuse and misuse of antibiotics, its lack of effectiveness, and the affordability to medicines and health care (van der heijden et al., ) . when talking about reducing antibiotic consumption, it is important to remind that up to two-thirds of overall antibiotic usage is for animal husbandry (done et al., ) . further, recent work states that the use of antibiotics in crops, particularly in lmics, might have been largely underestimated (taylor and reeder, ) . despite that evidences on the presence of common args distributed among animals and humans were published decades ago wegener et al., ; aarestrup, ; aarestrup et al., ) , and although the use of antibiotics as growth promoters has been banned in different countries (cox and ricci, ) , they are still allowed in many others (mathew et al., ) . of relevance is the fast increase of antibiotic consumption for animal food production in china ( % in ) and other brics countries . as stated previously, in these countries, increased income has produced a fast increase in meat products demand, due to changes in diet of their population. in addition, the increasing international competitiveness in meat production of these countries has fostered the rampant development of their industrial farming. together with the fact that legislation on antibiotics use remains weak, this situation increases the risk of emergence of ar linked to animal production. nevertheless, the problem is not restricted only to lmics, because antibiotics consumption rose as well in the highincome countries as the united states ( %) , where approximately % of the antimicrobials purchased in were applied in livestock production as non-therapeutic administration (done et al., ) . the development of intensive methods of fish production has also contributed to the rise in the use of antimicrobials and the selection of resistance determinants that can be shared among fish and human bacterial pathogens (cabello et al., ) . economic development has facilitated as well more global transport, waste disposal, and tourism, favoring ar spread within and between different geographical areas (ruppe et al., a; ruppe and chappuis, ) . however, economic growth can also reduce the ar burden, especially when it enables the development of regulations and infrastructures that might reduce the risks of infection and ar spread. this is particularly relevant in the case of public health interventions on food, water, and sewage. because ar pathogens are mainly introduced in natural ecosystems through the release of human/animal stools (karkman et al., ) , the best way of reducing this impact is through the use of wastewater treatment plants, which are still absent in several places worldwide. indeed, it has been described that drinking water is a relevant vehicle for the spread of arbs in different countries (walsh et al., ; fernando et al., ) and that raw wastewater irrigation used for urban agriculture may increase the abundance of mobile args in the irrigated soil (bougnom et al., ) . notably, the analysis of args in wastewaters has shown that the prevalence of args in the environment in each country might be linked to socioeconomic aspects mainly related to economic development, as general sanitation, particularly the availability of drinking and wastewater treatments, malnutrition, number of physicians and health workers, human overcrowding, or external debt grace period (hendriksen et al., ) . the field of ar has mainly focused in the mechanisms of selection; the main driver for the increased burden of ar would be then the use of antibiotics itself. however, these results indicate that transmission, even in the absence of direct human-to-human contact, might be, at least, equally relevant. in this situation, an important element to reduce the ar burden will be to break the transmission bridges among different ecosystems that could be reservoirs of args. even when wastewater-treatment plants are available, the presence of arbs in drinking, fresh, and coastal waters, as well as in sediments nearby industrial and urban discharges, has been described in several countries (ma et al., ; leonard et al., ) . as in the case of fecal contamination markers, a reduction in the amount of args to non-detectable levels would be extremely difficult even when advanced water treatment procedures are applied. a standard definition of polluting arb/arg markers, as well as their acceptable levels, is then needed. this would be required not only for potable water, but also for water reutilization, as well as for land application and release of sewage effluents, because in all cases the reused water/sewage may carry arbs and args, together with pollutants, such as antibiotics, metals, biocides, or microplastics, which, as above stated, may select for ar (baquero et al., ; moura et al., ; yang et al., ; zhu et al., ; larsson et al., ; imran et al., ; wang et al., ) and may even induce hgt. the examples discussed above justify that human health in general and ar in particular are closely interlinked with economic development (sharma, ) . economic differences are also found at individual level, because there is a positive relationship between economic status and health (tipper, ) . in addition, social behavior might also impact ar, a feature discussed in the following section. different socioeconomic factors can modulate the spread of infective bacteria in general and of ar in particular. among them, the increasing crowding of humans and foodborne animal populations favors transmission at the local level (one health), whereas trade of goods and human travel (figure ) favor worldwide transmission (global health) (laxminarayan et al., ; hernando-amado et al., ) . besides these global changes in social behavior, linked to economic development, more specific socioeconomic factors (income, education, life expectancy at birth, health care structure, governance quality), sociocultural aspects (inequalities, uncertainty avoidance, integration of individuals into primary groups, gender biases, cultural long-term orientation), and personality dimension highly influence antibiotic use and ar transmission (gaygısız et al., ) . for instance, although the governance quality seems to be the most important factor associated with a proper antibiotic use, western countries with distinct national culture patterns show different levels of antibiotics consumption (kenyon and manoharan-basil, ) . a better understanding of human social responses facing ailments, especially epidemics and antibiotic use, requires then a more detailed analysis of the differences between collectivistic (individuals living integrated into primary groups) and individually long-term oriented societies (oriented to future individual rewards) (hofstede, ; gaygısız et al., ; kenyon and manoharan-basil, ) . consistent with the sociological elements of ar, many of the aspects influencing ar reviewed above depend on social norms (figure ) . in the classic view of the psychoanalyst erich fromm presented in his book "escape from freedom" (fromm, ) , human individual behavior is oriented to avoid being excluded from a higher social group. indeed, not following social common rules can be eventually considered as a mental disorder; a sociopathology. a social norm is defined as a predominant behavioral pattern within a group, supported by a shared understanding of acceptable actions and sustained through social interactions within that group (nyborg et al., ) . in democratic societies, laws usually derive from already accepted social norms; otherwise, they would be changed, and in that sense, the establishment of accepted social norms for fighting ar is a prerequisite to implement the global approaches, based on worldwide rules, which are required for tackling this relevant problem. interestingly, the ar problem is a bottom-up process, where small emergent changes (in some type of individual patients, in some groups, in some locations) cumulatively escalate to gain a global dimension. frequently, that occurs by crossing tipping points, that is, points where the local ar incidence becomes significant enough to cause a larger, eventually global, health problem. because of that, the implementation of solutions should be adapted to the control of critical tipping points in the small groups of individuals to disrupt the bottom-up processes. however, as ar spread can occur everywhere and at any time, global surveillance and mechanisms of control should be implemented to prevent a top-down process of global ar expansion. individual selfishness for ar is the cornerstone of social norms. this concept was coined and developed by one of us over a decade ago (baquero, ) . let us imagine that each individual is aware that each consumption of an antibiotic increases the personal risk of himself/herself or for his/her closer relatives (frequently exchanging microorganisms) of dying because of an antibiotic-resistant infection. the situation is analogous to the consumption of cholesterol-rich or highly salted food, or drinks with excess of sugar, concerning individual health. however, in the case of ar, it requires the understanding of the impact of individual actions at the global level. in this respect, anti-ar social actions should resemble more antitobacco and even general pollution/ecological campaigns. at the individual level, there is inertia that precludes changing habits, until a tipping point is crossed and health is compromised. the conclusions of studies mainly based on long-term cohort analysis, such as the framingham program for the influence of diet or smoking on personal cardiovascular disease (mahmood et al., ) , have become social norms that are naturally imposed by the ensemble of individuals. this creates a kind of societal culture, leading to appropriate individual behaviors, in occasions without the need of specific laws (diet), in occasion favoring the implementation of such laws (antismoking). however, we lack similar studies on issues such as these dealing with personalfamiliar risks that have successfully shifted social norms, driven by groups of individuals and based on the promotion of individual behaviors in the case of ar. despite that quantitative models on how individual antibiotic use may impact ar at the population level are still absent, it is worth mentioning that a reduced antibiotic consumption has also begun to occur in a number of countries just as a result of a change in individual behavior (edgar et al., ) , and some tools and indicators to address these changes have been suggested (ploy et al., ) . the "tragedy of the commons" metaphor, first proposed in the xix century (lloyd, ) and later on discussed in (hardin, ) , has been used for addressing the sociology of ar, by showing how individual selfishness promotes antibiotic use, increases resistance, and influences the health of the community by impairing antibiotic efficacy (baquero and campos, ; foster and grundmann, ) . ensuring the prestige of individuals that follow the social rules is needed to counteract the tragedy of the commons. nevertheless, it is important noticing that the tension between individual freedom and social rules that is inherent to the construction of democratic societies (tocqueville, ; hobbes, ; rousseau, ; spinoza, ) also applies here. one example of this situation is vaccination, considered in the last century as one of the most important advances to fight infectious diseases and now being the focus of antivaccination campaigns (megget, ) , a movement that has been considered by the who as one of the top global health threats of . it is commonly accepted that social norms are mainly created by learning and education, a rational path that promotes health (chen and fu, ) . also, the increasing activities of "personalized medicine, " including antibiotic stewardship, follow the same trend (gould and lawes, ) . however, the antivaccination movement is an example of how the narrative, as well as the use of decentralized, social information channels such as the internet search, blogs, and applications to facilitate communication such as twitter, facebook or whatsapp, is of particular relevance in the construction of social norms, not necessarily based on scientific and rational grounds (jacobson et al., ; scott and mars, ) . the impact of social norms goes beyond human societies as human activities alter natural ecosystems; consequently, humans cannot be aliens of nature. we should then shape a socioecological system, linking the individuals, the groups, and the entire society, as well as natural ecosystems, also potentially damaged by ar, in a common multilevel adaptive system based on social norms and policies at the individual, local (one health), and global (global health) scale (levin et al., ) . the recent crisis of covid- illustrates the influence of social norms in the individual behavior. each one of the individuals, protecting himself/herself, also protects the others. a person not wearing on face mask is frowned upon, and on the contrary, somebody attaching to the rules increases reputation. the individual adopts the right behavior being influenced by the judgment. of others. in addition, different political regimes (democracy or autocracy), as well as their organization (centralized, federal), together with the capacity of the health services to support the norms and their efficacy to communicate the chosen policy to the citizenry, may shape the individual responses to social norms (greer et al., ; häyry, ; kavanagh and singh, ) . notwithstanding, two reasons that have been proposed to explain the low prevalence of covid- in japan were related with social norms more than with biological issues. these reasons, which are not common to other countries, were the socially accepted use of face masks and the mandatory vaccination of all the population against tuberculosis, which might protect from sars-cov- infection (iwasaki and grubaugh, ) , a feature that is still to be confirmed. the loss of social prestige of individuals taking antibiotics without prescription, as well as the pharmacies delivering these drugs or do not respect environmental protection, or the overconsumption of antibiotics in hospitals or in farms, or even in certain countries, is progressively constituting a "social norm, " converted in rules able to reduce ar emergence and spread. of course, family and school education, as well as governmental campaigns, including the use of social media (grajales et al., ) reinforces such social norms, which could allow the support of the society for the implementation of different interventions, some of them described below. controlling resistance not only requires establishing local interventions, which could be relatively easily implemented, but would also require global interventions that every country should follow, despite their disparate regulatory systems. local and global interventions are necessarily intertwined; for example, the use of a new drug to treat a single individual depends on regulations at the county level (one health approach), but the worldwide prevalence and transmission of resistance to this drug, as well as the regulations of its use, should be established internationally (global health approach). three main interventions to tackle ar have been historically considered: first, reduction of the antibiotic selective pressure by decreasing antimicrobials use; second, reduction of transmission of arbs using improved hygienic procedures that prevent spread; third, development of novel antimicrobials with limited capacity to select arbs or the design of new treatment strategies based on use of non-antibiotic-based approaches or, more recently, on the exploitation of trade-offs associated with ar evolution (imamovic and sommer, ; gonzales et al., ; barbosa et al., ; imamovic et al., ) . these interventions have been basically limited to local initiatives, applied mainly to hospitals and, more recently, to farms. however, ar has emerged and spread globally, in bacteria from different environments, so the health and dynamics of the global microbiosphere could be affected by antibiotics. in a sense, ar is affecting the planetary health (lerner and berg, ) , and the needed interventions for tackling this problem cannot be restricted to hospital settings (figure ) . the proposed reduction in the use of antibiotics (blaskovich, ) must be compensated with alternative approaches for fighting infectious diseases. in this regard, strategies based on improving the capability of the immune system for counteracting infections (levin et al., ; traven and naderer, ) or the use of non-antibiotic approaches to prevent them, such as vaccines (jansen and anderson, ) , may help to reduce the burden of ar infections. indeed, vaccination against haemophilus influenzae and streptococcus pneumoniae has been demonstrated to be an effective intervention for reducing ar (jansen and anderson, ) . however, while vaccination has been extremely useful to prevent viral infections, it has been less promising in the case of bacterial ones. recent approaches, including reverse vaccinology, may help in filling this gap (delany et al., ; ni et al., ) . moreover, vaccination should not be restricted to humans, because veterinary vaccination can also contribute to animal wealth and farm productivity (francis, ) . besides, the use of vaccines in animal production reduces the use of antibiotics at farms/fisheries, hence reducing the selection pressure toward ar. other strategies to reduce antibiotic selective pressure include the use of bacteriophages (a revitalized strategy in recent years) (viertel et al., ; forti et al., ) , not only in clinical settings, but also in natural ecosystems (zhao et al., b) , as well as the use of biodegradable antibiotics (chin et al., ) or adsorbents, able to reduce selective pressure on commensal microbiome (de gunzburg et al., . besides reducing the chances of selecting arbs, the use of antibiotics adsorbents may preserve the microbiomes, reducing the risks of infections (chapman et al., ) . importantly, the procedures for removing antibiotics should not be limited to clinical settings, but their implementation in wastewater treatment plants would reduce selection of ar in non-clinical ecosystems (tian et al., ) . concerning the development of new antimicrobials (hunter, ) , while there is a basic economic issue related to the incentives to pharmaceutical companies (sciarretta et al., ; theuretzbacher et al., ) , the focus is on the possibility of developing novel compounds with low capacity for selecting ar (ling et al., ; chin et al., ) . for this purpose, multitarget (li et al., ) or antiresistance drugs, such as membrane microdomain disassemblers (garcia-fernandez et al., ) , are also promising. furthermore, antimicrobial peptides, with a dual role as immunomodulators and antimicrobials, may also help fight infections (hancock et al., ) . in fact, some works figure | local and global intervention strategies to tackle ar and knowledge gaps that could help improve existing ones. most interventions for reducing antibiotic resistance are based on impairing the selection of arbs/args, which is just the first event in ar spread. our main goal, as for any other infectious disease, figure | continued would be reducing transmission. this does not mean that selective pressure is not relevant for transmission. indeed, without positive selection, hgt events are not fixed, allowing the enrichment of some args that are consequently more prone to diversification, both because they are more abundant and more frequently subjected to selection (davies, ; martinez, a,b; salverda et al., ) and because they can explore different landscapes when present as merodiploids in multicopy plasmids (rodriguez-beltran et al., ) . therefore, reducing the selective pressure, either due to antibiotics or by other coselecting agents as heavy metals, still stands as a major intervention against ar emergence and transmission. to address this issue, we need to know more on the amount of pollutants, their selective concentrations, and their mechanisms of coselection and cross-selection in different ecosystems. this is a general example illustrating the gaps in knowledge in the ar field that need to be filled as well as strategies that may help in tackling this problem. the figure includes several other examples of the gaps of knowledge (red) that require further studies and the interventions (blue) that may help to tackle ar. have shown that arb frequently present collateral sensitivity to antimicrobial peptides (lázár et al., ) and that, importantly, some antimicrobial peptides present limited resistance or crossresistance (kintses et al., ; spohn et al., ) . from a conservative point of view, based on the use of the drugs we already have, it would be desirable to fight ar using evolution-based strategies for developing new drugs or treatment strategies. regarding this, the exploitation of the evolutionary trade-offs associated with the acquisition of ar, as collateral sensitivity, could allow the rational design of treatments based on the alternation or the combination of pairs of drugs (imamovic and sommer, ; gonzales et al., ; barbosa et al., ; imamovic et al., ) . in addition to interventions that reduce the selective pressure of antibiotics or that implement new therapeutic approaches, reducing transmission is also relevant to fight infections. the development of drugs or conditions (as certain wastewater treatments) able to reduce mutagenesis or to inhibit plasmid conjugation may also help in reducing the spread of resistance (thi et al., ; alam et al., ; lin et al., ; lopatkin et al., ; valencia et al., ; kudo et al., ) . besides specific drugs to reduce the dissemination of the genetic elements involved in ar, socioeconomic interventions to break the bridges that allow transmission between individuals and, most importantly (and less addressed), between resistance entities (hernando-amado et al., ) are needed (figure ) . more efficient animal management, not only allowing less antibiotics use but also reducing animal crowding (and hence ar transmission), as well as improved sanitation procedures, including the universalization of water treatment, will certainly help in this task (berendonk et al., ; manaia, ; hernando-amado et al., ) . notably, wastewater treatment plants are usually communal facilities where the residues of the total population of a city are treated. hospitals are the hotspots of ar in a city; hence, on-site hospital (and eventually onfarm) wastewater treatment may help to reduce the pollution of communal wastewater by antibiotics and arbs (cahill et al., ; paulus et al., ) , hence reducing ar transmission. concerning trade of goods, it is relevant to remark that, although there are strict regulations to control the entrance of animals or plants from sites with zoonotic of plant epidemic diseases (brown and bevins, ) , there are no regulations on the exchange of goods from geographic regions with a high ar prevalence, a feature that might be taken into consideration for reducing the worldwide spread of ar. once arbs are selected and disseminated, interventions based on the ecological and evolutionary (eco-evo) aspects of ar lehtinen et al., ) should be applied to restore (and select for) susceptibility of bacterial populations, as well as to preserve drug-susceptible microbiomes in humans and in animals . eco-evo strategies include the development of drugs specifically targeting arbs. for that, drugs activated by mechanisms of resistance, vaccines targeting high-risk disseminating resistance clones or the resistance mechanisms themselves (kim et al., ; ni et al., ) , or drugs targeting metabolic paths that can be specifically modified in arbs ) might be useful. the use of bacteriovores such as bdellovibrio to eliminate pathogens without the need for antibiotics has been proposed; although its utility for treating infections is debatable, it might be useful in natural ecosystems (shatzkes et al., ) . more recent work suggests that some earthworms may favor the degradation of antibiotics and the elimination of arbs (wikler, ) , a feature that might be in agreement with the finding that arbs are less virulent (and hence might be specifically eliminated when the worm is present) in a caenorhabditis elegans virulence model ruiz-diez et al., ; paulander et al., ; olivares et al., ) . however, the information on the potential use of worms for reducing ar in the field is still preliminary and requires further confirmation. noteworthy, ar is less prone to be acquired by complex microbiomes (mahnert et al., ; wood, ) , a feature that supports the possibility of interventions on the microbiota to reduce ar. among them, fecal transplantation (chapman et al., ; pamer, ) or the use of probiotics able to outcompete arbs (keith and pamer, ) has been proposed as strategies for recovering susceptible microbiomes. the recent crisis of covid- (garrett, ) resembles the pandemic expansion of args and clearly shows that pandemic outbreaks cannot be solved by just applying local solutions. further, unless all population is controlled, and comprehensive public-health protocols are applied to the bulk of the population, such global pandemics will be hardly controlled. the case of covid- is rather peculiar, because we are dealing with a novel virus. very strict interventions have been applied, mainly trying to control something that is a novel, unknown, disease; we have been learning along the pandemic and still ignore what will come further. ar is already a very well-known pandemic affecting humans, animals, and natural ecosystems (anderson, ; verhoef, ) . in this case, we have tools that might predict the outcome, and likely because the degree of uncertainty is lower than in the case of covid- , we have not applied clear, common, and comprehensive procedures to reduce the spread of ar. it is true that we know the evolution of antibiotics consumption and ar prevalence in several countries, and also interventions, mostly based on social norms, have been applied. social norms have reduced the unnecessary prescription of antibiotics, or pharmacy sales without prescription, and the use of antibiotics for fattening animals has been banned in several countries, being still allowed in several others. nevertheless, these actions are not general, and more aggressive, global actions are still needed. coming back to the covid- example, while the aim of health services worldwide is to detect any possible source of sars-cov- , surveillance of infections (eventually by arbs) is not universal. in other words, it does not apply to all citizens in all countries. the reasons can be just political such as the inclusion of immigrants in public health services (scotto et al., ) or the consequence of limited financial resources and technical capacity that countries such as those belonging to the lmic category can face (gandra et al., ) . the problem is not only on citizens, because different non-human reservoirs, such as wastewater, drinking water, or freshwater, may jointly contribute to ar dissemination (hendriksen et al., ) . in this regard, it is important to highlight that low quality of water is regularly associated to poverty. universalization of health services, sanitization, access to clean water, and in general reduction of poverty are relevant step-forward elements for reduction of the burden of infectious diseases in general and of ar in particular. the time has come to tackle ar, and this cannot be done just by taking actions at the individual or even country level, but by taking convergent actions across the globe. as stated by john donne ( ) in his poem, "no man is an island, " written after his recovery from an infectious disease (likely typhus): "no man is an iland, intire of itselfe; every man is a peece of the continent, a part of the maine; if a clod bee washed away by the sea, europe is the lesse, as well as if a promontorie were, as well as if a manor of thy friends or of thine owne were; any mans death diminishes me, because i am involved in mankinde; and therefore never send to know for whom the bell tolls; it tolls for thee." this reflection on how infectious diseases in general should be faced by the society was published at , but the idea behind still applies nowadays, especially for ar. all authors have contributed to the concept of the review and in its writing. jm was supported by grants from the instituto de salud carlos iii [spanish network for research on infectious diseases (rd / / )], from the spanish ministry of economy and competitivity (bio - -r) and from the autonomous community of madrid (b /bmd- ). work in tc and fb laboratory was supported by grants funded by the joint programming initiative in antimicrobial resistance (jpiamr third call, starcs, jpiamr -ac / ), the instituto de salud carlos iii of spain/ministry of economy and competitiveness and the european development regional fund "a way to 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bacteriophage and bacteriophage cocktails for controlling antibioticresistant bacteria in soil-plant system microbial mass movements from "one medicine" to "one health" and systemic approaches to health and well-being key: cord- -hth nf b authors: tsukagoshi, hiroyuki; ishioka, taisei; noda, masahiro; kozawa, kunihisa; kimura, hirokazu title: molecular epidemiology of respiratory viruses in virus-induced asthma date: - - journal: front microbiol doi: . /fmicb. . sha: doc_id: cord_uid: hth nf b acute respiratory illness (ari) due to various viruses is not only the most common cause of upper respiratory infection in humans but is also a major cause of morbidity and mortality, leading to diseases such as bronchiolitis and pneumonia. previous studies have shown that respiratory syncytial virus (rsv), human rhinovirus (hrv), human metapneumovirus (hmpv), human parainfluenza virus (hpiv), and human enterovirus infections may be associated with virus-induced asthma. for example, it has been suggested that hrv infection is detected in the acute exacerbation of asthma and infection is prolonged. thus it is believed that the main etiological cause of asthma is ari viruses. furthermore, the number of asthma patients in most industrial countries has greatly increased, resulting in a morbidity rate of around - % of the population. however, the relationships between viral infections, host immune response, and host factors in the pathophysiology of asthma remain unclear. to gain a better understanding of the epidemiology of virus-induced asthma, it is important to assess both the characteristics of the viruses and the host defense mechanisms. molecular epidemiology enables us to understand the pathogenesis of microorganisms by identifying specific pathways, molecules, and genes that influence the risk of developing a disease. however, the epidemiology of various respiratory viruses associated with virus-induced asthma is not fully understood. therefore, in this article, we review molecular epidemiological studies of rsv, hrv, hpiv, and hmpv infection associated with virus-induced asthma. acute respiratory illness (ari) is a major cause of morbidity and mortality worldwide (williams et al., ; sloots et al., ) . ari imposes a large burden on health, particularly in children. for community-based care, ari has been estimated at a cost of over us$ per case (ehlken et al., ) . the disease burden for ari is estimated at , , disability-adjusted life years and . million deaths (world health organization, ) . thus, ari has a huge impact on health and society. although severe lower respiratory tract infections have been observed, ari is most often associated with mild upper respiratory infection (uri). most ari cases in early childhood are confirmed as uri, leading to symptoms of the common cold with coryza and cough. in contrast, around one-third of infants with ari develop lower respiratory tract symptoms such as tachypnea, wheezing, severe cough, breathlessness, and respiratory distress (tregoning and schwarze, ) . in general, viruses are the most common causative agents of ari. more than different types of viruses are known to cause ari, with respiratory syncytial virus (rsv), human rhinovirus (hrv), human metapneumovirus (hmpv), and human parainfluenza virus (hpiv) most commonly identified in ari patients. indeed, together with these respiratory viruses, human enterovirus (hev), influenza virus (infv), human coronavirus (hcov), adenovirus (adv), and human bocavirus (hbov) account for around % of aris detected (kusel et al., ) . respiratory viral infections can have severe adverse outcomes in patients with established asthma and are associated with nearly % of asthma exacerbation episodes (nicholson et al., ; johnston et al., ; wark et al., ; heymann et al., ; grissell et al., ) . accumulating evidence indicates that the etiology of most cases of asthma, namely virus-induced asthma, is linked to such respiratory virus infections. in addition, rsv and hrv are the most frequently detected pathogens and may play an important role in viral induction and exacerbation of asthma. molecular biology techniques have developed rapidly over recent years. the application of molecular techniques to the study of virus-induced asthma enhances epidemiologic studies by improving our ability to classify these pathogens into meaningful groups (foxman and riley, ) . in this review, we focus on molecular epidemiological studies of respiratory viruses, including rsv, hrv, hmpv, and hpiv infections, associated with virus-induced asthma. in infancy, illnesses such as bronchiolitis share many clinical features with acute asthma, including wheezing, rapid breathing, prolonged expiratory phase inflammation, and respiratory www.frontiersin.org compromise. respiratory viruses are detected in the majority of asthma exacerbations in both children ( - %) and adults ( - %; johnston et al., ; grissell et al., ) . in addition, wheezing illnesses are also closely associated with respiratory viral infections in all age groups (gern, ) . fujitsuka et al. ( ) attempted to detect various respiratory viruses in japanese children with acute wheezing using pcr technology and found viruses in samples from . % patients: rsv or hrv alone were detected in . and . % patients, respectively and both rsv and hrv were detected in . % patients. other previous reports suggested that the prevalence of rsv and hrv is similar ( and %, respectively) in children less than years of age, but differs ( and %) in older children (johnston et al., ; grissell et al., ) . in addition, fujitsuka et al. ( ) suggested that rsv was the dominant species detected in patients with no history of wheezing and/or asthma, while hrv was dominant in patients with such a history. thus, the main causative viral agent of asthma depends on previous illness and age. around one-third of infants who have an acute wheezing illness go on to develop recurrent wheezing, indicating that viral respiratory illnesses in early life promote asthma. recently, the "two-hit" hypothesis has been proposed, whereby viral infections promote asthma mainly in predisposed children (gern, ) . infants who develop virus-induced wheezing episodes are at increased risk for subsequent asthma, but most acute wheezing illnesses in infancy resolve with no long-term sequelae. it has been recognized for years that rsv infections often produce the first episode of wheezing in children who go on to develop chronic asthma (lemanske, ) . indicators of heightened risk for developing asthma include wheezing episodes caused by hrv infections and the development of atopic features such as atopic dermatitis, allergen-specific ige for foods or aeroallergens (e.g., house dust, mites, or cat or dog dander), and blood eosinophilia (figure ) . once asthma has been established, hrv infections are the most common cause of figure | relationship between respiratory viral infections and development of asthma. host-pathogen interactions that determine the severity of respiratory illnesses, and risk for subsequent asthma was increased by respiratory virus infection, especially due to rsv, in infants. although most acute wheezing resolves within a relatively short time, a history of wheezing and host immunological conditions (e.g., atopic features) heightens the risk for asthma. once asthma is established, hrv infections are the most common causative agents of asthma in children. acute exacerbations, especially in children. as in infancy, atopy is an important risk factor for acute episodes of virus-induced wheezing (kusel et al., ) . many previous reports have suggested that such respiratory virus infections are deeply associated with virus-induced asthma (kusel et al., ; pierangeli et al., ; kuehni et al., ; fujitsuka et al., ; kato et al., ) . thus, it is entirely plausible that viral infections induction and/or exacerbation asthma in children. respiratory syncytial virus of genus pneumovirus and family paramyxoviridae causes ari in children (vardas et al., ; peter and james, ) . rsv infection may cause major problems in infants less than year of age and can lead to life-threatening aris such as bronchiolitis and bronchopneumonia (shay et al., ; leung et al., ; yorita et al., ) . epidemiological studies suggest that around % of infants have experienced an rsv infection by the age of year, and % by the age of years; host response to the virus varies greatly, but includes upper respiratory tract infections, typical bronchiolitis, and rsv-induced wheezy bronchitis (cane, ; kuehni et al., ). long-term prospective case-control and cohort studies have also linked rsv bronchiolitis to the development of wheezing and asthma later in childhood (sigurs et al., (sigurs et al., , (sigurs et al., , henderson et al., ) . thus, rsv infections may be associated with the initiation and/or exacerbation of asthma. the rsv genome encodes proteins (peter and james, ) . among these, the attachment glycoprotein (g) is a major structural protein that may be associated with both infectivity and antigenicity (johnson et al., ; rueda et al., ) . molecular epidemiological studies have shown that rsv can be classified into two phylogenetic subgroups, rsv-a and rsv-b (mufson et al., ) . the strains of subgroup a can be subclassified into eight genotypes (ga -ga and saa ), as can those of subgroup b (ba, gb -gb , and sab - ; parveen et al., ) . from phylogenetic analysis of the g gene of rsv, martinello et al. ( ) showed that rsv belonging to ga genotype may be associated with greater severity of illness in, for example, bronchiolitis and pneumonia. although ga genotype has been detected in the united kingdom, spain, and new zealand, it is not the most prevalent strain (cane et al., ; garcia et al., ; matheson et al., ) . martinello et al. ( ) therefore suggested that the association between greater severity of illness and ga genotype may be solely due to a transient shift in genotype-specific immune status within the community. in addition, correlations between certain strains and/or genotypes of rsv and slight differences in disease severity have been described previously (hall et al., ; walsh et al., ) . some genotypes such as subgroup a genotypes ga , ga , ga , ga , and na and subgroup b genotype ba have been detected throughout the world in recent years (zlateva et al., ; parveen et al., ; zhang et al., ; nakamura et al., ; rebuffo-scheer et al., ) . of these, na is a novel genotype known to be genetically close to ga genotype, while ga genotype and ba genotype are the most common genotypes of rsv subgroups a and b around the world and have persisted for many years (tran et al., ) . furthermore, a new genotype belonging to rsv-a, on , has been detected in some countries, including canada, korea, malaysia, south africa, and japan (eshaghi et al., ; lee et al., ; khor et al., ; tsukagoshi et al., ; valley-omar et al., ) . this genotype contains a unique tandem repeat ( nt sequence duplication) in the c-terminal rd hypervariable region of the g gene, and may be classified as a subdivision of na (eshaghi et al., ) . some reports have suggested that the severity of illness is not linked to subgroups or genotypes, but is associated with the quantity of rsv in nasopharyngeal aspirate (sullender, ; campanini et al., ) . a larger population study is needed to identify the different rsv genotypes circulating in different areas to gain a better understanding of the relationship between disease severity and rsv genotype. the g protein is a major antigen of rsv and amino acid substitutions may be related to changes in antigenicity. there are some reports of amino acid substitutions, and some positively selected sites in the c-terminal rd hypervariable region of the g gene have been estimated (botosso et al., ; yoshida et al., ; kushibuchi et al., ) . for example, yoshida et al. ( ) estimated some sites under positive selection in the region (asn ser, met glu, arg lys,and arg glu substitutions in rsv-a strains were estimated by the rel method, and asn tyr and leu pro substitutions of rsv-a, as well as leu pro substitution of rsv-b, were estimated by the ifel method). botosso et al. ( ) found and amino acid sites under putative positive selection in rsv-a and rsv-b, respectively. in addition, some unique positively selected sites were found in the g gene . these amino acid variations at these sites might play a key role in severe respiratory infection, such as bronchiolitis (goto-sugai et al., ) . furthermore, the rate of molecular evolution of the region might be high. for example, kushibuchi et al. ( ) estimated the evolutionary rate of rsv-a at . × − substitutions/site/year, while that of rsv-b was estimated at . × − substitutions/site/year. thus, it is suggested that this c-terminal rd hypervariable region in the g gene of rsv-a and -b evolved rapidly . based on host immunological conditions, it is suggested that host immunity such as tlr polymorphism is linked to symptomatic rsv infection (delgado et al., ) . thus, both the antigenicity of the viruses and host immune conditions may play important roles in the pathophysiology of severe respiratory infections such as bronchiolitis, pneumonia, and virus-induced asthma (awomoyi et al., ) . human rhinovirus are a group of positive-sense ssrna viruses belonging to genus enterovirus in the family picornaviridae (turner and couch, ) . although hrvs were previously thought to be mainly associated with the common cold causing mild respiratory symptoms, recent reports strongly suggest that hrvs may induce and/or exacerbate asthma (virus-induced asthma; chung et al., ; turner and couch, ; busse et al., ; gern, ; khadadah et al., ) . one report suggested that hrv wheezing illness within the first three years of life is significantly associated with the development of asthma at age years (jackson et al., ) . another report suggested that hrvs are major agents in the induction of wheezing and exacerbation of asthma (khadadah et al., ) . thus, hrvs are being re-evaluated as important agents of ari in humans (imakita et al., ; wos et al., ) . the basis for these lower respiratory symptoms has been a source of controversy in terms of the mechanisms of hrv pathogenesis. there are a variety of potential barriers to hrv infection of the lungs, including temperature-sensitive replication of the virus. for this reason, it is thought that the optimum propagation temperature of hrvs may be - • c in vitro (papadopoulos et al., ; schroth et al., ) . however, a recent study suggested that hrvs can propagate in lower airway tissues and this may be an important factor in the development of airway obstruction, coughing, and wheezing that can lead to bronchiolitis and pneumonia (mosser et al., ) . hrv has been concomitantly isolated with bacterial pathogens in - % of children and - % of adults with pneumonia (juven et al., ; templeton et al., ; jennings et al., ) . thus, it is not clear whether hrv is ever the causative agent for the disease. human rhinovirus were previously classified into two species, hrv species a (hrv-a) and species b (hrv-b), containing over serotypes (turner and couch, ) . however, a genetically heterogeneous third species, hrv species c (hrv-c), was discovered recently (lamson et al., ; mcerlean et al., ) . recent reports suggest that hrv-a, b, and c have a unique and wide genetic diversity simmonds et al., ; arakawa et al., ) . hrv-a and -c appear to be mainly associated with aris and virus-induced asthma, while hrv-b has been detected in a relatively small number of patients with aris (linsuwanon et al., ; wisdom et al., ; smuts et al., ) . our previous findings obtained from samples from children with aris in japan indicated that hrv-a and -c can be classified into many clusters in the phylogenetic tree, with % nucleotide divergence of the vp /vp coding region (mizuta et al., a; arakawa et al., ; kiyota et al., ) . in addition, kiyota et al. ( ) estimated that the rate of molecular evolution of the vp /vp coding region was rapid ( . × − substitutions/site/year) in hrv-c. these results suggest that hrv-a and -c detected in ari cases are the predominant strains and have varied genetic properties (wisdom et al., ; mizuta et al., a; arakawa et al., ) . thus, the association between hrv type and disease severity is not fully understood. there may be important differences in the susceptibility of individuals to the replication of hrv in lower airway tissues. parry et al. ( ) and gern et al. ( ) found that weak peripheral blood mononuclear cell (pbmc) th (ifn-γ) response to hrv infection is associated with increased viral shedding, and decreased proliferative response of pbmcs to hrv is associated with increased severity of symptoms. in addition, it was found that weak th responses (ifn-γ/il- mrna ratio) in sputum are also associated with greater severity of illness . furthermore, weak th responses to viral infection in adults with asthma have been associated with decreased lung function and greater airway responsiveness (brooks et al., ) . these results indicate that individuals with a weak th response to viruses, and perhaps individuals with asthma in general, may be more susceptible to hrv illnesses, and this association may be strongest in those with more severe disease (parry et al., ; gern et al., ; brooks et al., ) . other epidemiological and biological factors, such as allergy, atopic dermatitis, www.frontiersin.org or a family history of allergy, may be related to virus-induced asthma (green et al., ; singh et al., ) . recently it is suggested that variants at the q locus were associated with hrv induced asthma in children who had a history wheezing illnesses, although associations of q variants with asthma were restricted to children who had a history of hrv wheezing illnesses (calışkan et al., ) . human metapneumovirus is a recently identified rna virus belonging to the paramyxoviridae family, of genus metapneumovirus (collins and crowe, ) . hmpv is a major pathogen that causes ari in all ages (collins and crowe, ) . the first hmpv infection appears to take place within the first six months of life, after which infections may occur repeatedly and frequently (schildgen et al., ) . the nosocomial impact of hmpv is estimated to be as high as that for rsv. in an hmpv outbreak in japan, . % of elderly patients who shared the same day care room in a hospital were infected with hmpv (honda et al., ) . higher morbidity is observed in young children, the elderly, and immunocompromised adults (boivin et al., ; falsey et al., ; van den hoogen et al., ; sumino et al., ; williams et al., ; o'gorman et al., ) . hmpv is classified into two genotypes (a and b) and four subgroups (a , a , b , and b ) by phylogenetic analysis, using the f and g genes (biacchesi et al., ; van den hoogen et al., ) . subgroup a has been subdivided into two lineages, subgroup a a and a b (huck et al., ) . it has been suggested that these genotypes circulate in variable proportions in some areas (gerna et al., ; mackay et al., ) . although the molecular epidemiological information on hmpv has gradually accumulated, the detailed epidemiology remains unclear (mizuta et al., b; pitoiset et al., ; omura et al., ) . hmpv infections can occur throughout the year, but seasonality has been described in several studies, with the epidemiological peak occurring several months later than that observed for rsv epidemics (robinson et al., ; wilkesmann et al., ; madhi et al., ; aberle et al., aberle et al., , heininger et al., ). it remains unclear whether different hmpv subgroups are associated with differences in the clinical course of disease. several groups have suggested that hmpv subgroup a might be associated with more severe clinical disease (martinello et al., ; kaida et al., ; vicente et al., ; arnott et al., ) , while others have reported that subgroup b may cause more severe illness (esper et al., ; pitoiset et al., ) , and still other groups have found no evidence for differential severity caused by different hmpv lineages (agapov et al., ; manoha et al., ; larcher et al., ; xiao et al., ) . previous reports suggested that the substitution rates for the g gene ( . × − substitution/site/year) and the f gene ( . × − to . × − substitution/site/year) are high, and some positively selected sites have been found in the latter (de graaf et al., ; yang et al., ) . it may be that there is a correlation between some positively selected epitopes and disease severity. thus, the association between hmpv subgroup and disease severity is controversial. to gain a better understanding of host responses that may contribute to differences in clinical severity between hmpv subgroups, a more detailed analysis that includes host immunological status is needed. human parainfluenza virus belong to the paramyxoviridae family. there are two genera of hpiv, respirovirus (hpiv- and hpiv- ) and rubulavirus (hpiv- and hpiv- ; karron and collins, ) . hpiv is classified into four serotypes (hpiv - ) , all of which can cause various ari in humans such as uri, croup, bronchitis, asthma, and pneumonia (henrickson, ; karron and collins, ) . although hpiv type (hpiv ) is rarely reported, hpiv - are important causes of various ari, including the common cold, croup, bronchitis, bronchiolitis, and pneumonia in children, and they commonly re-infect both children and adults. while such infections are generally mild in healthy persons, they may cause serious diseases in children, such as asthma (henrickson, ; karron and collins, ) . although fewer hpiv strains have been detected compared with other respiratory viruses such as rsv, hrv, and hmpv, previous reports suggest that hpiv and are the dominant viruses in children with ari (reed et al., ) . indeed, serological surveys indicate that at least % of children have been infected with hpiv by years of age, approximately % have been infected by age , and at least % have been infected with hpiv by years of age (parrott et al., (parrott et al., , . hpiv and show high prevalence and are associated with up to % of acute lower respiratory tract infections in adults (azevedo et al., ; matsuse et al., ) . hpiv and hpiv , may be major agents of ari throughout the world, along with other viruses such as rsv, hrv, and hmpv (laurichesse et al., ; iwane et al., ; monto, ; do et al., ) . in addition, it is suggested that hpiv is a major causative agent of virus-induced asthma (henrickson and savatski, ) . several previous studies have reported that hpiv infections demonstrate clear outbreaks in autumn, mostly in september and november, every years (knott et al., ; hall, ; counihan et al., ) . other studies have reported that hpiv causes yearly outbreaks around the globe, mainly in the spring-summer season (knott et al., ; counihan et al., ; hall, ; mizuta et al., ) . a recent study suggested that four different types of hpiv cause similar clinical manifestations in patients, and the clinical presentation of hpiv infection may differ depending on patient age (liu et al., ) . henrickson and savatski ( ) analyzed the longitudinal evolution of the hn coding region in strains of hpiv isolated in the usa. these results showed that the antigenic and genetic subgroups are very stable. in addition, suggested that the evolution of the hn gene in the present hpiv isolates was relatively slow and that the gene is highly conserved. only a few reports on the molecular epidemiology of hpiv are available and it appears that the molecular epidemiology of hpiv is poorly understood. larger and more detailed studies on the association of hpiv with asthma are needed. hev was recently detected in asthmatic patients (hasegawa et al., ) . hev was found to be relatively acid resistant and thus could be distinguished from acid-sensitive hrv (schieble et al., ; kapikian et al., ) . hrv was recently reclassified frontiers in microbiology | virology as hev based on phylogenetic analysis and neutralization test, and some laboratories have confirmed its acid sensitivity ishiko et al., ; savolainen et al., ) . distinguishing between hrv and hev based on the acid sensitivity of isolates is therefore not appropriate for hev . the number of reports of an association between respiratory disease and hev infection has recently increased. one report of the phylogenetic analysis of hev based on partial vp gene sequences indicates wide genetic diversity (linsuwanon et al., ) . in addition, tokarz et al. ( ) showed the presence of multiple clades among the circulating strains, and that all strains are spreading rapidly worldwide and contributing to the prevalence rates of respiratory diseases. in addition, asthmatic individuals infected with hev also have the propensity to develop unstable asthma or an acute attack (hasegawa et al., ) . influenza virus is also a major causative agent of ari in both children and adults. furthermore, asthmatic patients were found among children and adults hospitalized with seasonal infv (dao et al., ; dawood et al., ) . although it is recognized that viral infections such as rsv or hrv may induce and/or exacerbate asthma, the effect of infv on asthma remains arguable (johnston et al., ) . although one study suggested that a(h n )pdm viruses impose greater risk factors on children than seasonal infv (tran et al., ) , infv vaccine was available before the influenza season since infv causes more severe illness than other respiratory viruses. therefore, it is suggested that infv vaccine be recommended for children with asthma (kloepfer et al., ) . although the level of detection of hcov, hbov, or adv is relatively low, these infections are also detected in children with acute wheezing (chung et al., ; jartti et al., ) . further studies are needed to clarify the clinical roles of hcov, hbov, or adv infections and those of other respiratory viruses. in particular, the prevalence of hcov, hbov, or adv infection in healthy control subjects, assessment of disease severity by other clinical variables, and the immunological effects should be investigated. infants with severe bronchiolitis have an increased risk of developing recurrent wheezing later in life (chung et al., ) . hrv may be detected concurrently with other viruses such as rsv, hmpv, infv, or hcov (richard et al., ; fujitsuka et al., ) . considering their ubiquity, it is interesting that the number of respiratory viruses detected concurrently with hrv strains is relatively low mackay, ) , supporting the concept that hrvs have a direct role in the clinical outcome of infection (miller et al., ) . in fact, hrv strains are co-detected with other pathogens in reproducible, but clinically undefined, patterns (brunstein et al., ) . the hrv partnership with host immunity may be a mutualistic one, inadvertently imparting an advantage to the host by protecting against more cytopathic respiratory viral pathogens while the host provides a vessel for hrv replication and transmission. respiratory viruses other than rsv and multiple viral infections may contribute to the severity of bronchiolitis and asthma. indeed, it was reported that dual infections of hmpv and rsv or hrv and rsv confer a -to -fold increase of severe disease in children admitted to pediatric intensive care units semple et al., ) . in contrast, other studies reported that co-infection with two respiratory viruses was not significantly associated with disease severity (van woensel et al., ; wolf et al., ) . thus, there is no consensus on the effects of co-infection on disease severity. the effect of dual infection may depend upon which viruses co-infect together. for example, although there was no increase in severity when hrv and/or adv were detected during rsv infection, co-infection with both hmpv and rsv increased the rate of intensive care unit admissions (aberle et al., ; semple et al., ) . thus, although dual infections and reinfections have been well documented in children, chronic infection with the development of quasispecies cannot be ruled out without obtaining more complete data using high performance detection methods (hall and mccarthy, ) . respiratory viral infections are a major cause of virus-induced asthma in early life. although antiviral therapy is not yet available for patients infected with respiratory viruses, the detection and identification of these viruses could help to explain serious respiratory illness, provide guidance for medical care, and prevent unnecessary treatment with antibiotics. based on the results of many related studies, we propose a two-step hypothesis of asthma development in children. the first step is mainly due to rsv infection: when rsv infects bronchial cells, the bronchial cells produce various cytokines and chemokines. these responses cause hyperresponsiveness in the bronchial cells. in other words, rsv infection might create a preparatory step as the first step in the development of asthma. hrv infection might then bring about the second step in the development of asthma. an infant with a history of wheezing caused by rsv infection may develop the heavy wheezing of asthma due to hrv infection followed by rsv infection. to understand the cause of asthma, we need to examine the larger complex picture of genetic susceptibility, immune components, environmental exposures, and the interactions between these elements. aberle single versus dual respiratory virus infections in hospitalized infants: impact on 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prevalence and clinical and molecular characterization of human metapneumovirus in children with acute respiratory infection in china genetic diversity and evolution of human metapneumovirus fusion protein over twenty years severe bronchiolitis and respiratory syncytial virus among young children in hawaii molecular epidemiology of the attachment glycoprotein (g) gene in respiratory syncytial virus in children with acute respiratory infection in japan in / genetic variability of group a and b respiratory syncytial viruses isolated from provinces in china molecular evolution and circulation patterns of human respiratory syncytial virus subgroup a: positively selected sites in the attachment g glycoprotein this study was supported in part by research on emerging and re-emerging infectious diseases, labour, and welfare programs from the ministry of health, labour, and welfare, japan. the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. key: cord- - oc ykds authors: gendy, sherif; chauhan, ashvini; agarwal, meenakshi; pathak, ashish; rathore, rajesh singh; jaswal, rajneesh title: is long-term heavy metal exposure driving carriage of antibiotic resistance in environmental opportunistic pathogens: a comprehensive phenomic and genomic assessment using serratia sp. srs- -s- date: - - journal: front microbiol doi: . /fmicb. . sha: doc_id: cord_uid: oc ykds the carriage of both, heavy metal and antibiotic resistance appears to be a common trait in bacterial communities native to long-term contaminated habitats, including the savannah river site (srs). there is widespread soil contamination at the srs; a united states department of energy (doe) facility with long-term contamination from past industrial and nuclear weapons production activities. to further evaluate the genomic and metabolic traits that underpin metal and antibiotic resistance, a robust mercury (hg) and uranium (u)-resistant strain- srs- -s- , was isolated. minimum inhibitory concentration of this strain revealed resistance to hg ( μg/ml) and u ( mm), the two main heavy metal contaminants at the srs. metabolic assessment of strain srs- -s- using biolog metabolic fingerprinting analysis revealed preference for carbohydrate utilization followed by polymers, amino acids, carboxy acids, and esters; this physiological activity diminished when hg stress was provided at and μg/ml and completely ceased at μg/ml hg, indicating that continued release of hg will have negative metabolic impacts to even those microorganisms that possess high resistance ability. development of antibiotic resistance in strain srs- -s- was evaluated at a functional level using phenomics, which confirmed broad resistance against . % of the antibiotics tested. evolutionary and adaptive traits of strain srs- -s- were further assessed using genomics, which revealed the strain to taxonomically affiliate with serratia marcescens species, possessing a genome size of , , bp, , proteins (cds), genes for transfer rna (trna), and an average g + c content of . . comparative genomics with closest taxonomic relatives revealed distinct genes in srs- -s- , with multiple functions related to both, antibiotic and heavy metal resistance, which likely facilitates the strain’s survival in a metalliferous soil habitat. comparisons drawn between the environmentally isolated serratia srs- -s- with other strains revealed a closer functional association with medically relevant isolates suggesting that propensity of environmental serratia isolates in acquiring virulence traits, as a function of long-term exposure to heavy metals, which is facilitating development, recruitment and proliferation of not only metal resistant genes (mrgs) but antibiotic resistant genes (args), which can potentially trigger future bacterial pathogen outbreaks emanating from contaminated environmental habitats. the carriage of both, heavy metal and antibiotic resistance appears to be a common trait in bacterial communities native to long-term contaminated habitats, including the savannah river site (srs). there is widespread soil contamination at the srs; a united states department of energy (doe) facility with long-term contamination from past industrial and nuclear weapons production activities. to further evaluate the genomic and metabolic traits that underpin metal and antibiotic resistance, a robust mercury (hg) and uranium (u)-resistant strain-srs- -s- , was isolated. minimum inhibitory concentration of this strain revealed resistance to hg ( µg/ml) and u ( mm), the two main heavy metal contaminants at the srs. metabolic assessment of strain srs- -s- using biolog metabolic fingerprinting analysis revealed preference for carbohydrate utilization followed by polymers, amino acids, carboxy acids, and esters; this physiological activity diminished when hg stress was provided at and µg/ml and completely ceased at µg/ml hg, indicating that continued release of hg will have negative metabolic impacts to even those microorganisms that possess high resistance ability. development of antibiotic resistance in strain srs- -s- was evaluated at a functional level using phenomics, which confirmed broad resistance against . % of the antibiotics tested. evolutionary and adaptive traits of strain srs- -s- were further assessed using genomics, which revealed the strain to taxonomically affiliate with serratia marcescens species, possessing a genome size of , , bp, , proteins (cds), genes for transfer rna (trna), and an average g + c content of . . comparative genomics with closest taxonomic relatives revealed distinct genes in srs- -s- , with multiple functions related to both, antibiotic and heavy metal resistance, which likely facilitates the strain's survival in a metalliferous soil habitat. heavy metal contamination is a global environmental problem shown to cause both ecological and public health problems (tchounwou et al., ) . however, ecosystems with pervasive and long-term heavy metal contamination, such as the savannah river site (srs), located in aiken, sc, united states, are known to harbor metal-resistant microorganisms, which have acquired genomic features to resist and circumvent metal toxicity (ye, ; agarwal et al., ; pathak et al., pathak et al., , gendy et al., a,b) . the two main heavy metal contaminants within the impacted srs soils include mercury (hg) and uranium (u) (sowder et al., ) . our ongoing culture-dependent and culture-independent work on the srs metalliferous soils has demonstrated several different bacterial groups involved in metal cycling processes, such as arthrobacter spp., burkholderia spp., bacillus spp., bradyrhizobium spp., pseudomonas spp., lysinibacillus spp., paenibacillus spp., stenotrophomonas spp., and serratia spp. (agarwal et al., a,b; gendy et al., b) . many of these groups have been demonstrated to cycle hg by activity of their mer operon, which consists of regulatory proteins-merr and merd, transport proteins-merp, t and e, as well as protein(s) with reductase activity-mera pathak et al., ) . in reference to u cycling processes, several different genes have been shown to render u resistance and biomineralization, such as the gene encoding uranium binding complex (ubc), uranium response in caulobacter (urca), phosphatase genes (phoy; phok; and phon) and pib-type atpase (martinez et al., ; hillson et al., ; nilgiriwala et al., ; nongkhlaw et al., ; yung and jiao, ; kulkarni et al., ; thorgersen et al., ) . however, no specific bacterial operon has been shown to be active under u stress to date. despite this advantage of microbially based heavy metal bioremediation, studies have also unequivocally shown that bacteria can acquire antibiotic resistance when exposed to longterm contamination, especially with hg and u (benyehuda et al., ) , including our previous work gendy et al., a,b; pathak et al., ) . this builds upon significant evidence that exists on the srs soils which unequivocally shows higher levels of antibiotic resistance in contaminated srs soils relative to reference soils wright et al., ; thomas et al., ) . furthermore, both, empirical and anecdotal evidence from srs soils strongly suggest recruitment of antibiotic resistances in many human pathogens that are native to these soils [e.g., burkholderia, ralstonia, massilia, acinetobacter, and pseudomonas ]. despite the fact that many of these srs native soil bacteria are bioremediative being armed with an arsenal of metal resistance genes (mrgs), but they can also pose public health risks by virtue of their acquired antibiotic resistance(s), via the activity of antibiotic resistant genes (args), thus strongly negating their beneficial properties. thus, heavy-metal induced development and carriage of args, or the 'resistome' , within the native soil microbiota, can lead to the emergence and rapid spread of multidrug-resistant (mdr) bacteria, also referred to as "superbugs" (davies and davies, ) , leading to potential future pathogenic outbreaks. in fact, antimicrobial resistance is projected to cause approximately million deaths, annually by year (o'neill, ) , outpacing cancer, which is the current leading cause of human mortality. it is also pertinent to mention here that secondary infections by nosocomial, or hospital acquired pathogens is on the rise; at least % of the deaths reported from the ongoing covid- outbreak were caused from secondary infections (antibiotic resistance: the hidden threat lurking behind covid- , ). specifically, respiratory disease caused initially by the covid- viral agent has been shown to rapidly ensue in pneumonia, thus predisposing patients to secondary bacterial infections, and exacerbating underlying cardiac and pulmonary conditions leading to death. therefore, it is critical to obtain a better understanding on the state of antimicrobial resistance prevalent in environmental representatives of bacterial pathogens, because the environment is a well-known reservoir of pathogens. a central question in our ongoing work is focused on assessing those genomic features that underpin antimicrobial resistances in opportunistic bacterial pathogens in the environment, so that appropriate mitigation strategies to combat these pathogens can be obtained. among the bacteria genera that are listed above and relevant to the srs ecosystem, one genus that deserves further study because of its double life strategy is serratia species, from the enterobacteria family. notably, recent genomics analysis has resulted in the division of serratia spp. into the following broad strain categories based on their functional activities: pathogenic, environmental and symbiotic strains (sandner-miranda et al., ) . furthermore, the environmental strains are divided into three sub-groups: environmental strains that are symbiotic and associated with soil and plants, environmental strains that have been isolated from water and environmental strains that were isolated from food sources. overall, it was demonstrated that pathogenic and environmental serratia isolates presented a high number of antibiotic resistant genes (args); especially for efflux systems (levy, ; nelson et al., ; armalytė et al., ; ben maamar et al., ) . the nosocomial pathogenic serratia strains possessed a higher degree of acquired resistance genes relative to environmental isolates. however, stress posed by long-term heavy metal contamination is known to exacerbate recruitment and proliferation of args in the native soil microbiota (cycoń et al., ; kraemer et al., ) , leading to the main question for this study: are environmental serratia species developing broad antimicrobial resistance activities? to address this overarching question, we performed this case study on environmental representatives of serratia spp., specifically on a newly isolated representative strain (gendy et al., b) , compared to a plethora of other serratia spp., having originated from both environmental and clinical settings. note that the environmental serratia species perform a variety of ecosystem services in the environment, including bioremediation of pentachlorophenol (singh et al., ) , dichlorodiphenyltrichloroethane (bidlan and manonmani, ) , diesel (rajasekar et al., ) , chlorpyrifos (cycoń et al., ) , as well as heavy metals to include, nickel (kannan and ramteke, ) , chromium (mondaca et al., ; campos et al., ) , molybdate (yunus et al., ; shukor et al., ) , lead, cadmium (cristani et al., ) , manganese (ii) and hg (françois et al., ) . some serratia spp., are also well-known plant growth promoting bacteria (pgpb), exhibiting positive impacts by increasing nutrition to the plants, reducing stress and enhancing overall plant productivity (singh and jha, ) . as stated above, despite these attractive environmental and agricultural traits of serratia spp., they are also known to possess pathogenicity, accounting for nosocomial infections of the respiratory tract, urinary tract, surgical wounds and even meningitis (khanna et al., ; abreo and altier, ; cristina et al., ) , specifically caused by serratia marcescens. therefore, this potential duallife strategy of serratia marcescens needs to be carefully assessed, specifically for the presence and activities of args, in context to their environmental applications, such as for u and hg bioremediation. with this backdrop, we pursued this study keeping in mind the following objectives which are summarized as follows: ( ) obtain an environmental serratia isolate resistant to hg and u; ( ) evaluate metabolic potential of the isolated strain and screen how metabolic activity is affected by stress posed by hg; ( ) evaluate the presence and functional activity of antimicrobial resistance using phenomics; and finally, ( ) survey the genomic potential of the isolated strain in context to both, metal resistant genes (mrgs) and antibiotic resistant genes (args). overall, this study provides a broader and comprehensive understanding on the myriad of evolutionary and adaptive traits possessed by an environmental isolate of serratia species, which can be opportunistic pathogens, with special emphasis on carriage of heavy metal and antibiotic resistances. acquired resistance to multiple antibiotics has been recognized as a global crisis, which needs urgent attention so that a major outbreak, similar to the ongoing global crisis brought about by covid- pandemic, can be avoided. studies such as this, suggests immediate remedial measures be adopted to obtain mitigation strategies against the development of multidrug resistant bacteria, such as the environmental serratia species, and promulgate strategies in the event a bacterial pathogen outbreak occurs and causes large-scale human fatalities, akin to the covid- , which is ongoing, as of this writing. as part of an ongoing study, soil samples were obtained from the srs mills branch (area ) which is found cocontaminated with total hg concentrations in the range of - ng/g dry weight (xu et al., ) as well as u. to isolate heavy metal resistant bacteria, soils were serially diluted and plated onto lysogeny broth agar (lba) containing µg/ml of mercuric chloride; the resulting colonies were picked and further isolated based on their distinct morphologies and pigmentation agarwal et al., agarwal et al., , a . plates were incubated aerobically at • c in the incubator, and resulting colonies were further isolated, yielding a robust hg resistant (hgr) strain, labeled as srs- -s- . strain identification was performed using s rrna sequencing, as recently reported (gendy et al., b) . hg resistance of strain srs- -s- was evaluated by dilution of overnight grown strain to od of . . this inoculum ( µl) was then added to µl of lb amended with incremental concentrations ( - µg/ml) of mercuric chloride. od was then evaluated every h for up to h at • c using the bioscreen c microbial growth analysis system (growth curves usa, piscataway, nj, united states), as shown previously . this minimum inhibitory concentration (mic) assay was performed three times, and the average values are reported. controls were also run on plates that did not contain hg and compared under the same growth conditions. to evaluate resistance against uranium, our recently developed plate mic method was used because u is known to cause precipitation in liquid media causing turbidity which interferes with monitoring of bacterial growth . briefly, strain srs- -s- was prepared taking a single colony inoculated into lb broth overnight and then diluted to an od of . . further, a -fold dilution series was prepared up to − dilution range and plated onto lb plates containing u in concentrations of , , , and mm, using uranyl nitrate hexahydrate. plates were incubated at • c and photographs were taken after hours of growth to identify mic against u. inoculum was prepared in lb broth with a single colony of strain srs- -s- , incubated at • c until od reached . . then the cells were washed five times with . % nacl to eliminate any nutrients originating from previous growth in lb broth. this cell suspension ( µl) was used as inoculum for establishing biolog r ecoplates tm , as suggested by the manufacturer (biolog inc., hayward, ca, united states), such that, each well contained a cell concentration of − , which was checked by microscopic evaluation of cell density. the biolog r ecoplates tm consists of -well microtiter plates with three replicates of common substrates utilized by soil microbiota, thus are ecologically relevant for microbial physiological and metabolic activity assessment (preston-mafham et al., ) . a separate set of ecoplates was established, containing hg at different concentrations to assess the strain's physiological response; these concentrations were , , and µg/ml mercuric chloride, respectively. the resulting absorbance was measured via plate reader at nm (spectrophotometer-spectramax-m ), every h over a -day period. obtained data was then plotted using microsoft excel and further analyzed. average well color development obtained from the biolog r ecoplates tm was estimated as shown before (gamo and shoji, ; deng et al., ; gryta et al., ) . phenotype microplate tm (pm) from biolog is an array of well microtiter plate, where each set of wells contain the same antibiotic in increasing increments along with the needed minimal medium components and specific dye. the arrays can provide resistance patterns against antibiotics belonging to different chemical classes, e.g., aminoglycosides, β-lactams, lincosamides, synthetic antibiotics, glycopeptides, tetracyclines, amphenicols, macrolides, sulfonamides, and rifamycins. the antibiotic resistance of bacteria was assessed using biolog pm c and pm b microplate tm plate assays, as suggested by the manufacturer; layout of antibiotics is as detailed by the manufacturer for the biolog r ecoplates tm (biolog inc., hayward, ca, united states) . to initiate the pm assay, strain srs- -s- was grown overnight at • c on an lb agar medium. a single colony was then picked and inoculated into ml of the inoculating fluid [if- a gn base inoculating fluid ( . ×), biolog inc.]. resulting cell density was measured by a plate reader according to the biolog protocol (biolog inc., hayward, ca, united states), followed by inoculation of the cell suspension into plates ( µl/well) and incubated at • c for hours. od was collected every min to determine color shifts resulting from microbial growth in the wells reflected of phenotypic differences and sensitivity/resistance to different antibiotics. collected data was organized and plotted in microsoft excel. data obtained from biolog r ecoplates tm over the -day period was analyzed using primer v (primer-e ltd.). statistical relationships were evaluated by running non-metric multidimensional scaling (nmds) analysis, where the different concentrations of substrates utilized in the presence and absence of hg amendments were log transformed [(log (x + )], hg treatments were used as variables, as appropriate and scaled by maximum data point. the whole genome shotgun project of serratia sp. srs- -s- reported in this study has been deposited at ddbj/ena/genbank under the accession vhnf ; bioproject: prjna ; biosample: samn . the version described in this research is version vhnf . . to understand the genomic characteristics of strain srs- -s- in reference to mrgs and args, a single colony of the bacterium growing on lb plate, amended with hg, was inoculated into liquid lb media and incubated overnight at • c in a shaker at rpm. after overnight growth, the media was centrifuged at , rpm for min to obtain a pellet, which was then used for dna extraction with zr fungal/bacterial dna kit (zymo research, irvine, ca, united states) and sequenced using an illumina hiseq instrument. assembly and annotation on the obtained contigs were performed, as reported in our recent study (gendy et al., b) . default settings were used in all the genomic analysis, unless otherwise stated. taxonomic affiliation was conducted using the onecodex workflow (minot et al., ) , which is based on the identification of typically - bp short sequences, that are found unique to a specific taxa within the inputted sequence reads. based on the collection of k-mers found in a given read, the whole genome sequence is then assigned to a taxon. phylogenomic analysis was run using the default value of k = for the taxonomic assessment of strain srs- -s- using the onecodex database. to obtain a physical map of the genome, cgview comparison tool was used with default settings (grant et al., ) . the genome, with a × coverage was annotated and the genes were predicted using rapid annotations using subsystems technology-rast (aziz et al., ) , prokaryotic genomes automatic annotation pipeline (pgaap), version . (tatusova et al., ) , the pathosystems resource integration center (patric, version . . ) (wattam et al., ) or integrated microbial genomes (img) system (chen et al., ) . average nucleotide identity (ani), and average amino acid identity (aai) was also obtained via edgar (blom et al., ) and ezbiocloud pipelines (yoon et al., ) . ani is a measure of the genomic resemblance of two different bacterial species and typically, ani values between genomes of the same species are % or beyond (goris et al., ) . the merr gene taxonomy in strain srs- -s- was evaluated using comparative functions embedded within the patric pipeline. venn diagram and phylogenomic comparisons were obtained using the edgar pipeline, version . (blom et al., ) . after edgar analysis, the newick phylogenomic tree file was downloaded and an amino acid based tree was constructed using megax (wattam et al., ; kumar et al., ) . island viewer (bertelli et al., ) was used to identify genomic islands (geis) (yung et al., ) , within the genome of srs- -s- . remnants of phage dna, also called prophage, present in the whole genome sequence of srs- -s- were located using the prophage hunter (song et al., ) . our recent studies suggest that the metalliferous srs soils may be serving as a reservoir for the recruitment and proliferation of metal and antimicrobial resistances in the native microbial communities gendy et al., a,b; pathak et al., ) , thus presenting risks to the ecological processes and public health. overall, this study further builds upon evidence to show that the srs contaminated soils inherently harbor higher antibiotic resistance, relative to the reference soils, and thus driving antimicrobial resistance to the native microbiota, including several known pathogens (e.g., burkholderia, ralstonia, massilia, acinetobacter, and pseudomonas wright et al., ; thomas et al., ) . to assess if carriage of metal resistant genes (mrgs), and antibiotic resistant genes (args) is a rampantly widespread trait, especially within opportunistic bacteria native to the srs soils, this study was conducted on soils collected from a different site ( ), relative to that reported by our group recently (pathak et al., ) . specifically, soils from a highly contaminated site-srs were serially diluted and spread plated onto lb agar plates supplemented with µg/ml mercuric chloride. site has been shown to be co-contaminated with a variety of different heavy metals, such as hg and u (edwards et al., ) . plates were incubated at • c, for hours and colonies that appeared on the plates were further purified resulting in a vigorous hg-resistant (hgr) strain, labeled as srs- -s- . identification of the isolated strain using s rrna gene sequencing revealed closest taxonomic affiliation with s. marcescens (gendy et al., b) . specifically, phylogenomic analysis, as stated later in this study, revealed . % of obtained genome reads affiliated with s. marcescens ( figure a) ; at the species level, % of the reads belonged to s. marcescens strain ww , followed by % of s. marcescens strain egd-hp ( figure b) . to determine the resistance potential of strain srs- -s- against hg, the bacteria was grown in lb broth, supplemented with different concentrations of hg; these results were presented in our recent study (gendy et al., b) . it was observed that strain srs- -s- could resist up to µg/ml hg, which revealed a strong hg resistance (hgr) phenotype of strain srs- -s- . to test for resistance against uranium (u), a plate mic method was used, which showed resistance of strain srs- -s- at , , and mm but strain was sensitive at mm u, respectively (figure ) . note that s. marcescens has previously been shown to be resistant to u under both aerobic and anaerobic environments (kumar et al., ; choudhary et al., ; nongkhlaw et al., ; newsome et al., ) . to further understand the hg and u-resistant ability of this strain, physiological and genomic studies were then performed. biolog r ecoplates tm , containing distinct nutrient sources, was used to evaluate the metabolic and physiological response of strain srs- -s- in the absence and presence of hg amendments, as a representative heavy metal contaminant. the cellular response in this assay was monitored as a function of color change of the tetrazolium salt when substrates in each well are utilized by cellular respiration, resulting in the production of a purple formazan dye. higher the substrate utilization, the more intense the color of the formazan produced and by inference, metabolic activity of the cell. kinetic growth response curves were obtained for each growth substrate and plotted to assess cellular phenotype comparisons by calculation of the average well color development (awcd), which revealed that serratia sp. srs- -s- utilized different sources of carbon in the following order carbohydrates > polymers > amino acids > carboxy acids > esters (figure a) , respectively. esters initially were preferred over carboxy acids and amino acids but after days, the above stated trend held up until the experiment ended at days. specific to different carbon sources, it was found that strain srs- -s- preferred the following carbon substrates: specific to different carbon sources, it was found that strain srs- -s- preferred the following carbon substrates: d-mannitol, i-erythritol, γ-amino butyric acid, l-asparagine, n-acetyl-d-glucosamine, and tween (data not shown). to understand how hg stress may impact metabolic potential, growth of strain srs- -s- was evaluated in the presence of increasing concentrations of hg, which showed that at both, and µg/ml hg concentrations, the strain's metabolic potential (awcd) became inhibited; the strain started to use substrates after a lag of almost days ( figure b) . however, at an hg concentration of µg/ml, the strain was unable to utilize any of the carbon substrates, despite the strain's potential to resist up to µg/ml hg based on mic values (gendy et al., b) . this difference in the mic and physiological activity response in the presence of hg amendments can be explained on the basis that the higher mic in lb broth, which is a richer nutrient source relative to those present in the biolog r ecoplates tm . regardless, this extensive physiological characterization demonstrated that serratia sp. srs- -s- used an average of . % of the different nutrient sources tested without hg stress (table ) ; conversely, in the presence of µg/ml hg, this response became inhibited at . % of total substrates; at µg/ml hg even fewer substrates were utilized-at an average of . %, while at a concentration of µg/ml hg, total physiological activity of the hg-resistant strain ceased ( figure b and table ) , despite the robust hg resistance ability of srs- -s- . interestingly, the utilization rates of amino acids, such as l-asparagine, l-alanine, and l-serine, relative to other substrates significantly decreased upon exposure across all treatments (data not shown), most likely due to binding of hg to amino acids which reduces their availability and hence physiological activity. it could also be that metabolic pathways for the utilization of these amino acids are hampered by hg toxicity. it has been well-established that mercury, the most toxic heavy metal toxicity, is mainly owed to its high degree of affinity to the sulfhydryl ligands in amino acids, binding to which causes alteration in protein structure along with loss of protein function. overall, this part of the study suggests that hg can interfere with the bacterial physiological response, even on an organism that is resistant to hg. therefore, continued addition of hg can have detrimental impacts to microbially mediated ecosystem services, such as biogeochemical cycling of nutrients. this is a significant finding from an environmental standpoint, which has not been demonstrated before, to our knowledge. statistical analysis of the strain's physiological response to hg amendment was assessed using primer v , which is shown in figure c . cluster analysis clearly showed that in the absence of hg, the strain's metabolic activity was a positive sign (+) indicates strain utilization of carbon source and the negative sign (−) indicates inability of the strain to utilize the substrate source. statistically different relative to hg treatments. specifically, the metabolic activity profiles at µg/ml hg and µg/ml hg clustered together and away from the highest tested concentration of µg/ml hg, mirroring the trends shown in figures a,b . this confirms the observation that hg can interfere with the physiological and metabolic response, even in an organism that harbors hg resistance. therefore, continued addition of hg will likely have significant detrimental impacts to microbially mediated nutrient cycling processes; even to those microorganisms that have developed tolerance/resistance against heavy metals, such as hg. arguably, microbiota that are unsuccessful in the recruitment of resistant gene determinants against heavy metals will have likely been removed from a contaminated soil habitat and ecosystem services rendered by the outcompeted bacteria are either lost, diminished or taken over by the surviving metal resistant bacteria. to further understand the gene determinants for heavy metal resistance, genomic studies were performed on strain srs- -s- . genomic sequences of strain srs- -s- , with a coverage of × were obtained (gendy et al., b) , and characterized by the following parameters: contig count ( ); total length ( , , bp); n ( bases); l ( ); an average gc content of . %; a coding sequence of , proteins (cds) and genes for transfer rna (trna) and genes for rrna, respectively. figure shows a circular genomic map of strain srs- -s- constructed with cgview comparison tool. gene prediction performed using patric, rast and ncbi pipelines revealed that strain srs- -s- harbored a total of protein coding genes; . % of these genes were annotated as protein coding genes with predicted functions; . % genes were annotated as protein coding genes with enzyme production and . % genes were associated with kegg pathways, respectively. moreover, . % of the protein coding genes were annotated with cogs, i.e., clusters of orthologous groups of protein; cogs represent an ortholog or direct evolutionary counterpart among bacterial genomes as they evolve over time. as stated before, phylogenomic analysis of strain srs- -s- relative to other sequenced serratia species revealed closest taxonomic affiliation with serratia species and other genera, such as salmonella ( figure a) . specifically, onecodex analysis resulted in the assignment of % contigs from strain srs- -s- to s. marcescens ww , followed by % to s. marcescens egd-hp and % with s. marcescens subsp. sakuensis, respectively ( figure b) ; these are all environmental strains not directly related to their hospital or nosocomial counterparts. similar result was demonstrated by edgar analysis (data not shown), in which a phylogenomic tree of strain srs- -s- was built with serratia genome sequences, build out of a core of genes per genome, in total. the core had amino acid residues/bp per genome, in total. furthermore, average nucleotide identity (ani) analysis mirrored the previous results such that s. marcescens sp. ww was the closest strain to serratia sp. srs- -s- with a . % value followed by s. marcescens sp. egd hp ( . %) and serratia sp. fs ( . %), respectively (supplementary figure s ) . note that the ani calculator estimates the average nucleotide identity using both best hits (one-way ani) and reciprocal best hits (two-way ani) between two genomic datasets, as calculated by goris et al. ( ) . typically, the ani values between genomes of the same species are above % and therefore, strain srs- -s- is assigned to s. marcescens. after the taxonomic assessment of strain srs- -s- , further functional characterization was performed using rast, which revealed the presence of subsystems with the following top six categories, genes in parenthesis: amino acid metabolism ( ); carbohydrate metabolism ( ); vitamin and cofactor metabolism ( ); protein metabolism ( ); membrane transport ( ) and fatty acids and lipids ( ), respectively. similar results were shown by patric based annotation also revealed the presence of genes (in parenthesis) for metabolism ( ); energy production ( ); protein processing ( ); stress response/defense/virulence ( ); membrane transport ( ); cellular processes ( ); dna processing ( ); genes were also found for resistance to antibiotics and toxic compounds. also identified are genes shown homologous with prophages, transposable elements and plasmids, indicating the receptivity or interactions of strain srs- -s- with mobile genetic elements. of particular relevance are several gene homologs that have previously been shown to be involved in the resistance against heavy metals/radionuclides (genes in parenthesis), including efflux systems ( ) and membrane transporters ( ), which likely facilitates survival of strain srs- -s- in a metalliferous soil habitat. these findings are similar to our previous genomic analysis of another serratia species-strain s b, isolated from the savannah river swamp system soils . note that the srss site is located at the confluence of four mile creek and the savannah river, which received liquid hg (hg) effluents from a chloralkali facility near augusta, ga, united states, until the s. strain s b, native to these hg-contaminated soils, was found to harbor a genomic size of approximately , , bases with , coding sequences and a gc content of . %. a total of subsystems were annotated from strain s b with genes (number in parentheses) for membrane transport ( ), stress response ( ), metabolism of aromatic compounds ( ), motility and chemotaxis ( ), among gene homologs shown to be involved in the resistance against heavy metals, similar to results presented here for strain srs- -s- . collectively, these analyses of serratia species strain srs- -s- revealed the presence of several genome-enabled metabolic and catabolic processes, which likely play a significant role in the colonization and strain's survival within the metalliferous srs soil habitat; similar to previous studies that we have conducted on long-term contaminated sites (olaniran et al., ; ojuederie and babalola, ; chauhan et al., ) . further analysis was performed using comparative genomics to evaluate evolutionary and adaptive traits of strain srs- -s- with the closest taxonomic strains-serratia species ww , egd_hp , vgh , and fs , as shown by edgar analysis (data not shown). this revealed the presence of distinct genes present only in strain srs- -s- relative to the other closely associated serratia strains (figure ) . similarly, distinctive genes were also identified in the other four strains analyzed, shown in parenthesis: serratia marcescens egd_hp ( ); s. marcescens vgh ( ); s. marcescens ww ( ), and s. species fs ( ), respectively. even though genes present in srs- -s- (# sector in figure ) , made up only about % of the total genome size of the strain, but this set of genes are representative of "distinct" determinants for efflux, metal resistance, universal stress proteins, cytochromes, drug resistance, transport proteins as well as transposases. moreover, many of the unique genes in srs- -s- were classified as hypothetical proteins, functions of which are unknown at this time. it is also noteworthy that the strain harbored several genes that have been implicated in diverse roles pertaining to plant promoting activities, such as: phosphate metabolism (phou, pstb, psta, pstc, and psts); spermidine synthase and n acetyltransferase genes (data not shown). these traits are similar to other serratia species (matteoli et al., ) , and may render a plant growth promoting role of strain srs- -s- in its native soil habitat, simultaneously reducing the metal toxicity. overall, this comparative genomic analysis confirmed a strong genomeenabled bioremediative as well as plant growth promoting potential of strain srs- -s- . using the integrated prediction method to identify geis, strain srs- -s- was shown to harbor different geis, ranging in size from , to , bp (figure ) , when s. marcescens ww was used as the reference genome. note that genomic islands is a universal trait in many environmental isolates we have studied to date pathak et al., ) ; geis are beneficial gene segments recruited by host bacteria from their external environment via horizontal gene transfer (hgt) mechanisms (pérez-pantoja et al., ) . typically, geis have been shown to be associated with host-beneficial adaptive traits such as bioremediation, virulence, antibiotic resistance and metabolism. overall, these gei-encoded traits can be binned under the following four major categories (bertelli et al., ) : ( ) metabolic islands (mis), which are a set of genes for secondary metabolite biosynthesis; symbiotic islands (sis), which are those set of genes that facilitate symbiotic associations with other micro and/or macroorganisms; pathogenicity islands (pais), coding for virulence or disease causing genes; and resistance islands (ris), rendering resistance for antibiotics and other bacteriostatic/bactericidal agents. overall, this analysis led to the identification of several genomic islands in strain srs- -s- , with gene functions previously identified for metal resistance, efflux and transposons. this strongly indicates a likelihood that geis are probably recruited via hgt to promote survival in a heavy metal contaminated soil habitat (bertelli et al., ) . these findings are similar to our previous reports from serratia strain b , which also revealed a repertoire of biodegradative genes, several occurring on genomic islands . the presence of these geis in srs- -s- also provides clues into the strain's genome plasticity, introduced by mobile genetic elements including integrases or transposases, which were also identified. notably, one mechanism for geis to be incorporated into bacterial genomes is through bacteriophage attack, which can leave phage remnant dna, or prophage, integrated into the bacterial genome. thus, bacteriophage attacks is a major mechanism that influences bacterial evolution, resulting in the recruitment of genomic traits beneficial to the host species, such as virulence factors, antibiotic resistance mechanisms and bioremediative functions (bernheim and sorek, ) . when presence of prophage was evaluated in strain srs- -s- , candidate genes were identified in this category, as shown in supplementary table s . specific details on the top phageassociated genes are as follows (sizes in parenthesis): salmonella phage fsl sp- ( , bp); salmonella phage ( , bp); nocardia phage nbr ( , bp); edwardsiella phage pei ( , bp) and klebsiella phage lv- ( , bp), respectively. in fact, different salmonella-specific prophages were found associated in the genome of strain srs- -s- followed by from klebsiella spp. conversely, only phage x was identified in the genome of strain srs- -s- that was related to serratia spp., which indicates strain srs- -s- was more responsive to salmonella-specific phage interactions relative to its own genus. because salmonella possess pathogenic traits, it is likely that pathogenicity of serratia spp. are transmitted from salmonella into serratia via hgt mechanisms, but this observation is based only on genomic analysis with no rigorous functional proof available as of this writing. this observation, however, also goes well in line with the taxonomic similarity between the strain's genome with salmonella spp., albeit at only . % (data not shown). in this context, genome-wide analysis of serratia species have recently established that the environmental serratia representatives have commonalities pertaining to the high number of antibiotic resistant genes relative to the hospital or nosocomial strains (sandner-miranda et al., ) and it appears that the environmental isolates are also equally potent in their antimicrobial activities. genomic mining of serratia sp. srs- -s- also revealed several genes that likely enable bacterial adaptability and survival to survive in a contaminated environment. for example, it was found that the strain contained several gene homologs for heavy metal and biocide resistance such as efflux proteins [e.g., resistance, nodulation, and proteinfamily (rnds); outer membrane channel tolc], and heavy metal-responding transcription regulators. furthermore, the srs- -s- strain genome had significant numbers of abc-type transport and heavy metal detoxication proteins, which could maintain homeostasis of metals -all of these adaptations likely contribute for environmental and evolutionary response of strain srs- -s- and survival in a metalliferous soil habitat. one major mechanism underpinning microbial resistance against both heavy metals and antibiotics is by efflux of these compounds outside of the cellular environment, as soon as the cells sense their presence (levy, ; nies, ; blanco et al., ; buffet-bataillon et al., ) . strain srs- -s- possessed at least genes related to different efflux groups such as abc-type efflux, multidrug efflux, rnd efflux system etc. (data not shown). therefore, efflux mechanisms may be one major response of strain srs- -s- to survive in a highly metalliferous soil habitat. toward this end, hgresistance is also likely efflux based in the strain. genome mining revealed that the strain did not support a complete mer operon but only harbored the transcriptional regulator, merr family (fig| . .peg. ) (supplementary figure s a) , along with the heavy metal sensor histidine kinase (hmhk) and the dna-binding heavy metal response regulator (hmrr). the merr gene in strain srs- -s- was taxonomically closest to s. marcescens ww and s. marcescens egd-hp (supplementary figure s b) ; these strains were also identified closest relatives in the phylogenomic based analysis shown in figures a,b . therefore, one central question arises from the lack of a complete mer operon in strain srs- -s- -what mechanism(s) render hg resistance to serratia marcescens strain srs- -s- ? to this end, previous body of information revealed that not all hgr bacteria support a complete mer operon. the mer operon has origins in geothermal environments (boyd and barkay, ), and has evolved from being a constitutively expressed system made up of simple genetic elements to a highly regulated and complex operonic system-the complete mer operon. we are tempted to speculate that hg resistance in strain srs- -s- is likely mediated via efflux mechanisms, as proposed elsewhere. in fact, studies have found energy-dependent efflux systems to render resistance to many heavy metals, including cadmium, zinc, nickel, cobalt, and copper (reyes et al., ) . in a seminal report, several mer negative but hgr bacteria exhibited decreased resistance to hg when amended with the protonophore-carbonyl cyanide m-chlorophenylhydrazone (cccp) (reyes et al., ) . it may also be that strain srs- -s- harbors genes which are not homologous to recognized mer genes and hgr is a mechanism driven by potentially novel genes; we are currently conducting more work to resolve these possibilities in strain srs- -s- as it relates to hg resistance. regardless of these possibilities, the presence of abundant mrgs in strain srs- -s- provides for a deeper understanding on soil survival strategies. to validate the phenomic response indicating carriage of broad antibiotic resistance in srs- -s- , a comprehensive survey of antibiotic resistance genes (args) was performed employing patric, card, and rast pipelines. this bioinformatic analysis revealed a plethora of args in serratia sp. srs- -s- . specifically, patric analysis revealed the presence of several genes classified under speciality category, that included genes for transporters, genes for drug targets, genes for virulence factors, and genes for antibiotic resistance, respectively ( figure a ). this points to the repertoire of speciality genes, many related to virulence and args, that are recruited by this soil-borne isolate as its arsenal againts multiple antimicrobials, likely shaped by long-term exposure to heavy metals, such as uranium and mercury. similar results were obtained by using card, which revealed resistomes for the following drugs (percent identity with the corresponding region is shown in parenthesis): fluoroquinolone ( . %), tetracycline's ( . %), cephalosporin ( %), penam ( %), carbapenem ( . %), cephamycins ( . %), fosfomycin ( . %), macrolide ( . %), monobactam ( . %), and aminoglycoside ( . %) (jia et al., ) , as shown in figure b . several other genes that possibly confer antibiotic resistance were also recognized, including antibiotic target protein (penicillin-binding protein mutations), antibiotic target alteration, antibiotic self-resistance gene, resistance-nodulationcell division (rnd), major facilitator superfamily (mfs), efflux pump complex etc. this overall arg evaluation of serratia sp. srs- -s- indicates that the strain has acquired resistance to multiple antibiotics, most likely exacerbated by exposure to hg, u and possibly other heavy metal contamination in the srs soils. these findings are in line with previous body of information on srs soils, which unequivocally show that the legacy contaminated srs soils serve as a repository for acquisition of both metal and antibiotic resistances wright et al., ; thomas et al., ) . however, this study is focused on serratia spp., which are opportunistic pathogens and brings out the necessity for further focused studies on this aspect rather than a more generalized assessment of the proliferation and abundance of mrgs and args within the srs soil habitat, as has been the case with most previous studies. to validate the functional activity of args in serratia sp. srs- -s- , phenotype microarray (pm) technique was used, which is a microtiter-plate-based substrate utilization assay (bochner and savageau, ; bochner, ; bochner et al., ) , similar to the biolog ecoplates (borglin et al., ) . note that the pm technique, also referred to as phenomics, offers the ability to obtain cellular response of axenic microbiota or environmental communities, as well as screening antimicrobial resistances (blanco et al., ) . the results obtained from the pm microplate tm analysis are shown in figures a,b . this data is compared in table , showing that only out of antibiotics tested, inhibited or ceased growth, indicating high antibiotic resistance. in fact, . % of the antibiotic/concentration tested did not affect growth, suggesting the broad arg potential of this strain. the antibiotics and concentrations (as provided by the manufacturer), that affected growth included the following: cloxacillin ( µg/ml), lomefloxcin ( . µg/ml), minocycline ( µg/ml), nafcillin ( µg/ml), enoxacin ( . µg/ml), nalidixic acid ( µg/ml), potassium tellurite ( . µg/ml), f- neomycin ( . µg/ml), ofloxacin ( . µg/ml), carbenicillin ( µg/ml), oxacillin ( , µg/ml), d,l-serine hydroxamate ( µg/ml), novobiocin ( µg/ml) d,l-serine hydroxamate ( µg/ml), dodecyltrimethyl ammonium bromide ( µg/ml), respectively. after correlating the findings from the antimicrobial resistance genes (args) analysis and phenotype microarray (pm) antibiotic profile of serratia sp. srs- -s- , it can be concluded that the serratia sp. srs- -s- harbors a suite of args against many antibiotics as well as metal resistant genes (mrgs). the antibiotic profile of serratia sp. srs- -s- was also mirrored in the genomic analysis (figures a,b) . biolog pm microplate tm array assay on serratia sp. srs- -s- yielded an extensive resistance pattern to antibiotics. the development of a broad spectrum of args in strain srs- -s- are very likely due to the stress imposed by the long-term contamination of srs soils with hg and u, thus presenting with serious public health and ecological risks. note that hg resistance is almost invariably found to co-occur with antibiotic resistance, specifically for ampicillin, tetracycline, erythromycin, and penicillin (ready et al., ) . therefore, presence of hg and u stressors present in the srs soils pose public health and ecological concerns from the metal driven antibiotic resistome or vice versa. serratia marcescens represents a recently recognized pathogenic species and hence remains largely understudied. furthermore, whole genome sequence projects on this pathogen have also only recently become available to the scientific community (abreo and altier, ) , thus limited understanding is available on the genome-wide adaptive traits of s. marcescens as a pathogenic agent. toward this end, this study reveals evolutionary and adaptive traits possessed by an environmental serratia species, and in particular, carriage of heavy metal and antibiotic resistance gene determinants. it is very likely that broad antibiotic resistances are being acquired by microbiomes, such as serratia, in sites that remain historically contaminated with heavy metals, which is a major public health concern. note that resistance of opportunistic pathogens to one or more antibiotics has been recognized as a global crisis, which needs immediate attention such that a major pathogenic outbreak such as the covid- , can be avoided. even though covid- is a viral disease but some preliminary estimates suggest that % of mortalities are occurring from nosocomial secondary infections. therefore, one broader recommendation from this study is to take immediate action against development of multidrug resistant bacteria and develop strategies should a bacterial pathogen outbreak occur that is similar in magnitude to the ongoing covid- , as of this writing. it is noteworthy that outbreaks related to s. marcescens and other serratia species have occurred sporadically since s (grohskopf et al., ; mahlen, ; yao et al., ) , mainly in neonatal and cardiac icus, orthopedic clinics and dialysis centers. there is a large body of information available on serratia epidemiology and resistance patterns and s. marcescens strains are typically resistant to all penicillins and susceptible to all carbapenems; a recent study, however, was found resistant to all tested antibiotics (bertrand and dowzicky, ) . however, not much is known on the antimicrobial patterns of environmental serratia members, and this study addresses this gap. more recently, pangenome-based analysis of s. marcescens strains from nosocomial and environmental origins as well as different countries, revealed consistently presence of high number of args (abreo and altier, ), likely due to stress imposed by environmental contaminants, as also suggested in this study on strain srs- -s- . to obtain a better understanding on the relationship between the environmentally relevant s. marcescens strain srs- -s- with some other medical/clinically relevant isolates, comparative whole genome analysis was run using the integrated microbial genomes (img) system, and results are shown in figure . specifically, we selected the closest taxonomic relatives of strain srs- -s- identified in previous analysis, such as s. marcescens ww , s. marcescens egd-hp , along with several strains isolated from bacteremia or clinical settings, such as s. marcescens umh , s. marcescens ; a total of s. marcescens strains were included in this analysis. remarkably, strain srs- -s- clustered closest to the strains umh and umh in a pca drawn on cog profiles for the selected genomes ( figure a) . conversely, strains ww and egd-hp -the closest taxonomic relatives of strain srs- -s- , clustered away from the cohort with which strain srs- -s- clustered, which were all medically relevant strains. as stated before, cogs represent families of orthologous protein-coding genes and is a reliable assessment of comparing protein phylogeny from microbial genomes (galperin et al., ) , and hence, is a functional assessment of a bacteria of interest. this observation was again confirmed when pca clustering was based on the presence of kegg pathway (ec) profiles (figure b) , which is based on the enzymatically catalyzed network of metabolic pathways and reactions, constructed upon the entirety of all available biochemical information and is not organism-specific. similar to the cog-based analysis, the closest association of strain srs- -s- was with strains umh and -many of these strains were active in bacteremia (anderson et al., ) . overall, this is strong evidence that strain srs- -s- , isolated from a metalliferous contaminated soil environment, is functionally more similar to medically relevant isolates. overall, this is succinct evidence that the environment is potentially serving as a niche for the development, recruitment and proliferation of pathogenic traits within serratia species, which needs to be evaluated to obtain mitigation strategies and avoidance of possible future pathogenic outbreaks emanating from ecological systems; as is indicative for the origin of the covid- viral pathogen from bats, which is likely the main reservoir for this pathogen (banerjee et al., ; andersen et al., ) . the datasets presented in this study can be found in online repositories. the names of the repository/repositories and accession number(s) can be found in the section titled "nucleotide sequence accession number". sg, ma, ap, and rr designed the experiments. sg, ap, and ac performed the genomic analysis. sg, ma, and rr performed the biolog and phenomic tests and analyzed the results. ac, sg, ap, ma, rr, and rj drafted the manuscript. 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interest.copyright © gendy, chauhan, agarwal, pathak, rathore and jaswal. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- -fz utxss authors: jheng, jia-rong; ho, jin-yuan; horng, jim-tong title: er stress, autophagy, and rna viruses date: - - journal: front microbiol doi: . /fmicb. . sha: doc_id: cord_uid: fz utxss endoplasmic reticulum (er) stress is a general term for representing the pathway by which various stimuli affect er functions. er stress induces the evolutionarily conserved signaling pathways, called the unfolded protein response (upr), which compromises the stimulus and then determines whether the cell survives or dies. in recent years, ongoing research has suggested that these pathways may be linked to the autophagic response, which plays a key role in the cell's response to various stressors. autophagy performs a self-digestion function, and its activation protects cells against certain pathogens. however, the link between the upr and autophagy may be more complicated. these two systems may act dependently, or the induction of one system may interfere with the other. experimental studies have found that different viruses modulate these mechanisms to allow them to escape the host immune response or, worse, to exploit the host's defense to their advantage; thus, this topic is a critical area in antiviral research. in this review, we summarize the current knowledge about how rna viruses, including influenza virus, poliovirus, coxsackievirus, enterovirus , japanese encephalitis virus, hepatitis c virus, and dengue virus, regulate these processes. we also discuss recent discoveries and how these will produce novel strategies for antiviral treatment. the endoplasmic reticulum (er) is a eukaryotic organelle in which an array of cell functions takes place. these include the transportation of cellular materials, provision of increased surface area for cellular reactions, and the production of proteins, steroids, and lipids. the er may be overloaded with molecular chaperones, folding enzymes, and massive protein products during normal processes, such as in the differentiation of b lymphocytes into antibody-secreting plasma cells (shaffer et al., ; ma et al., ) or in highly specialized cells for secretion (harding and ron, ) . in addition, dysfunction of the er, known as er stress, results from pathogenic stress signals, such as hypoxia (koumenis, ) , er-ca + depletion, viral infections, or agents that affect ca + balance (i.e., thapsigargin), protein glycosylation (i.e., tunicamycin), and er-golgi vesicular transport (i.e., brefeldin a), which lead to accumulation of misfolded and unfolded proteins (kaufman, ) . to reduce the adverse effects of accumulating misfolded or unfolded proteins, the cell operates an adaptive response known as the unfolded protein response (upr) to reduce the load of newly synthesized proteins within the er and eliminate inappropriately folded proteins through upregulation of er chaperone expression. in addition, proteins that fail to correctly fold are then deployed to the distal secretory pathway from the er by the er-associated protein degradation (erad) pathway of the upr (hampton, ; yoshida et al., ) . there are two erad models for protein degradation: ubiquitin-proteasome erad, designated as erad (i), and autophagy-lysosome erad, designated as erad (ii) (fujita et al., ; korolchuk et al., ) . both models depend on retrotranslocation of erad substrates from the er back to the cytoplasm with the help of the cdc p-p complex. most soluble misfolded proteins are cleared through the ubiquitinproteasome system, which involves action of a cascade of three canonical ubiquitin enzymes: e ubiquitin-activating enzyme initiates the reaction by using atp to covalently activate and then conjugate the ubiquitin to an e ubiquitin-conjugating enzyme. ubiquitin is then transferred from the ubiquitin-charged e to the lysine residue of a specific target or a growing ubiquitin chain by e ubiquitin ligase, which results in a multiubiquitin chaintagged substrate. proteins that are ubiquitinated with k -linked chains are specifically recognized by the s proteasome and subjected to degradation (hershko et al., ) . in contrast, erad (ii) degrades both soluble and insoluble misfolded protein aggregates in autolysosome. autophagy receptors and adaptors, called p /sqstm , nbr , hdac , and alfy, bind to proteins with k -specific monoubiquitination or polyubiquitin chains and then guide them to the concave side of developing autophagosomes (behrends and fulda, ) . notably, p also recognizes k polyubiquitin-tagged proteins for autophagic clearance upon proteasome dysfunction. in addition to the protective role of upr, prolonged and/or excess er stress typically activates caspase- , an er-resident caspase, leading to upr-mediated cell death (szegezdi et al., ) . basal autophagy plays a key role in maintaining cellular homeostasis through eliminating unwanted proteins and damaged organelles by cellular self-digestion in the lysosome to fulfill the demand for the building blocks required for cell survival (levine and klionsky, ; shintani and klionsky, ) . recently, the study of autophagy regulation has grown in different research areas, including regulation of cancer development and progression (mahoney et al., a) , lipid metabolism (singh et al., ) , degenerative diseases (wang et al., ) , and the control of viral pathogenesis . the first step of autophagy relies on the formation of an isolation membrane at the so-called preautophagosomal site (pas) where a system of evolutionarily conserved proteins (atg proteins) comes together. recent reports have revealed that the er serves as a subcellular platform for autophagy initiation (axe et al., ) . the elongation of the initial autophagic membrane requires continued processing by two ubiquitin-like protein-conjugation systems, the atg and lc systems, which modify the autophagy proteins, atg and atg /lc , respectively (geng and klionsky, ) . the autophagosome then fuses with endosomal and/or lysosomal vesicles to create an autolysosome, where digestion of intracellular components occurs (eskelinen, ) . in addition, autophagy can be induced by various physiological and pathological conditions such as nutrient deprivation, oxidative stress, and pathogen infections. the live-or-dead signal is modulated by upr and autophagy and several lines of evidence suggest there is communication between these two pathways (bernales et al., ; ogata et al., ; yorimitsu et al., ; salazar et al., ) ; thus, it is believed that these two pathways could be a therapeutic target in certain circumstances (figure ) . herein, we review recent findings, focusing on the regulation of the upr and autophagy involved in rna virus infection as a new antiviral strategy. viral virulence is determined by successful entrance, replication in the host cell, and release of mature virion. during the life cycle, er stress may arise from the exploitation of the er membrane, accumulation of misfolded proteins, imbalance of calcium concentration by viroporin, and the sabotage or depletion of the er membrane during virion release. details of viral effects are given as follows. many positive-strand rna viruses cause the rearrangement of host intracellular membrane compartments that house replication complexes. er, trans-golgi, or lysosomes are the likely origin of virally induced membranes (miller and krijnse-locker, ; korolchuk et al., ) . upon poliovirus (pv) and coxsackievirus b (cvb ) infection, clusters of vesicles have been considered to derive from er, although other cellular compartment marker proteins also colocalized with viral nonstructural proteins (schlegel et al., ; van kuppeveld et al., ) . consistent with these findings, our previous study indicates that enterovirus (ev ) nonstructural c protein, which participates in viral replication, is associated with the er membrane through direct interaction with er membrane protein reticulon (rtn ), which is required and sufficient for immediate early virus replication and translation (tang et al., ) . in the rtn sirna knockdown cells, synthesis of the c protein was ablated. however, in the rtn rescue cell line a , figure | diagram of the upr arms and their connection to autophagy. alteration of er functions results from stress signals by rna virus infection, by the exploitation of er membrane for viral replication, rapid accumulation of viral proteins, imbalance of calcium concentration by viroporin, and the sabotage or depletion of er membrane for viral release. this leads to the accumulation of misfolded and unfolded proteins, which triggers er stress. to alleviate this adverse effect, the cells operate an adaptive upr to reduce the load of the newly synthesized proteins in the er by activating the perk-eif α branch and eliminating inappropriate protein accumulation by upregulating er chaperone proteins through ire and atf branches. in addition, the incurable misfolded proteins undergo retrotranslocation from the er into cytosol for degradation by an erad mechanism. er stress can contribute to autophagy via activation of jnk, xbp , chop, and atf . red dash arrows indicate the final outcome of the activated pathways, such as apoptosis and autophagy, caused by viral infection. red solid arrows indicate the upr pathways. the synthesis of viral protein and rna was restored. moreover, the interactions between rtn and two ev c homologs of pv and cva have been confirmed (tang et al., ) . immunofluorescence studies reveal that replication of flaviviruses dengue virus (denv) and hepatitis c virus (hcv) may take place on perinuclear er membranes (el-hage and luo, ) . denv nonstructural protein (ns a) is a -kda hydrophobic protein containing five integral transmembrane segments that span the er membrane. functional analysis reveals that ns a involves both denv rna synthesis and virion assembly/maturation (xie et al., ) . furthermore, denv infection induces rockdependent vimentin rearrangement and subsequent er redistribution (lei et al., ) . in addition, the hcv er integral membrane protein, ns b, is responsible for rearranging the er membrane and inducing the formation of new er-derived membrane structures, and this is possibly negatively regulated by rtn -ns b interaction (lundin et al., ; wu et al., ) . the n-glycosylation pathway in the er modifies a mass of proteins at the asparagine residue of the consensus sequence asn-x-ser/thr, where x is any amino acid except pro (kornfeld and kornfeld, ; gavel and von heijne, ) . the modification influences protein folding and attributes various functional properties to the protein. thus, interference with host protein glycosylation by viral proteins competing for the modification process may cause er stress. viruses, including influenza a virus (iav), hepatitis virus, and japanese encephalitis virus (jev), use this host cell process to enhance viral pathogenesis through facilitating folding and trafficking, affecting receptor interaction, and modulating host immune responses (tatu et al., ; dubuisson and rice, ; zai et al., ) . hemagglutinin (ha) of iav is a type i transmembrane glycoprotein that determines viral antigenicity. throughout the glycosylation process, ha rapidly associates with calnexin in a monoglucosylated form. once folded, the ha monomers dissociate from calnexin and assemble into trimeric structures in the er or in the intermediate compartment (tatu et al., ) . hcv envelope glycoproteins e and e have been shown to cooperate for the formation of a functional noncovalent heterodimer (dubuisson et al., ; dubuisson and rice, ) . based on studies of hcv pseudoparticles, coexpression of both envelope glycoproteins has been shown to be necessary to produce infectious pseudoparticles (bartosch et al., ) . glycosylation also occurs in jev and wnv proteins, namely the precursor of membrane protein (prm), the envelope protein (e), and the nonstructural protein ns , which affects the efficiency of virus release and infection (hanna et al., ; zai et al., ) . typically, viroporins are composed by integral membrane proteins to form a hydrophilic pore, which targets different cellular compartments and ions, thus affecting various viral functions (nieva et al., ) . for example, iav m reduces the acidity of vesicular compartments to trigger virus uncoating. it is also required for viral assembly and release. in the case of er-targeting viroporins, rotavirus-encoded nsp modifies the calcium homeostasis by enhancing the calcium permeability of the er membrane. this may be associated with virus-induced cell death and subsequent release of nsp , which in turn causes activation of the phospholipase c-ip cascade in neighboring noninfected cells and is responsible for viral pathogenesis (tian et al., (tian et al., , dong et al., ) . on the other hand, b proteins of picornaviruses also participate in the remodeling of membrane structures and the formation of replication complexes (de jong et al., ) . among them, cbv b, pv , and rhinovirus b are present at the membranes of the er and golgi complex and are responsible for the release of ca + and h + from these organelles. rotavirus studies propose that the double-layered particle (dlp)-vp -nsp complex breaches the er membrane and penetrates into the er. the viral capsid protein, vp , re-envelopes the immature particle (dlp) after removal of the er membrane and nsp , and forms the infectious triple-layered particle (tian et al., ; trask et al., ) . the induction of individual branches or part of the upr by viruses was reported previously. viruses have also evolved different means to modulate the arms of the upr, which consequently expanded both the temporal and spatial superiority for virus replication or completion of the life cycle. it has been reported that viruses regulate the host translational machinery to promote viral protein synthesis by inhibiting the synthesis of proteins involved in host immune responses. in enteroviruses, a and c proteases target translation factors such as eif gi and poly(a)-binding protein (pabp) to impede host translation (lloyd, ) . moreover, modulation of the integrated stress response (isr), which is determined by phosphorylation of eif α to attenuate cellular translation, is another strategy for promoting virulence (figure ) (sonenberg and hinnebusch, ) . four eif α kinases have been identified: heme-regulated inhibitor (hri), which is a response to heme deficiency (chen, ) ; double-stranded rna-dependent protein kinase (pkr), which is induced by interferon (ifn) and activated by double-stranded rna (dsrna) during viral infection (meurs et al., ) ; general control nonderepressible- (gcn ), which is activated by serum and amino acid deprivation (harding et al., ) ; and finally, pkr-like er kinase (perk or pek), which is activated by unfolded proteins in the er (ron, ) . some researchers consider that eif α phosphorylation plays a role in hampering viral protein synthesis. for example, upon vsv infection, the induction of activated perk only correlates with eif α phosphorylation at the later stage of infection. in mef cells carrying a phosphorylation-insensitive eif α s a variant, viral protein synthesis increased compared with a wild-type control, indicating that eif phosphorylation is inhibitory to viral protein synthesis. as demonstrated by matrix (m) protein mutant virus (rm rm), a viral protein (m protein) is involved in counteracting the antiviral response of the phosphorylation of eif α (connor and lyles, ) . like vsv infection, chikungunya figure | eif pathway under viral infection. the m protein of vsv, the e and ns a proteins of hcv, and ns a of jev counteract the phosphorylation of eif α for viral replication. blue solid arrows indicate the direct target of the virus or viral proteins. iav also targets eif α by inducing p ipk, a cellular inhibitor of perk and pkr. ibv upregulates eif α-atf -chop-mediated apoptosis to benefit viral replication. august | volume | article | virus (chikv) induces perk activation but delays eif α phosphorylation. the expression of chikv nsp , which is the rna-dependent-rna polymerase, contributed to suppression of eif α phosphorylation, thus ensuring translation of viral proteins (rathore et al., ) . furthermore, viruses containing type i or type ii internal ribosomal entry sites (iress), such as pv, foot-and-mouth disease virus, mengovirus and emcv, require many canonical translation initiation factors for initial replication (beales et al., ; sarnow, ) . it is reported that pv switches translation mode from an eif -dependent to an eif independent one during the course of infection to ensure efficient proliferation. furthermore, studies have shown that the c terminal of the eif b fragment, cleavage by c proteases, and proteolytic activity of a pro can stimulate virus ires translation of enteroviruses (de breyne et al., ; redondo et al., ) . interestingly, it is reported that phosphorylation of eif α is required for activation of ires during cell differentiation (gerlitz et al., ) . thus, whether the phosphorylation level of eif α positively correlates with ires-dependent viral mrna translational efficiency remains to be determined. some viruses regulate the eif α pathway by interfering with the activation of eif α kinases. hcv ns a protein, containing an ifn sensitivitydetermining region (isdr), interferes with pkr activity by binding to a pkr dimerization domain (pkr residues - ) (gale et al., ) , while hcv e protein binds to perk and inhibits downstream eif α phosphorylation by acting as a pseudosubstrate (pavio et al., ) . interestingly, it is reported that ns a stimulates eif α phosphorylation in the absence of pkr, implying that ns a may activate other eif- α kinases to regulate eif α phosphorylation (tardif et al., ) . overexpression of hcv ns induces eif α phosphorylation (von dem bussche et al., ) . taken together, these studies indicate that hcv proteins modulate eif α pathway in a complex way, and the effect of regulation on virus replication cannot be established unequivocally. the n-terminal region of ns a of jev contains a sequence that is highly similar to hcv ns a isdr and also inhibits pkr-induced eif α phosphorylation (tu et al., ) . denv infection triggers and then suppresses perk-mediated eif α phosphorylation by elevating the expression of growth arrest and dna damage-inducible protein- (gadd ), which acts together with phosphatase (pp ) to dephosphorylate eif α-p (pena and harris, ) . influenza virus nonstructural protein ns interferes with dsrna binding to pkr, and the infection also induces and activates p ipk, a cellular inhibitor of pkr and perk. both strategies deployed by ns and p ipk prevent pkr dimerization and autophosphorylation, which limits eif α phosphorylation (lee et al., ; lu et al., ; yan et al., ) . in some circumstances, such as when the host immune system specifically recognizes foreign viruses and kills them with cytotoxic t lymphocytes, or when cell death is directly induced in virus infected cells to prevent completion of the replication cycle, apoptotic cell death is considered to be a host strategy for fighting against viral infections. atf is a transcriptional activator of the isr, which is involved in the expression of isr target genes such as c/ebp homologous protein (chop) and gadd (ma and hendershot, ) . chop was originally identified as a transcriptional factor eliciting er stress-induced apoptosis. in cells subjected to west nile virus (wnv) infection, eif α phosphorylation and chop-mediated apoptosis were induced. both viral protein expression level and virus titer are increased in chop-deficient cells (medigeshi et al., ) . on the other hand, a virus may induce apoptosis to facilitate replication or the spread of viral progeny. it is reported that coronavirus infectious bronchitis virus (ibv) upregulates eif α-atf -chop signaling in infected cells and that it relies on perk or pkr activation. knockdown of chop reduces ibv-induced apoptosis through activation of the extracellular signal-related kinase (erk). viral protein expression level is moderately suppressed in chop-knockdown cells, which suggests that upregulation of chop-mediated apoptosis during ibv infection probably promotes virus replication (liao et al., ) . in addition to regulation of cell death, it is reported that hcv induces the expression of chop at mrna and protein levels and is correlated with autophagy induction; knockdown of chop not only increases hcv pamp-mediated innate immune activation, but also elevates its inhibitory effect on virus replication (ke and chen, ) . however, upstream chop induction is a matter of debate. overexpression of hcv e and/or e induces the expression of chop in a perk-dependent manner (chan and egan, ) ; while upon hcv infection, chop protein is upregulated by perk, activating transcription factor (atf ), and inositol-requiring transmembrane kinase/endonuclease (ire ) collectively. atf is a type transmembrane protein of amino acids and is constitutively expressed as a -kda protein (p atf ). its c-terminal region is located in the er, whereas the n-terminal region is located on the cytosolic side (figure ) . upon er stress, atf is cleaved to an n-terminal -kda protein (p atf ) sequentially by the golgi site- and site- proteases (s p and s p) figure | atf pathway under viral infection. many rna viruses activate the upr pathway by cleaving atf to release the p fragment. the n-terminal p with transcription activity enters the nucleus to activate the expression of er stress and erad genes, such as grp /bip, chop, xbp , or edem. however, the p fragment was not detected in the ev infection. august | volume | article | (ye et al., ) . nuclear translocation of p atf , as a transcription factor, activates expression of er stress and erad genes including er chaperones, chop (aka gadd ), edem , and x-box-binding protein (xbp ) by targeting the cis-acting er stress response element (erse) (ccaat-n -ccacg) and upr element (upre) (gatgacgtg(t/g) nnn(a/t)t), although atf has a much higher affinity for erse (yoshida et al., ) . in addition to directly regulating gene expression, atf also modulates the innate immune response. under subtilase cytotoxin (subab) treatment, cleavage and degradation of grp /bip leads to activation of the akt-nf-κb pathway through atf activation (yamazaki et al., ) . based on its pivotal role of connecting the arms of the upr and converging the upr and immune response, many viruses preferentially regulate atf pathways to benefit replication. in wnv strain kunjin (wnv kun )-infected cells, expression of atf -target genes increases, but viral production decreases in atf knockout mef cells. moreover, in atf knockout mef cells, phosphorylation of eif α, downstream chop activity, and jak-stat phosphorylation induced by ifnα are upregulated upon infection, which implies that virusinduced atf activation is a prosurvival mechanism required for replication and inhibition of the antiviral signaling pathway (ambrose and mackenzie, ) . however, it is still unclear whether wnv kun ns a and ns b, potent inducers of the upr, inhibit ifnα-induced jak-stat signaling in an atf -dependent manner (ambrose and mackenzie, ) . other flavivirus infections, including hcv, jev, and denv , also induce cleavage of atf , nuclear translocalization of atf and increases in chaperone proteins expression. in hcv replication, silencing of atf reduces hcv intracellular mrna levels (ke and chen, ) . however, in jev-infected cells, knockdown of the atf -targeted gene, grp , by sirna did not affect jev viral rna replication, although it did impair virus assembly or release. in sucrose gradient, mature jev viruses that do not cofractionate with gpr displayed a significant decrease in viral infectivity, indicating that jev acts with gpr to promote its infectivity (wu et al., ) . notably, denv triggers atf signaling in a celltype-specific manner. in a cells, nuclear-localized atf was observed (umareddy et al., ) ; however, no activating events can be detected in human fibrosarcoma ftgh cells, therefore, gpr upregulation may be mediated in an atf -independent fashion (pena and harris, ) . this cell-type-specific regulation of atf , also observed in iav infection, p atf , and its target gene erp /grp expression (roberson et al., ) , has been shown to increase in murine primary tracheal epithelial cells infected with influenza a/pr/ / , which is known to be involved in influenza virus ha protein folding (solda et al., ) . knockdown of erp abrogates viral progeny production. however, atf activity is not induced in infected human tracheobronchial epithelial (htbe) cells (hassan et al., ) . although atf -mediated transcriptional activation is an ongoing research field, another role for atf in virus infection has emerged. we have previously demonstrated that ev infection results in the decline of p atf , while the grp promoter containing classical erse sites responsive to p atf in ev infected cells was not activated (jheng et al., ) . indeed, two potential c cleavage sites (glutamine-glycine; qg) located at adjacent amino acids - and - near the c terminus of p atf were computationally predicted. it would be interesting to investigate the role of viral c in the regulation of atf , for its possible contribution in manipulating virus infection. ire is an er-localized type i transmembrane protein containing an er luminal dimerization domain and cytosolic kinase and rnase domains (mori et al., ; sidrauski and walter, ) . during er stress, accumulation of unfolded proteins in the er stimulates ire oligomerization and autophosphorylation (figure ) . its endoribonuclease activity initiates an unconventional splicing of the xbp mrna, excising a -nt sequence and shifts the reading frame to produce a functional isoform xbp (s), which contains a c-terminal transactivation domain absent from the unspliced form, xbp (u). xbp (s) then translocates to the nucleus where it induces expression of target genes containing upre or erse. these target genes are involved in erad, chaperone protein production, and er membrane biosynthesis (shamu and walter, ; friedlander et al., ) . studies of the ire signaling pathway demonstrate its significant role in virus infection (figure ) . hcv glycoprotein e is an example of a virus-derived erad substrate. hcv infection activates the ire -xbp -edem pathway, where edem and edem , but not edem , interact with hcv e to accelerate its degradation. either knockdown of edems or treating cells with kifunensine (kif), a potent inhibitor of er mannosidase, interferes with the binding of edems with sel l, a component of erad complex, stabilizes e expression, and enhances virus replication and viral particle production. however, there is no interaction between edem proteins and the jev envelope protein and abolishing the erad pathway by kif does not affect jev production (saeed et al., ) . the results emphasize the pivotal role of the erad pathway in the life cycle of specific viruses. interestingly, upre reporter activity or erad of misfolded null hong kong α-antitrypsin is reduced in cells carrying hcv replicons, which lack structural proteins, even though upstream xbp splicing occurs (tardif et al., ) . this implies that hcv structural proteins play a key role in xbp -mediated upre activation, and this is supported by a related study demonstrating that hcv e and/or e activates the xbp -erad pathway (chan and egan, ) . furthermore, the ire signaling pathway also participates in viral protein retrotranslocation. hepatitis e virus (hev) orf is an n-linked glycoprotein which is cotranslationally translocated into the er while a significant fraction of it is also observed in the cytoplasm. based on the results of tunicamycin and kif treatment, it is believed that glycosylation and erad are essential for orf retrotranslocation from the er to the cytoplasm (surjit et al., ) . however, no ubiquitination of orf can be observed, and retrotranslocated orf protein was stable in the cytoplasm when the cells were treated with proteasome inhibitor mg , which suggests that erad is required for orf access to the cytoplasm. microarray analysis reveals that orf overexpression causes upregulation of hsp b, hsp , and hsp . hsp is an antiapoptotic heat shock protein that directly interacts with orf (john et al., ) . it is reported that expression of hsp enhances xbp mrna splicing and protects cells from xbp mrna splicing, studies demonstrated that ire activates ridd to promote the degradation of mrnas encoding er-targeted proteins to reduce the load of er client proteins during er stress. the mammalian ire -traf -jnk pathway, independent of xbp splicing, may lead to the activation of apoptosis after prolonged er stress. hcv and its structural proteins e and e play an important role in the activation of the ire -xbp -erad pathway. overexpression of orf of hev can upregulate antiapoptotic protein hsp to activate xbp splicing. however, further study is required to determine whether hev infection can activate xbp via hsp . denv infection activates chop and gadd expression downstream of ire -xbp signaling. however, apoptosis activation by jnk, but not chop, is essential for denv infection. er stress-induced apoptosis by association with ire (gupta et al., ) . thus, further investigation is needed to examine the correlations of orf , ire , and hsp in hev replication. under harsh er stress, the activation of ire -xbp can also lead to the induction and expression of chop. denv infection induces chop, and gadd expression is a downstream event of ire -xbp signaling. of note is that induction of chop does not lead to apoptosis markers such as decreased expression of bcl- or proteolytic cleavage of pro-caspase- , pro-caspase- , or parp, which indicates a role beyond guiding cell death in infected cells (pena and harris, ) . indeed, it has been reported that chop exhibits protective effects against radiation-induced apoptosis or has a role in autophagy induction (mayerhofer and kodym, ; ke and chen, ) . another er stress-induced cell death that relies on the ire -traf pathway is implicated in jnk activation (see figure ) . the role of this pathway is emphasized by denv infection; silencing of ire decreases the virus titer, but the viral progeny output is not affected by silencing of xbp (pena and harris, ) . however, jnk pathway inhibitors diminished virus yield significantly, which suggests that activation of jnk is essential for denv infection (ceballos-olvera et al., ) . our previous findings also demonstrated that ev phosphorylates ire , but inhibits the expression of xbp . the overexpression of xbp in cells appeared to inhibit viral entry, and therefore reduce viral rna and viral particle formation (jheng et al., ) . as previous studies have reported that picornavirus infections induce jnk activation (kim et al., ; peng et al., ) , further detailed studies of the ire -jnk activation in ev infection would extend our understanding of the contributions of ire -jnk in the virus life cycle. ire has also been linked to the mediation of the selective degradation of a subset of er-localized mrnas in a process known as regulated ire -dependent degradation (ridd) (hollien and weissman, ) . mutation or removal of the signal sequences in targeted mrnas prevents their decay (kimmig et al., ) . however, it has been observed that drosophila mrna smt , a homolog of a small ubiquitin-like modifier (aka sumo), lacks any er-targeting sequence, and is a noncanonical ridd target, which implies that unknown specific features other than er localization are involved in defining the ridd substrates (moore et al., ) . ridd has been suggested to play adaptive roles by reducing protein translocation load, such as decrease of proinsulin expression in pancreatic beta-cells faced with chronic high glucose, and protecting liver cells from acetaminopheninduced hepatotoxicity (lipson et al., ; hur et al., ) . alternatively, ridd has also been suggested to play destructive roles under unmitigated er stress because continued degradation of mrnas encoding secretory cargo proteins and proteins involved in er-resident protein folding occurs. in addition to ire -xbp activation, jev also induces activation of the ridd cleavage pathway (bhattacharyya et al., ) . the addition of stf , a specific inhibitor of ire rnase activity, to infected cells decreases the tg-induced xbp splicing and potential ridd target transcripts. it also decreases viral protein expression as well as mature progeny formation, but does not affect viral rna synthesis, which indicates that jev viral rna is not a substrate of ridd, and ridd activation is beneficial for viral infectivity. it is not clear whether other viral infections trigger ridd. to extrapolate from the study of hcv, hcv replicons activate the phosphorylation of ire but impede xbp activation (tardif et al., ) . depletion of ire attenuates replicon translation, which implies that ridd may enhance viral protein synthesis. thus, the study of hcv replicon may have potential for deciphering the role of ridd in hcv infection because it could uncouple xbp signaling from ire activation. autophagy is a vesicular process that results in the degradation of the sequestered component, which can then be recycled by the cell. in mammalian cells, a complete autophagy includes the following four steps. ( ) induction. induction is initiated by activation of the unc- -like kinase (ulk ) complex. the ulk complex contains ulk , focal adhesion kinase (fak)family-interacting protein of kd (fip ), atg and atg (mizushima, ) . ulk complex activity would be, at least, modulated by mtorc , akt, and ampk (inoki et al., ; bach et al., ; egan et al., ; kim et al., ) . mtorc is a serine/threonine kinase complex, which phosphorylates ulk and atg and also inhibits autophagy. akt and amp-activated protein kinase (ampk) phosphorylate tsc at different residues, which results in the gtp hydrolysis of rheb and indirectly antagonizes the mtorc signaling pathway. recently, the combination of bioinformatic and proteomic approaches has identified ulk as a direct target of ampk and as involved in autophagy induction. ( ) vesicle nucleation. the beclin -pi kc complex, generating pi p, is essential for recruitment of pi p effectors including dfcp , wipis upstream of atg proteins and lipids recruitment to the pas, which is required for autophagosome construction (proikas-cezanne et al., ; axe et al., ) . importantly, the activity of the beclin -pi kc complex depends on its subunit composition. complexes containing atg -like protein (atg l or barkor) or ultraviolet irradiation resistanceassociated gene (uvrag) activate autophagy (itakura et al., ) ; nevertheless, the run domain and cysteine-rich domain containing beclin -interacting protein (rubicon) act as negative regulators of autophagy (matsunaga et al., ) . ( ) vesicle expansion and completion. the cytosolic form of lc (lc -i) is cleaved by the cysteine protease atg , followed by conjugation with phosphatidylethanolamine (pe) assisted by the atg -atg -atg l complex, which functions as an e -like enzyme. lc -pe leads to pas expansion, and cytosolic cargos are then enclosed into double membrane vesicles called autophagosomes (geng and klionsky, ) . ( ) autophagosome maturation. an autophagosome matures into an autolysosome by sequential fusion with endosomes and with lysosomes, the contents of which are degraded by hydrolases therein. it is reported that autolysosome formation is related to uvrag and expression of lysosomal-associated membrane protein (lamp- ) (liang et al., ; fortunato et al., ). previous studies suggest that autophagy may be an important antiviral defense mechanism (talloczy et al., ; orvedahl et al., ) ; however, the role of autophagy in virus infection is complicated and may have opposite consequences for the viral pathogenesis. many viruses manipulate autophagy for their own benefit by the following mechanisms. the exploitation of autophagy has been identified in many rna viruses including pv, cvb , jev, and hcv wong et al., ; tanida et al., ; ke and chen, ; li et al., ) . increased amounts of autophagosomes, as well as colocalization of the autophagy marker protein lc and viral protein, were observed in virus-infected cells. in addition, cells treated with an autophagy inhibitor, or transfected with sirna specifically obstructed autophagic processes, which reduced virus replication or virus titer. for example, in pv infection, virus yield was correlated with the induction of autophagy. treating cells with sirna targeting lc or atg to block autophagy leads to reduced virus yield . in addition, based on the topology of a double membrane compartment, digestion of the inner membrane under the autolysosome formation would allow efficient fusion of the autophagosomal membrane with the cytoplasm membrane. thus, an emerging concept is that autophagy may also involve the nonlytic release of cytoplasm under autophagosome maturation, namely autophagic exit without lysis (awol), which may participate in the release of pv (kirkegaard and jackson, ; taylor et al., ) . virus-induced uncompleted autophagy was reported for cvb -, rotavirus-, and iva-infected cells (gannage et al., ; kemball et al., ; alirezaei et al., ; crawford et al., ) . in cvb infected pancreatic acinar cells, an increase in the number of double-membraned autophagy-like vesicles was observed upon infection. however, the accumulation of autophagy substrate p and the formation of large autophagy-related structures named megaphagosomes indicate that cvb blocks a later stage of the autophagic pathway (kemball et al., ) . further results highlight the impact of autophagy on cvb rna replication and translation (alirezaei et al., ) . it was reported that rotavirus nsp viroporin initiates autophagy to transport viral proteins to sites of virus replication for assembly of mature particles, which involves an increase of cytoplasmic calcium and subsequent activation of the camkk-β-ampk pathway. rotavirus also interferes with autophagy maturation; however, the mechanism is still unknown (crawford et al., ) . accumulated studies reveal that m , ha, and ns proteins of iav are involved in the induction of autophagy, while only m has been identified as playing a critical role in impeding fusion of autophagosomes with lysosomes (gannage et al., ; sun et al., ; zhirnov and klenk, ) . autophagy-mediated immune responses that benefit virus replication have been reported in vsv, hcv, denv, and jev (jounai et al., ; ke and chen, ; jin et al., ) . in vsv infection, the atg -atg conjugate targets rig-i/mda -mavsdependent type i ifn production by directly interacting with the mavs and rig-i, and negatively regulates mavs-mediated nf-κb and type i ifn promoters, and permits vsv replication. furthermore, through an unknown mechanism, hcv-or denv-induced complete autophagy negatively regulates type i ifn production and promotes hcv replication (ke and chen, ) . recently, research about jev has shown that in autophagyimpaired cells, virus infection induces aggregates of mavs and activation of ifn regulatory factor (irf ), markers for activation of innate immune responses, which suggests that autophagymediated immune responses are required for viral replication (jin et al., ) . as er proliferation, which paradoxically commits the cell to cell death or survival, is observed both in upr and autophagy, it is reasonable to propose a possible link between upr pathways and the autophagic response. indeed, many upr-related transcription factors manage atg expression ( table ) . as demonstrated previously, yeasts with mutations in the gcn -signaling pathway are defective in starvation-induced autophagy. gcn , which undertakes gcn -dependent transcriptional activation, is essential for autophagy induction (talloczy et al., ) . recently, august | volume | article | gomez et al., ; margariti et al., results of multiple genetic models showed that the perk-eif α-atf pathway affects cmyc-dependent tumorigenesis by evoking cytoprotective autophagy; while pharmacologic or genetic inhibition of autophagy resulted in enhanced myc-dependent apoptosis (hart et al., ) . thus, upr inhibition could provide new targets for the treatment of malignancies, characterized by cmyc overexpression. in addition, ire also mediates autophagy in huntington's disease under er stress. clearance of mutant huntingtin aggregates through autophagic flux was impaired via ire -traf signaling, which results in neuronal cytotoxicity (lee et al., ) . although studies on upr autophagy mainly focus on the regulation of eif α kinase and ire , transcriptional regulation of autophagic genes by atf and srebp , a membrane-bound transcription factor activated through proteolytic processing upon er stress, was noticed recently (ogata et al., ; seo et al., ; gade et al., ) . death-associated protein kinase (dapk ), a positive mediator of ifn-regulated growth suppressor, is principally regulated by transcription factor c/ebp-β, one of the genes that increases expression during er stress (chen et al., ) . dapk promotes autophagy by phosphorylating beclin , and therefore dissociating it from autophagy negative regulator bcl . an investigation found that activated atf could directly interact with c/ebp-β carrying an erk / target site; this heterodimer then coacts to activate the dapk promoter, which in turn induces autophagy. additionally, xbp , a downstream target of atf , is essential for c/ebp-β expression (chen et al., ) . the role of srebp in autophagy was disclosed through gene ontology analysis (seo et al., ) . further study shows that srebp- activates autophagy gene expression, such as lc b, atg b, and atg d, accompanied by increased lc puncta formation, while srebp- deficiency obtains an opposite result. in virus infection, hcv is a well-documented model illustrating upr autophagy regulation. induction of upr and incomplete autophagy was observed in cells transfected with hcv jfh rna. cells treated with sirna targeting perk, ire , and atf showed a suppression of lc conversion and a decrease of hcv rna replication (sir et al., ) . in the hcv infection system, hcv induces complete autophagy and -(s)- -amino- phenylpropanoylsilybin impairs autophagy dai et al., chop plays a leading role in upr autophagy signaling (ke and chen, ) . further efforts to decipher how hcv activates autophagy revealed that perk-eif α-atf and atf pathways activated chop expression in hcv core protein-transfected cells where the core protein had not been demonstrated to induce er stress previously. moreover, hcv core protein may promote atg and lc protein expression through transcriptional control by atf and chop, respectively (wang et al., ) . recent studies suggest that completed autophagy induced by chikv infection is mediated by the independent induction of the endoplasmic reticulum and oxidative stress pathways. knockdown of ire or treated cells with the ros inhibitor nacetyl-l-cysteine inhibits formation of autophagosomes as well as the conversion of lc -i to lc -ii. moreover, an additive inhibitory effect on autophagosome formation was observed in infected cells silenced for ire mrna and treated with n-acetyl-l-cysteine (joubert et al., ) . because upr and autophagy play a role in viral pathogenesis, the regulation of upr and autophagy may be an important strategy for the future development of new therapeutic approaches to combat viruses. for example, we have demonstrated that overexpression of grp to relieve er stress decreases ev replication (jheng et al., ) . thus, agents such as grp /bip inducer x (bix) (kudo et al., ) or chemical chaperone, tauroursodeoxycholic acid (tudca) (ozcan et al., ) , will be potentially useful in the treatment of ev ( table ) . there are other established strategies to inhibit viruses by modulating upr target eif α phosphorylation or ire , e.g., salubrinal is a small molecule that prevents dephosphorylation of eif α and , -dibromosalicylaldehyde, an ire inhibitor, may cause restriction of iva (boyce et al., ; volkmann et al., ) . there is emerging evidence that pharmacological agents that directly activate or deactivate autophagy influence virus replication. evodiamine and -(s)- -amino- -phenylpropanoyl-silybin have been identified as anti-iva agents aimed at multiple processes of autophagy (dai et al., (dai et al., , . additionally, chloroquine-suppressed hcv replication has been proved (mizui et al., ) . because upr and autophagy are closely related, combination treatment may show a synergistic effect of their application, which was demonstrated in cancer research. the combination of nelfinavir (which induces upr autophagy) and chloroquine enhances cytotoxicity against cancer cells (mahoney et al., b) ; therefore, the use of combination treatment with improved efficacy and decreased toxicity represents a promising strategy to fight viruses. although upr autophagy has been discussed in many research areas, its integrated response to virus infection is only now beginning to emerge. it needs to be experimentally proven whether virus-induced autophagy is associated with upr. furthermore, given what we know about the various means that viruses use to modulate upr or autophagy to advantage their own virulence, the development of specific inducers or inhibitors for these molecules is one of the major challenges in this field. pancreatic acinar cell-specific autophagy disruption reduces coxsackievirus replication and pathogenesis in vivo west nile virus differentially modulates the unfolded protein response to facilitate replication and immune evasion atf signaling is required for efficient west nile virus replication by promoting cell survival and inhibition of innate immune responses autophagosome formation from membrane compartments enriched in phosphatidylinositol -phosphate and dynamically connected to the endoplasmic reticulum the serine/threonine kinase ulk is a target of multiple phosphorylation events infectious hepatitis c virus pseudo-particles containing functional e -e envelope protein complexes the eif alpha/atf pathway is essential for stress-induced autophagy gene expression viral internal ribosome entry site structures segregate into two distinct morphologies receptor proteins in selective autophagy autophagy counterbalances endoplasmic reticulum expansion during the unfolded protein response regulated ire -dependent decay pathway is activated during japanese encephalitis virus-induced unfolded protein response and benefits viral replication a selective inhibitor of eif alpha dephosphorylation protects cells from er stress jnk phosphorylation, induced during dengue virus infection, is important for viral infection and requires the presence of cholesterol hepatitis c virus envelope proteins regulate chop via induction of the unfolded protein response human ccaat/enhancer-binding protein beta gene expression is activated by endoplasmic reticulum stress through an unfolded protein response element downstream of the protein coding sequence regulation of protein synthesis by the heme-regulated eif alpha kinase: relevance to anemias inhibition of host and viral translation during vesicular stomatitis virus infection. eif is responsible for the inhibition of viral but not host translation autophagy hijacked through viroporin-activated calcium/calmodulin-dependent kinase kinase-beta signaling is required for rotavirus replication a drug screening method based on the autophagy pathway and studies of the mechanism of evodiamine against influenza a virus identification of -(s)- -amino- -phenylpropanoyl-silybin as an antiviral agent for influenza a virus infection in vitro and in vivo cleavage of eukaryotic initiation factor eif b by enterovirus c proteases functional analysis of picornavirus b proteins: effects on calcium homeostasis and intracellular protein trafficking the rotavirus enterotoxin nsp mobilizes intracellular calcium in human intestinal cells by stimulating phospholipase c-mediated inositol , , -trisphosphate production formation and intracellular localization of hepatitis c virus envelope glycoprotein complexes expressed by recombinant vaccinia and sindbis viruses hepatitis c virus glycoprotein folding: disulfide bond formation and association with calnexin the autophagy initiating kinase ulk is regulated via opposing phosphorylation by ampk and mtor replication of hepatitis c virus rna occurs in a membrane-bound replication complex containing nonstructural viral proteins and rna maturation of autophagic vacuoles in mammalian cells impaired autolysosome formation correlates with lamp- depletion: role of apoptosis, autophagy, and necrosis in pancreatitis a regulatory link between er-associated protein degradation and the unfoldedprotein response two endoplasmic reticulum-associated degradation (erad) systems for the novel variant of the mutant dysferlin: ubiquitin/proteasome erad(i) and autophagy/lysosome erad(ii) an ifn-gamma-stimulated atf -c/ebp-beta-signaling pathway critical for the expression of death associated protein kinase and induction of autophagy critical role for transcription factor c/ebp-beta in regulating the expression of death-associated protein kinase control of pkr protein kinase by hepatitis c virus nonstructural a protein: molecular mechanisms of kinase regulation matrix protein of influenza a virus blocks autophagosome fusion with lysosomes sequence differences between glycosylated and non-glycosylated asn-x-thr/ser acceptor sites: implications for protein engineering the atg and atg ubiquitin-like conjugation systems in macroautophagy phosphorylation of initiation factor- alpha is required for activation of internal translation initiation during cell differentiation human x-box binding protein- confers both estrogen independence and antiestrogen resistance in breast cancer cell lines transactivation of atg b by c/ebpbeta promotes autophagy to facilitate adipogenesis hsp protects cells from er stress-induced apoptosis via enhancement of ire alpha-xbp signaling through a physical interaction er stress response: getting the upr hand on misfolded proteins n-linked glycosylation of west nile virus envelope proteins influences particle assembly and infectivity regulated translation initiation controls stress-induced gene expression in mammalian cells endoplasmic reticulum stress and the development of diabetes: a review er stress-mediated autophagy promotes myc-dependent transformation and tumor growth influenza a viral replication is blocked by inhibition of the inositol-requiring enzyme (ire ) stress pathway components of ubiquitin-protein ligase system. resolution, affinity purification, and role in protein breakdown decay of endoplasmic reticulum-localized mrnas during the unfolded protein response ire alpha activation protects mice against acetaminopheninduced hepatotoxicity tsc mediates cellular energy response to control cell growth and survival beclin forms two distinct phosphatidylinositol -kinase complexes with mammalian atg and uvrag subversion of cellular autophagosomal machinery by rna viruses inhibition of rnase l and rna-dependent protein kinase (pkr) by sunitinib impairs antiviral innate immunity endoplasmic reticulum stress is induced and modulated by enterovirus inhibition of enterovirus entry by transcription factor xbp japanese encephalitis virus activates autophagy as a viral immune evasion strategy hepatitis e virus orf protein activates the pro-apoptotic gene chop and anti-apoptotic heat shock proteins chikungunya virus-induced autophagy delays caspasedependent cell death the atg atg conjugate associates with innate antiviral immune responses stress signaling from the lumen of the endoplasmic reticulum: coordination of gene transcriptional and translational controls activation of the unfolded protein response and autophagy after hepatitis c virus infection suppresses innate antiviral immunity in vitro coxsackievirus infection induces autophagy-like vesicles and megaphagosomes in pancreatic acinar cells in vivo ampk and mtor regulate autophagy through direct phosphorylation of ulk coxsackievirus b infection induces cyr activation via jnk to mediate cell death the unfolded protein response in fission yeast modulates stability of select mrnas to maintain protein homeostasis topology of double-membraned vesicles and the opportunity for non-lytic release of cytoplasm the unfolded protein response signals through high-order assembly of ire assembly of asparagine-linked oligosaccharides mechanisms of cross-talk between the ubiquitin-proteasome and autophagy-lysosome systems er stress, hypoxia tolerance and tumor progression a molecular chaperone inducer protects neurons from er stress ire plays an essential role in er stress-mediated aggregation of mutant huntingtin via the inhibition of autophagy flux characterization and regulation of the , -dalton cellular inhibitor of the interferon-induced, dsrna-activated protein kinase rock is involved in vimentin phosphorylation and rearrangement induced by dengue virus development by self-digestion: molecular mechanisms and biological functions of autophagy autophagy is involved in the early step of japanese encephalitis virus infection beclin -binding uvrag targets the class c vps complex to coordinate autophagosome maturation and endocytic trafficking upregulation of chop/gadd during coronavirus infectious bronchitis virus infection modulates apoptosis by restricting activation of the extracellular signal-regulated kinase pathway the role of ire alpha in the degradation of insulin mrna in pancreatic beta-cells translational control by viral proteinases binding of the influenza virus ns protein to double-stranded rna inhibits the activation of the protein kinase that phosphorylates the elf- translation initiation factor topology of the membrane-associated hepatitis c virus protein ns b temporal orchestration of circadian autophagy rhythm by c/ebpbeta delineation of a negative feedback regulatory loop that controls protein translation during endoplasmic reticulum stress plasma cell differentiation initiates a limited er stress response by specifically suppressing the perk-dependent branch of the unfolded protein response autophagy and er stress play an essential role in the mechanism of action and drug resistance of the cyclin-dependent kinase inhibitor flavopiridol identification of endoplasmic reticulum stress-inducing agents by antagonizing autophagy: a new potential strategy for identification of anticancer therapeutics in b-cell malignancies xbp mrna splicing triggers an autophagic response in endothelial cells through beclin- transcriptional activation two beclin -binding proteins, atg l and rubicon, reciprocally regulate autophagy at different stages gadd restores resistance to radiationinduced apoptosis after thiol depletion west nile virus infection activates the unfolded protein response, leading to chop induction and apoptosis molecular cloning and characterization of the human doublestranded rna-activated protein kinase induced by interferon the role of atf stabilization and autophagy in resistance of breast cancer cells treated with bortezomib modification of intracellular membrane structures for virus replication inhibition of hepatitis c virus replication by chloroquine targeting virus-associated autophagy the role of the atg /ulk complex in autophagy regulation regulation of sumo mrna during endoplasmic reticulum stress a transmembrane protein with a cdc +/cdc -related kinase activity is required for signaling from the er to the nucleus viroporins: structure and biological functions autophagy is activated for cell survival after endoplasmic reticulum stress autophagy protects against sindbis virus infection of the central nervous system chemical chaperones reduce er stress and restore glucose homeostasis in a mouse model of type diabetes identification of an ire alpha endonuclease specific inhibitor with cytotoxic activity against human multiple myeloma protein synthesis and endoplasmic reticulum stress can be modulated by the hepatitis c virus envelope protein e through the eukaryotic initiation factor alpha kinase perk dengue virus modulates the unfolded protein response in a time-dependent manner activation of jnk / and p mapk signaling pathways promotes enterovirus infection in immature dendritic cells transcriptional up-regulation of ulk by atf contributes to cancer cell survival wipi- alpha (wipi ), a member of the novel -bladed wipi protein family, is aberrantly expressed in human cancer and is linked to starvationinduced autophagy differential unfolded protein response during chikungunya and sindbis virus infection: chikv nsp suppresses eif alpha phosphorylation aggregate-prone proteins with polyglutamine and polyalanine expansions are degraded by autophagy translation without eif promoted by poliovirus a protease influenza induces endoplasmic reticulum stress, caspase- -dependent apoptosis, and c-jun n-terminal kinase-mediated transforming growth factor-beta release in lung epithelial cells translational control in the endoplasmic reticulum stress response the unfolded protein response protects human tumor cells during hypoxia through regulation of the autophagy genes map lc b and atg role of the endoplasmic reticulum-associated degradation (erad) pathway in degradation of hepatitis c virus envelope proteins and production of virus particles cannabinoid action induces autophagy-mediated cell death through stimulation of er stress in human glioma cells viral internal ribosome entry site elements: novel ribosome-rna complexes and roles in viral pathogenesis cellular origin and ultrastructure of membranes induced during poliovirus infection genome-wide localization of srebp- in hepatic chromatin predicts a role in autophagy xbp , downstream of blimp- , expands the secretory apparatus and other organelles, and increases protein synthesis in plasma cell differentiation oligomerization and phosphorylation of the ire p kinase during intracellular signaling from the endoplasmic reticulum to the nucleus targeting autophagy enhances sorafenib lethality for hepatocellular carcinoma via er stress-related apoptosis autophagy in health and disease: a doubleedged sword the transmembrane kinase ire p is a sitespecific endonuclease that initiates mrna splicing in the unfolded protein response autophagy regulates lipid metabolism induction of incomplete autophagic response by hepatitis c virus via the unfolded protein response consequences of erp deletion on oxidative folding of obligate and facultative clients of the calnexin cycle regulation of translation initiation in eukaryotes: mechanisms and biological targets inhibition of autophagy ameliorates acute lung injury caused by avian influenza a h n infection cytoplasmic localization of the orf protein of hepatitis e virus is dependent on its ability to undergo retrotranslocation from the endoplasmic reticulum mediators of endoplasmic reticulum stress-induced apoptosis regulation of starvation-and virus-induced autophagy by the eif alpha kinase signaling pathway pkr-dependent autophagic degradation of herpes simplex virus type reticulon binds the c protein of enterovirus and is required for viral replication knockdown of autophagy-related gene decreases the production of infectious hepatitis c virus particles hepatitis c virus suppresses the ire -xbp pathway of the unfolded protein response hepatitis c virus subgenomic replicons induce endoplasmic reticulum stress activating an intracellular signaling pathway folding and oligomerization of influenza hemagglutinin in the er and the intermediate compartment role of microtubules in extracellular release of poliovirus the rotavirus nonstructural glycoprotein nsp possesses membrane destabilization activity the rotavirus nonstructural glycoprotein nsp mobilizes ca + from the endoplasmic reticulum structural insights into the coupling of virion assembly and rotavirus replication blocking double-stranded rna-activated protein kinase pkr by japanese encephalitis virus nonstructural protein a dengue virus serotype infection specifies the activation of the unfolded protein response inhibition of the vacuolar h(+)-atpase with bafilomycin reduces delivery of internalized molecules from mature multivesicular endosomes to lysosomes in hep- cells coxsackievirus protein b modifies endoplasmic reticulum membrane and plasma membrane permeability and facilitates virus release potent and selective inhibitors of the inositol-requiring enzyme endoribonuclease hepatitis c virus ns protein triggers endoplasmic reticulum stress and suppresses its own viral replication hepatitis c virus core protein activates autophagy through eif ak and atf upr pathway-mediated map lc b and atg expression induction of autophagy in axonal dystrophy and degeneration autophagosome supports coxsackievirus b replication in host cells reticulon interacts with ns b of the hepatitis c virus and negatively regulates viral replication by disrupting ns b self-interaction japanese encephalitis virus co-opts the er-stress response protein grp for viral infectivity membrane topology and function of dengue virus ns a protein activation of the akt-nf-kappab pathway by subtilase cytotoxin through the atf branch of the unfolded protein response control of perk eif alpha kinase activity by the endoplasmic reticulum stress-induced molecular chaperone p ipk er stress induces cleavage of membrane-bound atf by the same proteases that process srebps induction of lysosomal dilatation, arrested autophagy, and cell death by chloroquine in cultured arpe- cells endoplasmic reticulum stress triggers autophagy identification of the cis-acting endoplasmic reticulum stress response element responsible for transcriptional induction of mammalian glucose-regulated proteins. involvement of basic leucine zipper transcription factors a time-dependent phase shift in the mammalian unfolded protein response n-glycosylation of the premembrane protein of japanese encephalitis virus is critical for folding of the envelope protein and assembly of virus-like particles influenza a virus proteins ns and hemagglutinin along with m are involved in stimulation of autophagy in infected cells conflict of interest statement: the authors declare that the research was con the authors are in debt to all previous and current laboratory members for their contributions and involvement in the study of ev -induced er stress. this work was supported by the national science council [nsc- - -b- - ] and chang gung memorial hospital [cmrpd b and cmrpd a ]. key: cord- -omszep u authors: pochon, cécile; voigt, sebastian title: respiratory virus infections in hematopoietic cell transplant recipients date: - - journal: front microbiol doi: . /fmicb. . sha: doc_id: cord_uid: omszep u highly immunocompromised pediatric and adult hematopoietic cell transplant (hct) recipients frequently experience respiratory infections caused by viruses that are less virulent in immunocompetent individuals. most of these infections, with the exception of rhinovirus as well as adenovirus and parainfluenza virus in tropical areas, are seasonal variable and occur before and after hct. infectious disease management includes sampling of respiratory specimens from nasopharyngeal washes or swabs as well as sputum and tracheal or tracheobronchial lavages. these are subjected to improved diagnostic tools including multiplex pcr assays that are routinely used allowing for expedient detection of all respiratory viruses. disease progression along with high mortality is frequently associated with respiratory syncytial virus, parainfluenza virus, influenza virus, and metapneumovirus infections. in this review, we discuss clinical findings and the appropriate use of diagnostic measures. additionally, we also discuss treatment options and suggest new drug formulations that might prove useful in treating respiratory viral infections. finally, we shed light on the role of the state of immune reconstitution and on the use of immunosuppressive drugs on the outcome of infection. infections and still contribute to significant mortality with rates ranging between and % if infection progresses to the lower respiratory tract (boeckh, ; englund et al., ; renaud and campbell, ; protheroe et al., ; chemaly et al., ; spahr et al., ) . human pathogenic viruses frequently causing respiratory infections in the allogeneic hct setting include rsv, adv, ifv, piv, hmpv, rhv, hcov, and hbov (renaud and englund, ; abbas et al., ) . such respiratory virus infections contribute to morbidity and mortality for a number of reasons. firstly, hct recipients suffer from prolonged immunosuppression and a lack of both humoral and t cell-mediated immunity impairs viral clearance (kohlmeier and woodland, ; schmidt and varga, ) . in addition, these patients are usually antibody-depleted and the strength of the conditioning regimen and the ongoing immunosuppressive therapy diminishes the antiviral immune response further. moreover, the type of graft may have an impact on the immune response and pediatric hct recipients may also be immunologically naïve because of their age. secondly, viral factors shape the outcome. respiratory viruses vary in their pathogenicity, i.e., they cause more severe disease compared with other viruses. also, there is the capacity for certain strains of a given respiratory virus to display increased virulence as is the case with ifv and it is therefore important to identify viral and host genes that determine virulence. thirdly, a limited number of effective antivirals or the emergence of drug resistance also hamper successful treatment. finally, factors like persisting infections, co-infections and co-morbidities will also influence the outcome. in contrast to immunocompetent individuals, hct recipients present with prolonged viral shedding and have a higher rate of progression from uri to lri (chemaly et al., ; de lima et al., ; kim et al., ) . increased viral loads and protracted infections raise the likelihood of progression that differs between viruses. uri has been commonly defined as laboratory-confirmed viral nasal wash or swab with pharyngitis, cough, otitis, nasal discharge and/or congestion in the absence of infiltrates on chest x-ray or computed tomography (ct) scan and hypoxemia, and lri has been defined as laboratory-confirmed viral nasal wash or swab in the presence of infiltrates on chest x-ray or ct scan suggestive of a viral respiratory infection but not necessarily detection of viral nucleic acid in samples obtained from bal . in hct recipients, rsv is most frequently detected, followed by hmpv and piv. in - % of cases, these viruses progress to lri, often complicate the transplant course by causing airflow obstruction or bronchiolitis obliterans and also increase the rate of mortality up to % (chemaly et al., ) . several respiratory viruses share a number of risk factors associated with lri occurrence. it is worth noting that cmv also represents an opportunistic pathogen that is causally related to pneumonia and lri. however, cmv will not be discussed in this review since it is not primarily considered a respiratory virus. that said, cmv is a pathogen that frequently occurs as part of a simultaneous infection -indeed a respiratory viral infection can often precede bacterial and fungal infections -thus further complications associated with respiratory virus infections need to be acknowledged. in this review, we will focus on respiratory virus infections and, particularly, those that are commonly detected after hct. we highlight risk factors that facilitate progression from uri to lri and discuss diagnostic approaches. current treatment strategies along with new treatment options will be discussed. adenovirus is a member of the adenoviridae that circulates throughout the year. currently, human types are known which are further divided into seven species a-g (accessed on august ). alongside conjunctivitis and diarrhea, adv can cause pharyngitis, bronchitis and pneumonia but also lethal hepatitis or severe bloody colitis. pertinent to this review, adv is a pathogen associated with severe complications in immunosuppressed pediatric hct recipients including increased mortality (leen et al., ; feuchtinger et al., ; lion, ; feucht et al., ; hiwarkar et al., ) . in adult patients, adv infections are less commonly reported. however, it is possible this perception might be biased by reduced frequency of screening in adults. a study in adult allogeneic hct recipients reported an infection rate of . %. pneumonia occurred in % of cases and was the most common cause of death associated with adv (yilmaz et al., ) . an important consideration is that adv infections infrequently present with respiratory symptoms at the onset of infection; instead they are commonly detected by monitoring stool (lo et al., ; lion, ) . indeed, gastrointestinal shedding pre-transplant has been demonstrated to reflect increased risk of viremia after hct (kosulin et al., a) . human bocavirus was identified in as a human pathogen that causes respiratory tract infections in infants. it has been assigned to the parvoviridae and received its name because of sequence homology to two other members in the genus bocaparvovirus, bovine parvovirus infecting cattle and canine minute virus infecting dogs (allander et al., ) . currently, four hbov variants have been described. like hbov , hbov can enter an episomal state, allowing the establishment of a persistent infection (kapoor et al., ) . whereas hbov , hbov , and hbov cause gastroenteritis and are genetically diverse, hbov exhibits a stronger association with lri than enteritis, often in conjunction with other respiratory pathogens, and has limited genetic diversity although reinfections with different variants occur (arden et al., ; ditt et al., ; arthur et al., ; kapoor et al., ; wang et al., ; martin et al., ) . symptoms following infection include cough, dyspnea, pharyngitis, bronchitis, pneumonia, and diarrhea. several reports have suggested that hbov can cause severe infections on its own and is not a mere bystander virus ursic et al., ; edner et al., ) . likewise, hbov can cause life-threatening infections and prolonged shedding in immunocompromised patients, and for immunocompetent children a median shedding time of days has been reported (kupfer et al., ; allander, ; koskenvuo et al., ; de vries et al., ; martin et al., ) . this long shedding period might lead to more severe disease in immunocompromised children (schenk et al., ) . importantly, hbov does not exhibit seasonality and thus remains a threat throughout the year. human coronavirus belongs to the coronaviridae that are endemic in humans. annually hcov are responsible for - % of uri with pharyngitis and rhinitis in immunocompetent hosts. historically, two common hcov were known: hcov- e and hcov-oc . however, the emergence of severe acute respiratory syndrome-coronavirus (sars-cov) along with two further hcov (hcov-hku and hcov-nl ) has expanded the family (van der hoek et al., ; woo et al., ) . in contrast to the low incidence of bronchitis or pneumonia in healthy children, severe clinical features have been described in immunocompromised patients. both the presence of a respiratory co-pathogen (rsv) and host factors like young age < years and an immunocompromised status were reported to contribute to lri. however, it should be noted that only children with hct were included (ogimi et al., b (ogimi et al., , a . in separate studies, hcov have been associated with increased mortality and prolonged shedding in the hct setting (milano et al., ; renaud and campbell, ) . risk factors for prolonged shedding (at least days) in the upper respiratory tract were determined in a cohort of patients and included high viral load, myeloablative conditioning, and prior high-dose steroid use (ogimi et al., a) . of patients, samples were analyzed shown to contain evidence of hcov-oc ( %), hcov-nl ( %), hcov-hku ( %), and hcov- e ( %) infection. analysis for duration of shedding showed that none of the strains appeared to cause longer shedding compared with others. in addition, genomic approaches investigated whether viral genome evolution could identify genetic changes associated with prolonged shedding. identification of such changes could aid the development of new antiviral agents. single nucleotide polymorphisms could not be identified until day thirty after the onset of viral shedding. this finding might not be surprising given the protracted evolution rate of hcov. however, overt viral genome changes might occur at a later time point, and changes in genome composition might result in a modification of the treatment strategy (ogimi et al., a) . in another study, hcov were examined in bal (ogimi et al., b) . the median time to hcov lri occurrence was days after hct. among bal samples analyzed, % were hcov-oc , hcov-nl was detected in %, hcov- e in % and hcov-hku in %. although somewhat limited because of sample size, these data show a similar frequency and order of detected viral strains compared to those detected from nasal samples with the exception of hcov-hku which was predominant in nasal samples (ogimi et al., a) . the detection of rsvs did not change clinical outcome. influenza virus, an orthomyxoviridae member, exhibits high genomic variability due to an absence of proofreading activity in its rna -a process that contributes to antigenic drift. as some ifv strains are more virulent than others, the strain of ifv impacts on severity, depending on circulating strains, and this can be impacted upon by the composition of the vaccine and also the uptake of vaccine in the population. individuals with a compromised immune system such as the elderly and hct recipients are at increased risk of complications and death, and outbreaks in hct units have been reported (lalayanni et al., ; suyani et al., ) . in hct recipients, fever maybe absent in about % of cases as might classical symptoms such as sore throat, nasal congestion and discharge, cough, chills and myalgia . as with other respiratory viruses, ifv shedding in hct patients has been reported to be prolonged with a median of days; lymphopenia correlated with the duration of shedding, and steroid use > mg/kg increased ifv secretion (khanna et al., ; boudreault et al., ; engelhard et al., ) . infants may present with sepsis-like symptoms and pneumonia. complications in adults include pneumonia, myocarditis, encephalitis and guillain-barré syndrome. as with piv infections (see below), bacterial and fungal co-infections with ifv are known (nichols et al., b; engelhard et al., ) . disease progression to lri takes place in up to % and mortality ranges between and % in patients diagnosed with pneumonia. therefore, early treatment within days of presenting with symptoms has been favored (whimbey et al., ; nichols et al., b; chemaly et al., ; kmeid et al., ) . while risk factors have been validated for rsv and piv, equivalent quality indicators are limited for ifv. potential risk factors that might result in lri are lymphopenia and neutropenia as well as increased age (> years) while steroid use is unclear (ljungman et al., ; chemaly et al., ; choi et al., ) . ifv genome detection in blood was associated with hypoxemia, respiratory failure, and overall mortality (choi et al., ) . kmeid et al. ( ) applied an isi that had been developed for rsv to identify patients with ifv infection who might run at risk to develop progression to lri . according to variables such as age, neutrophil and lymphocyte counts, conditioning regimen, steroid use and time from transplantation, hct recipients were grouped into low, moderate, and high risk immunodeficiency categories. a high risk score was applied if the score was between and and was likely to include anc < /mm (score ) and/or alc < /mm ( ), age ≥ years ( ), myeloablative regimen ( ), gvhd ( ), steroid use within the last days ( ) and recent (within the last days) or pre-engraftment allogeneic hct ( ). patients with a high risk score had a significantly higher probability of developing lri compared to the low risk group (kmeid et al., ) . human metapneumovirus, formerly a member of the paramyxoviridae now belonging to the pneumoviridae, was identified in as an agent capable of causing respiratory disease in children (van den hoogen et al., ) . in immunocompetent individuals, hmpv causes symptomatic uri and lri predominantly in children or adults above years of age (boivin et al., ) . no specific symptoms that distinguish hmpv from other viral respiratory infections exist. it has been reported that disease is more severe in case of a rsv co-infection but these data are considered controversial. in immunocompetent children, one study revealed that hmpv with rsv co-infection increased bronchiolitis (semple et al., ) , however, this could not be substantiated in other studies that investigated single infections and co-infections for hmpv and rsv (moe et al., ; yan et al., ) . for both hmpv and rsv, a and b types occur that can be distinguished by different primer sets in pcr (renaud et al., ) . immunocompromised children are at higher risk of developing lri with higher risk of requiring intensive care treatment with increased mortality (chu et al., ) . in a study of hct recipients, hpmv infection was documented in the majority of patients but did not cause any symptoms or disease (debiaggi et al., ) . however, in a study that included episodes of uri and lri, of the episodes in which hpmv was detected occurred in hct recipients, resulting in an infection rate of approximately % of all hct patients . another study revealed hpmv in bal samples from of patients who became symptomatic within a month after engraftment, and fatality reached up to % if bal was positive for hmpv (englund et al., ) . progression to lri has been reported to take place between and % (renaud and campbell, ) . for hmpv, risk factors for mortality include length between hct and infection; neutropenia, lymphopenia, low monocyte count at diagnosis, and a steroid dose before diagnosis of ≥ mg/kg (seo et al., ) . in the latter study, the presence of co-pathogens contributed to higher risk. glucocorticoid application and lymphopenia have been identified as risk factors that contribute to progression from uri to lri (seo et al., ) . in a systematic review, hmpv progressed to lri in % of cases and the mortality rate increased from to % when lri was present but no hmpv-associated risk factors for increased mortality could be identified (shah et al., b) . parainfluenza virus is a member of the paramyxoviridae that circulates throughout the year. piv causes lri in approximately % of healthy children (bicer et al., ) and is also responsible for life-threatening lri in hct recipients (srinivasan et al., ; shah et al., a) . four piv serotypes are known. piv serotype (piv- ) is the most frequent occurring serotype and has been responsible for infections after hct (cortez et al., ; nichols et al., a; maziarz et al., ; hodson et al., ) . in one study comprising patients with piv infection, . % had an infection with piv- , and % of these patients developed pneumonia (nichols et al., a) . another study reported piv infections in up to % of cases during the first months post hct . in - % of patients who experience uri, infection progresses to lri with a median time of days. virus-associated mortality is around % after piv infection but increases to % if infection progresses to lri (nichols et al., a; srinivasan et al., ; chemaly et al., ; ustun et al., ; seo et al., a; shah et al., a) . piv risk factors for progression to lri are lymphopenia, steroid use and co-infections with other respiratory agents, however, myeloablation and infections in the early post-transplant period were variables with unclear association (seo et al., b; shah et al., a) . pulmonary co-pathogens, e.g., aspergillus fumigatus, are often present in patients with pneumonia and highly contribute to mortality (nichols et al., a; ustun et al., ) . respiratory syncytial virus, now also belonging to the pneumoviridae, is a frequent pathogen in infants and children that causes uri with rhinitis and laryngitis but also bronchitis and pneumonia if progression to lri occurs. in hct recipients, rsv infections occur up to % khanna et al., ; avetisyan et al., ; shah and chemaly, ; waghmare et al., ) . in approximately half of the hct recipients with uri caused by rsv, infection will progress and result in pneumonia that is associated with average mortality rates around % without effective treatment (nichols et al., b; boeckh et al., ; khanna et al., ; shah and chemaly, ; seo et al., ; waghmare et al., ) . risk factors for progression to lri include advanced age, lymphopenia, myeloablative regimen, steroid use, gvhd and pre-engraftment infection and occur at a mean of % (nichols et al., b; martino et al., ; khanna et al., ; shah and chemaly, ; hirsch et al., ) . further, smoking history, conditioning with high-dose total body irradiation with - cgy and specifically an absolute lymphocyte count ≤ /mm at uri onset were significantly associated with progression to lri. in contrast, lung function, steroid use, lymphocyte engraftment dynamics, rsv subtypes and subtypespecific neutralizing antibody levels were not associated with progression, revealing transplant-related rather than viral factors which might put a focus on the characterization of rsv-specific t cells (kim et al., ) . in addition, absolute lymphocyte counts of > /mm at uri onset were protective and no advance to lri was observed. another study that examined risk factors for rsv rna detection in serum or plasma identified mechanical ventilation, neutropenia, monocytopenia as well as thrombocytopenia as associated factors, however, lymphopenia and steroid use did not increase the risk of rsv rna plasma detection (waghmare et al., ) . for that study, a median time of days to lri onset was described. as described for ifv, an isi had been originally proposed to stratify rsv-infected patients into groups based on their risk of progression from uri to lri and to evaluate rsv-related mortality . this stratification should help to identify patients who might profit from antiviral therapy such as aerosolized ribavirin. this index is based on age, absolute neutrophil and lymphocyte counts, acute or chronic gvhd, myeloablative conditioning, corticosteroid use and time of infection. however, validation of this index needs to be performed in multicenter studies. rhinovirus, a member of the picornaviridae, comprises more than serotypes and are divided into the three species a, b and c. there is no seasonal preference for rhv, however, more infections might occur during spring and autumn. rhv are the most frequently detected community-associated respiratory viruses that cause cough, a runny nose but also bronchitis or pneumonia in hct recipients (ison et al., ; milano et al., ) . symptomatic rhv infections occur in - % of hct recipients and initial high viral loads are associated with prolonged shedding (parody et al., ; milano et al., ; ogimi et al., b) . a recent study showed that median shedding time among patients with rhv infection was . days, and / ( %) of hct recipients had prolonged rhv shedding (at least days; range, - days) that was similar for all three species but the majority of patients ( %) shed rhv species a. risk factors for prolonged rhv shedding pointed to a high initial viral load as defined by a ct value below the median ( . ) (ogimi et al., b) . infection control measures should consider these long infection periods. progression to lri is increasingly being detected and fatal outcomes due to rhv infections have been described (gutman et al., ; jacobs et al., ; ogimi et al., b) . patients who reveal respiratory tract infection symptoms before hct should be tested for respiratory pathogens by multiplex pcr. campbell et al. analyzed data from adult and pediatric patients and identified % of patients to be positive for at least one respiratory virus pre-transplant. symptomatic patients had lower survival days after hct compared with patients with negative test results and increased overall mortality. since this risk was increased even if only rhv was detected, the authors proposed to consider deferral of hct in symptomatic patients with any respiratory virus positive, not just only in case of rsv, ifv, piv, or hmpv detection. asymptomatic patients with a virus detected did not exhibit higher mortality and hct might proceed as planned; however, it is unclear if these patients actually shed replicating virus or if only viral nucleic acid is being detected. there was no higher incidence of bronchoscopy in the symptomatic versus the asymptomatic group. any decision to delay transplant has to consider hct factors such as underlying disease, the conditioning regimen associated with it and donor logistics (campbell et al., ) . in a follow-up study that focused on pediatric hct recipients, kim et al. ( ) detected respiratory viruses pre-transplant in of patients. rhv were detected in % of cases, followed by a group of viruses consisting of adv, rsv, hmpv pfv, and piv ( %). pre-transplant detection was associated with increased hospitalization during the first days, and hcov was only detected pre-transplant in % of cases. since the conditioning regimen might have an impact on the incidence of respiratory virus infections pre-transplant, patients with myeloablative and non-myeloablative conditioning were compared. the incidences for respiratory virus infections were similar among both groups. however, in contrast to patients receiving non-myeloablative conditioning, lri are significantly increased during the first days post hct when myeloablative regimen was given (schiffer et al., ) . immediately after hct in the neutropenic phase, patients frequently develop febrile episodes and are especially prone to bacterial infections. therefore, they are often treated with antibiotics to protect them from life-threatening bacterial infections. this treatment alters microbiota composition and has an impact on immune responses (ichinohe et al., ; abt et al., ; dyer et al., ). an immunodulatory compound associated with microbiota is butyrate, a short chain fatty acid that is important in maintaining gut microbiome equilibrium as well as in controlling immune responses in organs such as the lung (donohoe et al., ; chang et al., ) . in a recent study, haak et al. ( ) investigated the influence of butyrate on microbiota composition and its influence on respiratory viral infections and the associated risk to develop lri in hct recipients. patients who received antibiotic treatment resulting in altered microbiota colonization and diminished butyrate-producing bacteria had a higher risk of developing lri indicating that the presence of butyrate-producing bacteria appears to be protective (haak et al., ) . a recent analysis from the seattle group has shown that antibiotic exposure before the onset of viral respiratory infection increases the risk of progression from uri to lri in case of hmpv, piv and rsv infection (ogimi et al., a) . however, a certain class of antibiotics that poses a particular risk could not be identified. another recent study described significant lower overall survival for patients with respiratory virus infection accompanied by bacterial co-infection that contributed to increased mortality (pinana et al., ) . risk factors for developing bacterial co-infection following a respiratory viral infection were steroid use ≥ mg/kg/d and cmv dnaemia requiring antiviral therapy. mortality-associated risk factors such as lymphopenia < . × /ml, cmv dnaemia requiring antiviral therapy at the time of viral lri diagnosis and oxygen support at the time of bal were used to establish a risk score in order to stratify patients to help predict mortality, no matter if a co-infection was present or not. this new risk score was compared to other scores including the isi described above, however, the application of isi criteria did not prove useful to predict mortality in this cohort. currently, isi does not include co-infections as a variable, and future studies are needed to validate and compare different risk scores (pinana et al., ) . the profound lymphopenia after hct has been described as a risk factor in the study above and multiple other studies, and it has recently been suggested that cd t cells may protect against a secondary infection (schmidt and varga, ) . the fourth european conference on infections in leukemia (ecil- ) developed guidelines for diagnosis and treatment on several respiratory viruses (hirsch et al., ) . hct candidates and recipients with uri or lri should be tested for respiratory viruses to guide infection management and possible deferral of transplantation. specimens should be taken from the site of infection; for uri, pooled swabs should be analyzed and for lri, a tracheal aspirate or ideally a bal should be performed. however, patient circumstances often do not permit invasive procedures. these guidelines favored a first-line screening for ifv a and b, rsv, and piv, however, with more advanced multiplex pcr assays becoming available, all agents might be examined at once (waghmare et al., ) . over the past years, more respiratory infections in hct recipients have been reported due to the development and use of new diagnostic methods. routine molecular diagnostics of respiratory viruses nowadays includes multiplex pcr approaches that have improved detection of respiratory agents in hct recipients (leber et al., ; sam et al., ) . multiplex assays covering the relevant respiratory viral agents are preferred over laborious and time-consuming viral culture and direct fluorescence antibody assays because of its sensitivity, specificity and rapid turnaround time. in addition, viral nucleic acid quantification is important because determination of a viral load might indicate prolonged viral shedding in case of rhv (ogimi et al., b) . however, this does not provide information about active replication and viral culture techniques are not well established, e.g., for hcov. a problem with virus detection is a lack of standardization among assays since different primers, probes and techniques are used (huang et al., ) . nasal respiratory swabs are routinely performed pre-transplant and should be collected in case of suspected respiratory infection. a large prospective study associated respiratory virus detection before transplantation with prolonged hospitalization and decreased survival at day (campbell et al., ) . rhv detection after a routine pretransplant screening can result in fatal infection, even if the patient is asymptomatic and presents with regular findings on chest ct (milano et al., ; campbell et al., ; waghmare et al., ) . if any respiratory virus is identified by multiplex pcr pre-transplant, hct should be postponed peck et al., ; hirsch et al., ) . however, as occurs in leukemia relapse cases, the time window from remission to hct can be narrow, thus making a decision difficult. irrespective of uri or lri, transplant should be delayed if possible, probably even in cases where data are scarce as is the case in hcov and hbov infections. while episodes of both uri and lri have shown a higher mortality risk in symptomatic adults in contrast to asymptomatic patients (abandeh et al., ) , transplant delay should be recommended in symptomatic adults. however, in pediatric patients who tend to shed virus more frequently than adults and acquire viruses more frequently, transplant delay should be considered on a case by case basis in asymptomatic patients. in aiding to determine progression from uri to lri, radiologic signs are considered an important component although inter-observer variability is high and radiologic signs are sometimes difficult to interpret (elemraid et al., ; franquet, ) . it is important to perform useful radiologic diagnostics in symptomatic patients with suspected lri. chest x-rays are generally not recommended because lack of sensitivity but ct should be performed to evaluate lri. in children, chest x-rays do not provide specific information in case of hmpv infection where common findings include hyperinflation, atelectasis and perihilar opacities (hilmes et al., ) . in an attempt to try and provide a standardized framework a scoring tool named the rsi has been established to quantify severity of radiologic findings and correlate them with outcome in piv lri. infiltrate expansion, when longitudinally assessed by rsi, was predictive of mortality in patients with piv-associated disease and discrepant results were obtained when ct and chest x-ray findings were compared. early signs suggest that the rsi appears to be a useful tool to predict mortality but needs to be validated in future studies (sheshadri et al., ) . computed tomography findings associated with lri include bronchial wall thickening and diffuse or patch-like ground glass opacities as a sign of interstitial infiltrates (franquet et al., ; herbst et al., ) . however, radiologic approaches often do not show clear signs of infection in symptomatic patients. additionally, the occurrence of simultaneous infections means correlating a specific virus with ct findings is difficult. to address this, a retrospective study by kim et al. ( ) examined ct scans from lri patients with bal specimens and single infections of either rsv, ifv or piv in order to identify viruscharacteristic ct lesions. patch consolidations of at least one centimeter or more than one segmental level were only observed in piv infections. ground-glass opacities were identified in all ct scans when ifv was detected and were less frequently seen with piv (detected in % of cases) and rsv ( %). bronchial wall thickening was prominent with ifv and rsv in about two thirds of ct scans analyzed whereas it was seen in only one third in piv infections. anatomically, rsv infection was localized in the upper and middle lobes ( %), piv preferentially in the lower lobes ( %) and ifv infection resulted in a diffuse pattern (kim et al., ) . however, due to the retrospective nature of the study by kim et al., no distinction between early and late infection stage ct findings could be made which might have revealed different patterns. other studies similarly identified ground-glass opacities for ifv infections (franquet et al., ; kanne et al., ) and bronchial wall thickening combined with nodules and tree-in-bud in rsv infections (mayer et al., ) . another small study also detected bronchial wall thickening in patients with confirmed rsv infections but also groundglass opacities, and these findings were equally seen in hmpv infections where lesions appeared to be more asymmetrical (syha et al., ) . although ct findings appear to be non-specific, they might help to differentiate between pathogens causing lri if a thorough diagnostic workup including multiplex pcr with representative specimens is employed to largely rule out simultaneous infections. infection control measures are of high importance and should be in place in case of suspected or confirmed respiratory virus infection (boeckh, ) . since these viruses are transmitted by close contact with infected individuals or contaminated material, hand hygiene is of utmost importance since nosocomial infections have occurred that must be avoided (hoellein et al., ) . the use of universal surgical mask might also be useful to prevent respiratory viral infections after hct (sung et al., ) . patients with documented respiratory virus infection should be isolated with restricted contact and strict protection measures should be applied (gloves, gowning, masks, eye protection) to visitors and healthcare workers (hirsch et al., ) . since hct recipients might shed these viruses for a long time because of their impaired immunity, precautions should be taken if a patient is discharged. for example, a patient should be escorted through the outpatient clinic to avoid direct contact with and spread to other patients, and prolonged isolation might be necessary (tomblyn et al., ; hirsch et al., ) . this might be especially important for rhv in case high viral loads are detected since shedding time in these patients exceeds days (ogimi et al., b) . however, piv- and hmpv infections might go unrecognized in immunocompetent persons, thus making it difficult to prevent infection (nichols et al., b; debiaggi et al., ; shah et al., b; birger et al., ) . each time a respiratory tract infection is proven after hct, a reduction of immunosuppressive treatment should be the response. a steroid dose greater than or mg/kg/day has been proven to be an independent risk factor for overall mortality in lri with rhv (seo et al., ) , rsv (shah et al., a) , hmpv, and piv (waghmare et al., ) . however, no correlation between progression or mortality and steroid dose was found for ifv (waghmare et al., ) . adenovirus pneumonia is usually observed in adv disseminated disease. preemptive treatment is recommended as soon as viremia is higher or equal to copies/ml coupled with lymphocyte counts below /mm and cd t lymphocyte counts below /mm (hiwarkar et al., ) . this is particularly important in high risk patients and those who received cord blood graft or are under steroid treatment (lindemans et al., ) . high adv levels in stool have also been associated with gastrointestinal adv disease (feghoul et al., ) -an observation that underpins a current clinical trial addressing the usefulness of a preemptive treatment based on stool adv detection . so far, no recommendation has been made for adv detection in upper airways. specific treatment of adv pneumonia should be given as early as possible after diagnosis (neofytos et al., ; lindemans et al., ) . several studies have reported successful cidofovir treatment of adv infections in immunocompromised hosts after hct, combined with withdrawal of immunosuppression (e.g., at mg/kg once every week for weeks, followed by a maintenance dose of mg/kg once every fortnight, or three times weekly at mg/kg) (lindemans et al., ; wy ip and qasim, ) . cidofovir inhibits incorporation of deoxycytidine triphosphate https://clinicaltrials.gov/ct /show/nct into viral dna by the viral dna polymerase, leading to viral dna chain termination. probenecid co-prescription is useful to prevent or diminish cidofovir nephrotoxicity due to its accumulation in renal tubules. cidofovir is the current standard of care treatment for adv infections and diseases. however, cidofovir does not clear the virus in the absence of t cell immune reconstitution (hiwarkar et al., ) . another novel compound is cmx (hexadecyloxypropyl cidofovir, brincidofovir, chimerix), an orally bioavailable lipid conjugate of cidofovir with good oral bioavailability which achieves higher intracellular levels of active drug compared with cidofovir. brincidofovir lacks nephrotoxicity making it an attractive alternative. in phase i and phase ii trials as well as in retrospective studies, brincidofovir has been shown to be highly efficacious in controlling and clearing adenoviraemia (grimley et al., ; hiwarkar et al., ; ramsay et al., ; averbuch et al., ) . gastrointestinal toxicity is the major side effect observed and might be associated with epithelial apoptosis and crypt injury, as might be observed with gvhd (detweiler et al., ) . further, a small compound inhibitor named hbx has been reported to be effective against adv infections. a cellular component important for adv replication is ubiquitin-specific protease (usp- ), a deubiquitinating enzyme of the ubiquitin proteasome pathway. usp- interacts with e b- k, a regulator of adv replication. since usp- promotes adv replication, it represents a potential drug target because blocking its activity could aid in the treatment of adv infections (ching et al., ) . hbx and a derivative were generated that were able to block adv (c type) replication. with the exception of adv types a and a , hbx effectively blocked in vitro replication of all adv tested, making it a promising new antiviral candidate (kosulin et al., b) . for adv, there is no proven role for ganciclovir, foscarnet or immunoglobulin therapy in immunocompromised patients. importantly, donor lymphocyte infusions and more recently specific anti adv cytototoxic t cells should be considered in case of adv disease after hct (see below). there is anecdotal evidence of successful treatment of adv with ribavirin (schleuning et al., ) but most studies have not been supportive; (lankester et al., ) and conclude that cidofovir combined with immunotherapy is more efficient and should be preferred as the first treatment option. of note, ribavirin is active in vitro on species c isolates (morfin et al., ) . there is currently no recommendation of specific antiviral therapy due to the lack of effective agents against these viruses and the lack of clinical trials (hirsch et al., ) . several agents against rhv have been described in the context of immunosuppression: oral or intranasal pleconaril, a capsid binder, was effective in randomized trials to mildly reduce the duration and severity of colds in immunocompetent adults but its development was halted after fda rejection partly due to concerns regarding virus resistance (senior, ) . vapendavir is another capsid binder under development for the treatment of rhv infections of asthmatic adults (waghmare et al., ) . to avoid transmission of ifv, vaccination of family members, household contacts and hct recipients is recommended. however, hct recipients might not mount an adequate immune response if vaccination takes place during immunosuppression. available antivirals for ifv infections include the m ion channel inhibitors such as amantadine that exclusively act on ifv-a. however, neuraminidase inhibitors (nai) are preferred for prophylaxis and treatment of ifv infections since resistance to m inhibitors is frequent (englund et al., ; fiore et al., ; von lilienfeld-toal et al., ) . post exposure prophylaxis with oral oseltamivir, mg bid for days for adults and children whose weight is above kg, is the current treatment of choice. oral oseltamivir, inhaled zanamivir and iv peramivir are nais that were shown to be effective in hct recipients (vu et al., ; tomblyn et al., ; casper et al., ; choi et al., ; engelhard et al., ) . treatment within h after the occurrence of symptoms will result in better outcome (ljungman et al., ; nichols et al., b; tomblyn et al., ; choi et al., ; engelhard et al., ) ; although treatment should be initiated as early as possible, protracted and also prolonged treatment have been shown to have favorable effects. the duration of therapy should extend the treatment period recommended for immunocompetent hosts to circumvent reoccurrence and might last for days in hct recipients. also, longer therapy in case of unsuccessful clearance might be necessary (engelhard et al., ) . the most commonly described mutation resulting in oseltamivir and peramivir resistance is conferred by the h y mutation in influenza a h n that can be treated with inhaled zanamivir (memoli et al., ; engelhard et al., ) . in europe during the - winter season, high resistance rates up to % of ifv-a (h n ) to oseltamivir have been reported (meijer et al., ) . new agents with potential efficacy against oseltamivir-resistant viruses are being tested like inhaled laninamivir which is available in japan, jnj- , a non-nucleoside inhibitor of the rna polymerase protein pb , nitazoxanide, an antiparasitic agent with activity against ifv, and antibodies (medi , vis ) (shahani et al., ) . in a retrospective analysis of patients including hct recipients, one-third of patients with piv infection had lower respiratory tract disease and independent risk factors of progression were neutropenia, apache ii score ≥ , and respiratory co-infections within a month of piv infection. in this study, treatment with aerosolized ribavirin and/or ivig did not prevent progression to pneumonia and did not affect duration of illness or survival (chemaly et al., ) . a meta-analysis performed in reinforced this evidence of inefficiency of ribavirin for piv: indeed, pi-lri progression was not significantly https://clinicaltrials.gov/ct /show/nct different in hct recipients who were treated with ribavirin at uri stage, and piv-associated mortality rate was slightly higher in patients treated with ribavirin-based therapy at lri stage than in those who were not treated (shah et al., a) . a recombinant sialidase fusion protein inhibitor named das has been used to treat severe piv infections after hct (waghmare et al., ; dhakal et al., ; salvatore et al., ) . das cleaves the neu ac α( , )-gal and neu ac α( , )-gal sialic acid linkages on the surface of respiratory cells that are used by ifv and piv for attachment and entry, thereby inhibiting the latter. the largest cohort included patients after hct, of who received das for piv-associated pneumonia but unfortunately lacked a control group (salvatore et al., ) . thirteen out of patients responded to treatment, and the three patients who did not respond had a viral, bacterial, or fungal co-infection, respectively. the drug was well tolerated. piv loads were recorded in of patients using nasopharyngeal swabs. a > log decrease in piv load was seen in of patients accompanied by a partial or complete clinical response. high-dose ivig had no effect on mortality after piv lri in a retrospective analysis of patients with piv infection after hct (seo et al., a) . therefore, no specific treatment for piv infection is currently strongly recommended. however, current ecil- guidelines suggest treatment with aerosolized ribavirin or off-label use with systemic ribavirin. for infections other than piv and rsv, ribavirin use is not recommended (hirsch et al., ) . in a study among patients with uri from the fred hutchinson cancer center (seo et al., ) , the probability of progression to lri within days was %, and approximately % of the patients with uri who progressed to lri did so within weeks after uri. even if several small reports support the use of ribavirin with or without ivig for hmpv infection, a larger study showed no protective effect of ribavirin to reduce hmpv progression and mortality (renaud et al., ) . to date, antiviral therapy is not recommended to cure or to prevent infection progression even in patients at higher risk of hmpv progression. no licensed therapeutics or vaccines exist (wen and williams, ) . therefore, treatment for hmpv remains so far nonspecific and mainly supportive. mab , a monoclonal antibody against a fusion protein of hmpv, demonstrated interesting results in hamster and mice models, and further investigation in clinical trials is warranted (ulbrandt et al., ; hamelin et al., ) . in , international guidelines recommended aerosolized or systemic (oral or iv) ribavirin with ivig in patients with rsv uri undergoing allogeneic hct, allogeneic hct recipients with risk factors for progression to lri, and allogeneic hct patients with lri (hirsch et al., ) . however, the additional benefit of ivig remains controversial. in a retrospective study that reviewed rsv uri or lri infections in hct recipients, a combined ivig and ribavirin treatment was beneficial (shah et al., ) . further, given significant exponential cost increases in aerosolized ribavirin between and and lack of evidence that aerosolized ribavirin is more efficient, early oral ribavirin treatment is currently more frequently considered (waghmare et al., ) and is well tolerated (e.g., mg/kg/day in three divided doses for days) (gorcea et al., ) . a clinical trial comparing rsv treatment with oral versus inhaled ribavirin is ongoing . however, in france, aerosolized ribavirin is currently not available. lymphopenia is a specific risk factor that has been associated with progression to lrti in several studies. other risk factors such as total body irradiation, smoking history, stem cell source other than peripheral blood stem cells and oxygen requirement might be considered waghmare et al., ) . a lower virulence of rsv-b compared to rsv-a has been reported (kelly et al., ) . as stated above, the isi might be helpful to identify patients who would benefit from antiviral therapy (shah et al., a) . data from studies with the rsv-specific monoclonal antibody palivizumab have shown controversial efficacy, apart from the fact that it is very costly in adults. in larger studies of hct recipients with rsv lri, adjunctive palivizumab did not lead to outcome improvement waghmare et al., ) . therefore, rsv-specific monoclonal antibody is not recommended as a treatment option and might be only discussed https://clinicaltrials.gov/ct /show/nct for very young (age < years) allogeneic hct recipients with lri or at high risk for progression to rsv lri (e.g., palivizumab mg/kg body weight) (hirsch et al., ) . moreover, several agents are under development against rsv infections (shahani et al., ) . firstly, there are fusion inhibitors like gs- which reduced the viral load and the severity of clinical disease in a study of healthy adults (devincenzo et al., ) , mdt- and alx- . secondly, agents targeting the rsv polymerase exist: al- which had an interesting antiviral activity compared to placebo in healthy adults inoculated with rsv (devincenzo et al., ) and favipiravir which is currently being tested in phase iii clinical trials in the united states, europe, and latin america. thirdly, substances targeting the viral nucleocapsid protein have been investigated. a small interfering rna called aln rsv inhibiting the synthesis of the rsv nucleocapsid protein has demonstrated a benefit in phase i to iib clinical trials by decreasing the infection rate, reducing symptoms and the incidence of bronchiolitis obliterans. another drug, rsv- , targets the n terminal portion of the nucleocapsid protein. it was tested on hct recipients, however, no data have been published (simões et al., ) . finally, polyclonal high-titer anti rsv antibodies might be an option (ri- ) . current strategies recommended for the treatment of respiratory viral disease after hct, and molecules that are under investigation, are summarized in table . before the era of specific antiviral manufactured t cells, donor lymphocyte infusions (dli) were used and recommended in case of disseminated adv infections after hct (bordigoni et al., ; taniguchi et al., ) . the high incidence of gvhd after dlis limits its use in patients with previous history of gvhd. anecdotal use of dli has been reported for threatening rsv lri (kishi et al., ) . there are currently three strategies to manufacture viral specific t cells (vst) for clinical use: the first one is direct selection of antiviral t cells, either by multimers specific for a virus-derived peptide in the setting of class i hla molecule, or by column selection of ifn-γ expressing t cells (with immunomagnetic beads) after viral antigen stimulation (feuchtinger et al., ; peggs et al., ; icheva et al., ) . the second one is ex vivo expansion of t cells cocultured with antigen presenting cells pulsed, infected or transfected with viral peptide/protein/viral lysate or plasmid (peggs et al., ; gerdemann et al., gerdemann et al., , . the third one is genetic modification of t cells which incorporate high affinity vsts receptor or chimeric antigen receptor genes (schub et al., ; cruz et al., ; bollard and heslop, ) . so far, in the setting of community acquired viral respiratory infections, adoptive immune therapy has been developed and investigated in clinical trials only for adv infections. this strategy is promising as it has been reported to offer a way to cure severe adenoviral infections in case of resistance to antiviral drugs. adenoviral clearance has been obtained after infusions of adv-vst generated by interferon [ifn-γ-based immunomagnetic isolation (and further in vivo expansion) from initial donor or third party donor] in of hematopoietic stem cells transplanted patients in a french phase i/ii trial (qian et al., ) . similar results have been published earlier (feucht et al., ) from patients who received adv-vst for adv disease or viremia. of evaluable patients had in vivo expansion of th -vst (median time days); and patients responded to vst infusions and adv clearance occurred in . of note, adv-vst infusions were not associated with acute toxicities or significant onset of gvhd. the main disadvantage of this technique is that high doses of immunosuppressive drugs and especially steroids should be avoided before infusions as this drug hampers in vivo expansion of specific t cells. currently, multiviral (including adv) t cell manufacturing and banking from a third-party donor is a promising strategy as it might offer an immediate way to prevent adv infections after hct or to cure % of adv infected patients after hct . ex vivo expanded cytotoxic t cells against piv- antigens should be further tested in clinical trials (mclaughlin et al., ; aguayo-hiraldo et al., ) . the outcome in patients who experience progression to lri is worse compared to patients with uri regardless of the virus involved (chemaly et al., ) . in a french cohort of adult patients infected with viral respiratory infections, ( %) died within months. among these, eight patients ( %) died of viral pneumonia along with bacterial and/or fungal pneumonia. interestingly, the virus group and the time from hct had no impact on mortality. two factors were independently associated with increased overall mortality: steroid dose over mg/kg body weight and a lymphocyte count lower than . g/l (wolfromm et al., ) . the fact that lri associated death risk does not depend on the virus type is supported by seo et al. ( ) who reported an overall mortality probability at day as high as % in patients after hct who developed rhv lri, a rate that was comparable to rsv, piv and ifv lri in an adjusted model. among children, in a cohort of patients, ( %) died of respiratory failure (choi et al., ) . lri in the absence of uri and adenoviral infection has been described to be associated with a poorer outcome in another cohort of patients with specific overall mortality of % (lo et al., ) . bronchiolitis obliterans syndrome and obstructive airflow decline have been associated with piv and rsv infections within the first months after allogeneic hct which persisted at year after transplant (versluys et al., ) . further, the occurrence of a lri within days post-transplant was an independent predictive factor for late onset noninfectious pulmonary complications diagnosed in the first months after transplantation in an observational prospective cohort study on patients (bergeron et al., ) . therefore, functional pulmonary explorations and long-term surveillance are warranted after community acquired viral infections which occurred early after hct. to better understand risk factors of pulmonary complications in children after hct, a multicentric french trial is ongoing . early treatment with β mimetics and inhaled steroids might be useful and should be proposed in case of persistent obstructive syndrome after respiratory viral infections resolution in hct recipients. this review has limitations because we present results from small case series that have restricted meaningfulness. further, it includes recommendations that were in part made before the era of t cell repleted haploidentical transplantations. this is an area of intense research and these new developments might impact the epidemiology and outcomes of viral infections after hct. respiratory virus infections continue to cause disease both in the pre-transplant as well as in the post-transplant period and should be taken very seriously, especially in children who https://clinicaltrials.gov/ct /show/nct tend to shed virus for longer periods. this long shedding time has implications for infection control. therefore, both personnel and patients and their families should be properly instructed in hand hygiene. in case of pre-transplant infections, hct should be deferred but the underlying disease, the conditioning regimen and donor availability need to be considered. in pediatric patients who also tend to acquire viruses more frequently, we favor transplant delay in elected cases, even in asymptomatic patients. without doubt, disease progression from uri to lri influences the outcome in hct recipients. there are a number of risk factors shared by respiratory viruses that are associated with lri occurrence; lymphopenia appears to be a specific risk factor in this case. other risk factors shared with increased mortality are steroid use at the time of lri diagnosis, oxygen requirement at the time of bal, and severity of disease based on apache ii score. in addition, myeloablative conditioning, gvhd, bacterial as well as fungal pulmonary co-infections (especially with ifv and piv infections) seem to worsen the outcome. is there a possibility to reduce the risk of progression from uri to lri? there might be a chance if co-infections can be reduced or diagnosed and treated promptly. these co-infections could be more easily avoided if they were nosocomially acquired by transfer from personnel or visitors. therefore, people who have access to the patient should be aware of this problem. also, shortening of the lymphopenic interval could reduce disease progression. this might be achieved with a stricter withdrawal of immunosuppressive drugs, e.g., steroids, or a milder form of conditioning. early and specific diagnostics are of the essence. it would be best to obtain diagnostic samples straight from the area of infection, i.e., from bal material and not a nasal swab in case of a lri, however, this might be sometimes impractical and not be tolerated by the patient. unless a specific viral agent is suspected and a single or duplex pcr are ordered, e.g., during an outbreak, novel multiplex assays should be employed that also identify a bacterial co-infection. what is more, novel multiplex assays that are able to quantify viral loads clearly have the potential to help identify which patients are at increased risk and who might need treatment urgently -particularly since supporting measures like radiological imaging techniques often do not point to a specific pathogen and provide non-specific results with regard to identification of the pathogen responsible. however, while chest x-rays discover inflammatory processes with delay, ct findings might help to differentiate between pathogens. all these diagnostic steps should be carried out after full evaluation of the circumstances and in an orderly fashion. the isi and the rsi (tables , ) might be helpful to identify patients at risk but await validation in clinical trials. eventually, only few antivirals are available and licensed to treat respiratory virus infections. several drugs have been tested in hct recipients and promising results have been published. novel antiviral compounds are urgently required that might be used in combination with antiviral lymphocytes as currently being developed for cmv, ebv, adv, or bk antivirus ctls in us (nct , nct ) and european international trials (the trace trial). the production of these cells should be confined to specialized laboratories, either low risk: - score, moderate risk - score, high risk - score anc, absolute neutrophil count; alc, absolute lymphocyte count; gvhd, graftversus-host disease; modified after shah et al. ( ) . to calculate the rsi score, the predominant pattern for each lung zone is multiplied with the extent of the volumetric radiologic involvement, e.g., if ground-glass opacities are detected in all six lung zones with all having a volumetric score of , the rsi score would be ( × × ). the maximum score is . modified after sheshadri et al. ( ). university-based or commercial, however, they should be easily accessible. the expansion of this approach to target respiratory viruses might be similarly useful to halt disease progression from uri to lri. finally, the application of car t cells to combat respiratory viral infections might be an option in the future, especially in combination with tumor targeting car t cells. however, pulmonary toxicity should be considered due to cross-reactivity of car t cells with non-targeted proteins, and also ards that often occurs in severe lri might be worsened by cytokine release syndrome. while viral pneumonia still remains a concern in hct patients, new drug developments, along with sophisticated t cell approaches, should diminish complications caused by respiratory viruses. cp and sv wrote and edited the manuscript. this work was supported by intramural funds from the robert koch institute. outcomes of hematopoietic sct recipients with rhinovirus infection: a matched, case-control 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rongguang title: crystal structure of refolding fusion core of lassa virus gp and design of lassa virus fusion inhibitors date: - - journal: front microbiol doi: . /fmicb. . sha: doc_id: cord_uid: l r gte the envelope glycoproteins gp and gp of lassa virus (lasv) bind to the host cell receptors to mediate viral infection. so far, no approved vaccines and specific treatment options against lasv exist. to develop specific fusion inhibitors against lasv, we solved the crystal structure of the post-fusion helix bundle ( -hb) formed by two heptad repeat domains (hr and hr ) of gp . this fusion core contains a parallel trimeric coiled-coil of three hr helices, around which three hr helices are entwined in an antiparallel manner. various hydrophobic and charged interactions form between hr and hr domains to stabilize the overall conformation of gp fusion core. based on the structure, we designed several peptides spanning the hr domain and tested their antiviral activities. we found that the longer hr peptides were effective in inhibiting lasv gpc protein-mediated cell–cell fusion under low ph condition. these results not only suggest that lasv infects the target cell mainly through endocytosis, including micropinocytosis, and membrane fusion at low ph, but also provide an important basis for rational design of lasv fusion inhibitors. lassa fever is an acute viral hemorrhagic illness occurring in west africa, having posed a serious public health threat in many countries (sogoba et al., ; shaffer et al., ) . its case-fatality rate is % for overall infection and ∼ % for severe cases among patients hospitalized. this mortality rate will increase sharply during epidemics or in pregnant women (asogun et al., ) . the etiologic agent of lassa fever is lassa virus (lasv), belonging to the arenavirus family. arenavirus has more than members divided into two groups: the new world viruses (or tacaribe complex) and the old world viruses (or lcm-lassa complex). the new world family mainly contains venezuelan hemorrhagic fever (vhf), junín virus (junv), machupo virus (macv), and bolivian hemorrhagic fever (bhf), while the old world viruses includes, for example lujo virus (lujv), lymphocytic choriomeningitis virus (lcmv), morogoro virus (morv), and lasv. these arenaviruses have both geographical and genetic differences. lassa virus is an enveloped, single-stranded rna virus. the two rna segments in its genome encode four viral proteins, including zinc-binding protein (z), rna polymerase (l), nucleoprotein (np), and the surface glycoprotein precursor (gp, or spike protein). gp is cleaved into envelope glycoproteins gp and gp (cao et al., ) . gp that is responsible for receptor binding (including α-dystroglycan, heparin sulfate, dc-sign, etc.) and gp that mediates membrane fusion interact with each other to form a stable trimer complex on the lasv viral envelope (li et al., ; hastie et al., ) . upon receptor binding, lasv enters the target cell via clathrin-and dynaminindependent endocytosis with subsequent transport to late endosomal compartments, where fusion occurs at low ph (vela et al., ; rojek et al., ) . a recent study has also found that after gp binds to cell receptor α-dystroglycan, lasv enters into the target cell through the unusual micropinocytosis pathway and membrane fusion under low ph condition (oppliger et al., ) . so far, no approved vaccines and specific treatment modalities against lasv are available. given that the first peptide-based antiviral drug enfuvirtide (t ) inhibits human immunodeficiency virus (hiv) fusion with and entry into the target cell by targeting the heptad repeat domain in the viral envelope glycoprotein gp (qi et al., ; su et al., ) , the heptad repeat domain in gp of lasv may also serve as a target for the design of lasv fusion inhibitors. we have previously demonstrated that the n-terminal heptad repeat (hr ) domain binds to c-terminal heptad repeat (hr ) domain to form a stable six-helix bundle ( -hb) to mediate viral entry; therefore, peptides derived from hr should specifically bind to the homotrimeric hr , interfering with the formation of -hb and, hence, blocking viral entry (zhu et al., (zhu et al., , xia et al., ) . therefore, it is essential to determine the -hb core structure and characterize the interaction sites in the hr and hr domains, in order to design the hr -derived peptides against lasv infection. here, we solved the crystal structure of the post-fusion -hb formed by lasv hr and hr domains. based on the structure, we designed several peptides spanning the hr domain and tested their antiviral activities. concurrent with the preparation of the present manuscript, another group reported a post-fusion structure of lasv using the insect baculovirus expression system (shulman et al., ) , which allows us to compare the structural differences of -hb cores formed by the hr and hr domains in their truncated version of lasv gp spanning residues - with those in our lasv hr -t-loop-hr construct. these comparisons provide more comprehensive knowledge for better understanding the entry mechanism of lasv and designing of peptide-based lasv fusion inhibitors. the t cell line was obtained from atcc (manassas, va, united states), and the huh- cell line was from the cell bank of the chinese academy of sciences (shanghai, china). these two cell lines were propagated in dulbecco's modified eagle's medium (dmem) supplemented with % fetal bovine serum (fbs). peptides (lasv/hr - , lasv/hr - , lasv/hr - , lasv/hr - , and lasv/hr - ) were synthesized by solid-phase peptide synthesis at syn inc. (shanghai, china) . recombinant plasmids encoding the lasv gpc protein were synthesized by syn inc. the gene coding for hr -t-loop-hr construct of lasv gp (residues - , with a c s mutation and gk truncation) was amplified by pcr and cloned into vector pet- a with an artificially introduced prescission protease cleavage site at its n-terminus for stable expression. the fusion protein was overexpressed in escherichia coli bl . cells were grown to od ≈ . in lysogeny broth (lb) media supplemented with µg/ml kanamycin at • c and were induced by mm iptg for h at • c for expression. cells were harvested by centrifugation at × g for min at • c and were lysed by high pressure homogenizer twice after resuspension in buffer containing mm tris-hcl, ph . , and mm nacl. the inclusion body was harvested by centrifugation at , x g for min and resuspended in buffer containing mm tris-hcl, mm nacl, ph . , m urea, and mm dtt. then the trimeric lasv -hb protein was refolded using limit dilution method. briefly, the denatured protein was diluted at : volume ratio into renaturation buffer ( mm tris-hcl, mm nacl, ph . , mm arginine, mm gsh and . mm gssg) very slowly ( . ml/min), and stored for week at • c. refolded protein was isolated by ni-affinity chromatography and purified by anion exchange chromatography (hitrap q fast flow ml, ge healthcare), as well as gel filtration chromatography (superdex / gl, ge healthcare), and then concentrated to mg/ml for crystallization or storage at − • c for further use. crystals were obtained at • c for days using the hanging drop vapor diffusion method by mixing equal volume of protein solution [lasv- -hb: mg/ml] and reservoir solution, [ . m ammonium acetate, . m sodium citrate: hcl, ph . , . % (w/v) peg , and % (v/v) glycerol]. then crystals were flash-frozen after immersing in paraffin oil for about s, followed by transfer to liquid nitrogen for further data collection. the datasets were collected at beamline bl- u , shanghai synchrotron radiation facility, at a wavelength of . Å. the crystals were kept at k during x-ray diffraction data collection. data were indexed and scaled with hkl (otwinowski and minor, ) . phases were solved by the molecular replacement method using phenix.phaser (mccoy, ) . all refinement procedures were carried out with phenix.refine (zwart et al., ) and coot (emsley and cowtan, ) . table shows the detailed statistics of data collection and refinement. coordinates and structure factors have been deposited in the protein data bank with accession number jgy for crystal structure of fusion core of lasv gp protein. biolayer interferometry (bli) is a common technique for detecting interactions between substances. the working principle of this method is that the molecules are immobilized on the surface of the sensor to form a biolayer, thereby causing interference against light waves passing through the sensor, which was then detected in the form of phase displacement, so that any change in the number of molecules on the surface of the sensor can be detected. based on this principle, the mobile phase cannot have a non-specific combination with the sensor. the sensor type selected in this experiment was super streptavidin (ssa) biosensor, and all the tests were performed using octet red . the biotinylation of protein for immobilization onto ssa was carried out according to the following procedure. the activated biotin reagent was prepared in dmso at a concentration of mg/ml. the peptides were mixed with biotin reagent, and the molar ratio of peptide to biotin reagent was : . the reactions were incubated at room temperature for h. then free biotin was removed by dialysis. biotinylation of protein for immobilization onto ssa was at a concentration of µg/ml. the loading time was s. the concentration of mobile phase in the initial test was µm. the concentration gradient in the further test was , , . , and . µm. the association time was s, and the dissociation time was s. the secondary structure of peptides lasv/hr - (residues - ) and lasv/hr (residues - ), as well as their mixture, were determined by cd spectroscopy. briefly, the peptides were diluted in phosphate-buffered saline (pbs) (ph . ). after incubating at • c for min, cd spectra were acquired on a jasco spectropolarimeter (model j- ; jasco, inc., easton, md, united states) from to nm at room temperature, using the optical path length of . cm and bandwidth of nm. the baseline was determined using pbs. the α-helical content was calculated from the cd signal by dividing the mean residue ellipticity [θ] at nm by the value expected for % helical formation (− , degrees cm dmol − ). lassa virus pseudoviruses were constructed as described previously (li et al., ; wang et al., ) . briefly, t cells were seeded in a -cm tissue culture dish. when t cells grown in a -cm dish reached % confluence, cells were cotransfected with plasmids pcdna . -lasv-gpc (encoding gpc protein of lasv) and pnl - .luc.re (encoding env-defective, luciferase-expressing hiv- capsid protein) at a ratio of : using vigofect (vigorous biotechnology, beijing, china). the supernatant was replaced with fresh dmem at - h post-transfection and harvested after incubation for an additional h. cell debris was removed by centrifuging at rpm for min, followed by filtration through a . µm filter. a lasv pseudovirus inhibition assay was performed in a manner similar to other envelope virus assays (lu et al., ; channappanavar et al., ; xia et al., ) . briefly, huh- cells were placed ( cells/well) into a -well plate and incubated overnight at • c. lasv pseudovirus was incubated with serially diluted peptides for min at • c, followed by the addition of huh- cells. the cells were incubated with or without pseudovirus as virus control and cell control, respectively. at h post-infection, the culture was replaced with fresh medium, followed by an additional incubation for h. cells were lysed, and cell lysates were transferred to a -well costar flatbottom luminometer plate (corning costar, new york, ny, united states), followed by the addition of luciferase substrate (promega) to measure luminescence using an infinite m pro (tecan, grödig, austria). lassa virus gpc protein-mediated cell-cell fusion was performed as previously described (cosset et al., ) . briefly, plasmid paav-ires-lasv-egfp encoding the lasv gpc protein was transfected into t cells ( t/lasv/egfp) using the transfection reagent vigofect (vigorous). when gfp was obviously expressed on most t cells, t/lasv/egfp cells were digested and mixed with huh- cells at ratio of : . the mixture was incubated at × cells/well in wells of a -well plate for h. the peptides were serially diluted with low ph (ph ) dmem and then added to the mixture of t/lasv/egfp cells and huh- cells. after min of exposure in the low ph medium for triggering the fusion between the t/lasv/egfp cells and huh- cells, the cells were restored to neutral medium and cultured for to h at • c. the t/lasv/egfp cells fused or unfused with huh- cells were fixed with % pfa and counted under an inverted fluorescence microscope (nikon, tokyo, japan). the fused cells showed much larger size and weaker fluorescence intensity than the unfused cells because of the diffusion of egfp from one cell to more cells. the gp protein contains a n-terminal fusion peptide (fp) (residues - ), a n-terminal hr domain (residues - ), a linker t-loop domain (residues - ), a c-terminal hr domain (residues - ), a transmembrane domain (residues - ), and an intracellular domain (residues - ) ( figure a) . by multiple sequence alignment with other representative arenaviruses, like lcmv, junv, and macv, lasv showed a highly conserved t-loop domain and a variable hr (figure b) . two ends of the hr domain are conserved, but its middle region is relatively unique, which may be helpful to adapt the variable hr domain of lasv gp . to reveal the interaction between hr and hr domains of lasv, stable -hb covering hr , t-loop and hr domains (residues - ), was constructed for crystallographic study. the denaturation and renaturation were used to acquire stable -hb from inclusion body to mimic its post-fusion state. during refolding process, the wild type lasv -hb protein mainly formed precipitations or high polymers. after the single mutation of c s was introduced, the trimeric lasv -hb protein was obtained ( figure c) . however, the large-scale of crystal screening for this protein could not yield high-quality crystals. then we continued to try different truncations and deletions in -hb, and finally found that a deletion of gly and lys in the t-loop region could yield protein crystal with enough quality for structure determination. the overall structure of the hr -t-loop-hr domain showed a canonical -hb structure (figure a) . taking a rod-like shape with a length of ∼ Å and a diameter of ∼ Å, the lasv fusion core contains a parallel trimeric coiled-coil of three hr helices (gray in figure a) , around which three hr helices are entwined (green in figure a ) in an antiparallel manner. between these two domains, the t-loop forms a helix linker hovering outside (cyan in figure a) . according to the structure shown in electrostatic potential surface (figure b) , the hr region of lasv can be divided into frontiers in microbiology | www.frontiersin.org two parts: a linear n-terminal region and a helical c-terminal region. the linear n-terminal region binds to two adjacent hr helices through hydrophobic interactions, consistent with other fusion core structures, as previously determined (lu et al., ) . some obvious charged interactions take place between the helical c-terminal region of hr and hr trimer core, and these will be discussed later. another group deposited a postfusion structure of lasv in pdb (entry code omi), using a baculovirus expression system, instead of our renaturation method. compared with this structure, the hr helical region in our structure exhibited a significant shift of . ∼ . Å toward the hydrophobic grooves of two adjacent hr helices, showing much stronger hydrophobic interaction (figures c,e) . lymphocytic choriomeningitis virus and lasv both belong to the old world family and have high sequence homology ( figure b) . comparing the fusion core structure of lcmv (pdb entry mko) with that of lasv, we found that their hr domains overlap with each other very well, but their t-loop and hr helical regions have obvious differences ( figure d) . the t-loop region of lcmv fusion core moves closer to the hr region, and its hr helical region slopes away from hr of lasv. when further comparing the -hb structures of lcmv and lasv with those of other viruses, we found several unique features for arenavirus gp protein ( figure f) . the hr and hr regions of hiv form a short regular α-helix structure, while coronaviruses, like mers-cov and mhv, have a short helical hr region, but longer helical hr region. for arenavirus of lasv or lcmv, a special t-loop region is situated between hr and hr domains, packing along the linear hr region. the three hr helices of lasv are closely packed against each other by hydrophobic force in a parallel manner. the buried area for each hr domain reaches Å , indicating a much stronger interaction (figure a) . at the c-terminal of hr , it is interesting that two hydrophilic interactions occur among the hr trimers. the asp and lys of one hr domain bind to the residues with opposite charges in other two hr domains. as shown in figure b , these two residues are highly conserved among different arenaviruses, suggesting that this additional charged interaction may play an important role in enhancing the stability at the end of hr trimer. the t-loop region and hr domain are well packed against the hydrophobic grooves of a central three-helical coiled coil with an interface of Å ( figure b) . leu , ile , and ile of t-loop bind to the c-terminal end of hr trimer. in the linear hr domain, trp , val , leu , and phe are deeply buried in the hr hydrophobic groove. even in the helical hr domain, many hydrophobic residues with long side chains are involved in binding to hr , including ile , met , ile , met , and leu ( figure b) . moreover, at the junction region between linear and helical parts of hr , several strong hydrogen bonds and salt bridges are noted. for hydrogen bonds, the hydroxyl group of ser in hr interacts with glu in one hr helix (∼ . Å distance), while its carbonyl oxygen interacts with lys in another hr helix (∼ . Å distance) (figure b) . for salt bridges, asp interacts with arg (∼ . Å distance), and glu interacts with lys and gln (∼ . Å and . Å distances, respectively). these relatively concentrated hydrophilic interactions constitute an anchoring point in the middle of hr domain, stabilizing the linear and helical region of hr and also the whole -hb conformation. to confirm the secondary structure before and after the formation of fusion core, the α-helical ratios of hr peptide, hr peptide, and their mixture were analyzed by circular dichroism. in solution, the single hr and hr peptides showed relatively low α-helical ratio of and %, respectively. however, when they were mixed together to form -hb, the α-helical ratio largely increased to % (figure c) , which is consistent with the crystal structure. it suggests that the two peptides undergo a significantly conformational change when they interact with each other to form -hb fusion core. to elucidate the interactions between hr and hr regions of lasv, as observed in the crystal structure, the hr -derived peptide (hr - ) and hr -derived peptides (hr - ∼hr - ) were synthesized to measure their binding affinities using bli. hr - peptide showed a significant non-specific adsorption on the sensor, making it impossible to measure affinity data. all other peptides showed weak non-specific binding to the sensor at the concentration of µm. then, hr - was used as the immobilized molecule to detect the affinity between hr - and hr - ∼ hr - . the results showed that hr - and hr - peptides could strongly bind to hr - peptides in a dose-dependent manner (figure a) , while hr - and hr - could not. both hydrophobic and hydrophilic interactions between hr - and hr - peptide are seen in the crystal structure ( figure b) . the major hydrophobic interactions in hr - peptide ( - ) include phe , ile , and met , which were buried in the hydrophobic groove of hr - trimeric core. several hydrogen bonds were also observed between hr - and hr - . the side chain oxygen of ser binds to glu in one hr helix, while its main chain oxygen binds to lys in another hr helix ( figure b) . then asp binds to arg , and glu interacts with lys and gln . however, despite these potential interactions, bli results showed weak interaction between hr - and hr - peptides. it is possible that a short peptide like hr - may not form the right conformation to bind to its target; otherwise, the missing hydrophobic interactions mediated by met and leu might reduce the binding affinity. almost no hydrophilic interaction occurs between the hr - peptide and hr - peptide in the structure. their hydrophobic interactions are mainly provided by met , ile , met , and leu ( figure b ). based on bli testing, no strong interactions were found between hr - and hr - peptides. therefore, the missing hydrogen bonds and hydrophobic residues largely reduce hr - s interactions with viral hr core. in the bli test, both hr - and hr - peptides exhibited interactions with hr - peptide. the structure also showed many strong hydrophobic and hydrophilic interactions between hr - and hr - peptides, both of which have identical residues providing hydrophobic interactions with hr - , including phe , ile , met , ile , met , and leu ( figure b) . they also share the same amino acids involved in hydrophilic interactions, including ser , asp , and glu . compared with hr - and hr - , longer peptides like hr - and hr - exhibited the optimal conformation folding and figure | interactions within the lasv -hb fusion core. (a) three hr helices are tightly packed together in the fusion core with a buried interface of Å for each hr . hydrophilic interactions are also at the end of the trimers. (b) interactions for t-loop or hr domain against hr trimer core. the buried interface for each hr domain with hr core is Å . important hydrophobic and hydrophilic residues are indicated. (c) the circular dichroism spectroscopy of hr peptides. had sufficient key residues for interaction with the hr - trimer. because of the strict restriction on the use of highly pathogenic viruses in our bsl- facilities, we were not able to get live lasv for testing the anti-lasv activity of the hr -peptides. we thus tested the potential inhibitory activity of these peptides against lasv pseudovirus entry into the target cells. unexpectedly, none of the hr -derived peptides exhibited significant inhibitory activity on the entry of the lasv pseudovirus into the target cell at the concentration as high µm (figure a) . these results suggest that the hr -peptides may not interact with the gp protein, which mediates the attachment of lasv to the target cell, the first step of viral entry, and that lasv may not get into the cell through the cytoplasm membrane fusion under neutral ph condition, the second step of entry of some class i enveloped viruses, such as hiv and mers-cov (qi et al., ; su et al., ; xia et al., ) . next, we assessed the potential inhibitory activities of these hr -derived peptides against lasv gpc protein-mediated cellcell fusion under low ph condition. at µm, peptide hr - could completely inhibit the cell-cell fusion, while peptides hr - and hr - could inhibit about - % and % cellcell fusion, respectively, and the peptides hr - and hr - showed no inhibitory activity (figure ba) . further analysis indicated that the peptides hr - and hr - inhibited cellcell fusion in a dose-dependent manner with the ic (the half maximal inhibitory concentration) values of . and . µm, respectively (figure bb) , while other hr -derived peptides had no detectable inhibitory activity. these results suggest that the longer hr peptides could inhibit cell-cell fusion because of their higher affinity to bind with the hr groove ( figure b ) and that lasv may enters into the target cell through micropinocytosis and membrane fusion under low ph condition. we solved the crystal structure of the post-fusion -hb formed by hr and hr domains of lasv gp protein and then designed several hr -derived peptides to study their binding affinities against hr peptide and inhibitory activities for viral entry. we found that the longer hr peptides, , and hr - ( -mer: - ), had higher binding affinity with hr peptide than the shorter hr peptides, , possibly owing to their rich hydrophobic and hydrophilic interactions (figure ) . moreover, the longer peptides of hr - ( -mer: - ) and hr - exhibited more obvious inhibitory activities against lasv g protein-mediated cell-cell fusion. these results suggest that the longer hr -derived peptides, which have stronger affinity against the hr domain, also have more potent inhibitory activity. on the contrary, the shorter hr peptides, hr - and hr - , do not interact with hr peptide, thus showing no cell-cell fusion inhibitory activity. in the cell-cell fusion process, the gp protein of lasv binds to its target receptor to expose the gp subunit. then, the hr domain binds to the hr domain to form the -hb fusion core to mediate the formation of membrane fusion pores. at this time, the hr domain becomes a very exposed target for binding and blocking by an hr -derived peptide. therefore, the binding affinity of hr peptides is positively related to the inhibitory activity against lasv gpc proteinmediated cell-cell fusion under low ph condition. however, all these hr -derived peptides showed weak or no inhibitory activity against lasv pseudovirus entry into the target cell. it confirms that the hr -peptides do not interact with the gp protein to block its interaction with the receptor on the target cell, the first step of viral entry. these findings also suggest that lasv enters the target cell using the fusion pathway different from that utilized by some other class i enveloped viruses, such as hiv and mers-cov (qi et al., ; su et al., ; xia et al., ) , which get into the host cells via cytoplasm membrane fusion under neutral ph condition. several groups have shown that lasv enters the host cell via clathrin-and dynamin-independent endocytosis and membrane fusion occurs at low ph (vela et al., ; rojek et al., ) . oppliger and coworkers (oppliger et al., ) have recently reported that lasv enters the target cell through macropinocytosis. since our results are consistent with those in the reports above, we proposed an infection model of lasv (figure ) . specifically, lasv enters the target cells via endocytosis, including macropinocytosis. in the membrane fusion process as revealed by the cell-cell fusion assay, the gp trimer of lasv binds to its receptor(s), e.g., α-dystroglycan, to trigger the conformational change of gp . then hr is exposed to bind with hr to form -hb, resulting in the fusion between viral envelope and endosomal membrane. under these conditions, hr -derived peptides could bind to the hr target to block viral infection. however, during the entry process of live and pseudotyped lasv, the hr domain of viral gp can only be exposed in the endosomal compartment and interacts with the hr domain of viral gp to mediate membrane fusion under low ph condition, making it impossible for hr peptides to enter the endosomal compartment to block membrane fusion there (figure ) . therefore, hr peptides must be modified, for example, by adding tat cell penetration sequence (miller et al., ) or hydrocarbon stapling motif , so that they can enter into the endosomal compartment inside the cell to interact with hr domain of the viral gp domain and inhibit the membrane fusion at low ph there. in the bli test, hr - and hr - peptides exhibited different binding affinity with hr - peptide, even though they shared the same hydrophilic and hydrophobic residues to interact with nhr fusion core in the structure (figure ) . hr - peptide figure | proposed infection model of lasv. lasv can enter the target cell via endocytosis, including macropinocytosis, and membrane fusion at low ph. hr -peptides or their analogs, if they can enter the endosomal compartment inside the host cell, can interact with the hr domain in viral gp and block the -hb formation, thereby inhibiting membrane fusion at low ph. their fusion inhibitory activity can be assessed using the lasv gpc protein-mediated cell-cell fusion assay under low ph condition ( figure b ). has several additional residues in both n-terminus (sylne) and c-terminus (rqgktpl) compared with the hr - peptide. although these residues are not involved in the nhr-chr interactions in the crystal structure, they may help stabilize the overall structure of the entire hr peptide. thus, compared with the hr - peptide, the more stable hr - peptide has higher affinity in binding hr - peptide (figure ) , as well as higher inhibitory activity in cell-cell fusion ( figure b) . therefore, the hr - peptide will be modified for further development as an anti-lasv drug candidate. the datasets generated for this study can be found in the protein data bank with accession number jgy for crystal structure of fusion core of lasv gp protein. yz, sj, and sy designed the experiments. yz, sj, and rz wrote the manuscript. xz and bc performed the protein purification and crystallization. cw, qw, and wx participated in the viral experiments. molecular diagnostics for lassa fever at irrua specialist teaching hospital, nigeria: lessons learnt from two years of laboratory operation identification of α-dystroglycan as a receptor for lymphocytic choriomeningitis virus and lassa fever virus protective effect of intranasal regimens containing peptidic middle east respiratory syndrome coronavirus fusion inhibitor against mers-cov infection characterization of lassa virus cell entry and neutralization with lassa virus pseudoparticles coot: model-building tools for molecular graphics structural basis for antibody-mediated neutralization of lassa virus an lasv gpc pseudotyped virus based reporter system enables evaluation of vaccines in 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model for rational design of hiv- fusion inhibitors with improved antiviral activity arenavirus entry occurs through a cholesterol-dependent, non-caveolar, clathrin-mediated endocytic mechanism discovery of hydrocarbon-stapled short alpha-helical peptides as promising middle east respiratory syndrome coronavirus (mers-cov) fusion inhibitors development of smallmolecule viral inhibitors targeting various stages of the life cycle of emerging and re-emerging viruses peptide-based membrane fusion inhibitors targeting hcov- e spike protein hr and hr domains a pan-coronavirus fusion inhibitor targeting the hr domain of human coronavirus spike improved pharmacological and structural properties of hiv fusion inhibitor ap over enfuvirtide: highlighting advantages of artificial peptide strategy rational improvement of gp -targeting hiv- fusion inhibitors: an innovatively designed ile-asp-leu tail with alternative conformations automated structure solution with the phenix suite key: cord- -hwy a authors: reslova, nikol; michna, veronika; kasny, martin; mikel, pavel; kralik, petr title: xmap technology: applications in detection of pathogens date: - - journal: front microbiol doi: . /fmicb. . sha: doc_id: cord_uid: hwy a xmap technology is applicable for high-throughput, multiplex and simultaneous detection of different analytes within a single complex sample. xmap multiplex assays are currently available in various nucleic acid and immunoassay formats, enabling simultaneous detection and typing of pathogenic viruses, bacteria, parasites and fungi and also antigen or antibody interception. as an open architecture platform, the xmap technology is beneficial to end users and therefore it is used in various pharmaceutical, clinical and research laboratories. the main aim of this review is to summarize the latest findings and applications in the field of pathogen detection using microsphere-based multiplex assays. high-throughput multiplex detection techniques are designed for the rapid, sensitive and specific testing of large numbers of analytes (nucleic acid assays, immunoassays, enzyme assays, or receptor-ligands) in a single biological sample. these techniques enable analysis of large numbers of samples. on the other hand, there are also classical single reaction detection methods based on determination of nucleic acids such as polymerase chain reaction (pcr) (dunbar, ; taylor et al., ) , quantitative real-time pcr (qpcr) (wuyts et al., ; iannone et al., ) , reverse transcription pcr (rt-pcr) (weis et al., ) and reverse transcription quantitative pcr (rt-qpcr) (bustin, ) , or antibody-based tests like enzyme-linked immunosorbent assays (elisa) (engvall and perlmann, ; vanweeme and schuurs, ) represent nowadays the "gold diagnostic standard" in many laboratories. despite the previous implementation of these methods for routine rapid, sensitive, specific and cost-effective molecular diagnostics, their ability to simultaneously detect multiple analytes in a single reaction is limited and this limitation has yet to be overcome. the increasing amount of proteomic, transcriptomic and genomic sequence data from a large number of organisms accessible in public databases represents an exceptional opportunity for the development of new, multiplex detection technologies. the luminex r xmap technology (x = analyte, map = multi-analyte profiling) that was invented in the late s represents such a platform that can benefit from all the advances in dna research (angeloni et al., ) . although pcr allows multiplex amplification of several targets in a single run xmap as a methodology represents a significant step forward, and was designed with the aim of creating a high-throughput bioassay platform, enabling rapid, cost-effective, and simultaneous analysis of multiple analytes within a single biological sample. as an open architecture platform, the xmap system holds many benefits for the end user and therefore it is used in pharmaceutical, clinical and research laboratories (dunbar and li, ) . the main aim of this review is to summarize the state-of-the-art of xmap technology applications in the detection of viral, bacterial, parasitical and fungal pathogens from different matrices. the principle of xmap technology is based on the concept of a liquid (suspension) array. in contrast to the conventional microarray technology where the identity of the analyte is characterized by its position on the glass slide, the xmap technology uses different sets of microspheres in a liquid suspension as determiners of analyte specificity. microsphere sets are internally dyed with two spectrally different fluorophores. the spectral signature is unique for each microsphere set and is determined by different concentrations of internal dyes, producing a -member array of spectrally distinct microsphere sets (figure ). integration of a third internal dye has allowed the expansion of up to -member microsphere sets (dunbar and li, ) . the surface of each microsphere set allows a simple chemical coupling of various reagents specific to a particular bioassay, such as nucleic acid assays, immunoassays, enzyme assays or receptor-ligand assays. a further fluorescent reporter (e.g., streptavidin-r-phycoerythrin, alexa , cy ) is coupled to a target molecule, which allows its detection after specific capture on the microsphere surface. there are different types of commercially available microspheres (table ) , and their selection is generally figure | the xmap technology based on internally dyed microspheres. different concentrations of red and infrared fluorophores were used to create distinct microsphere sets. each set is able to conjugate to a specific target molecule (yellow and orange lines = nucleic acid; green star = fluorescent reporter). determined by the type of instrumentation used for detection and the particular analyte of interest (dunbar and li, ; houser, ) . basic microspheres are . µm polystyrene beads whose surface is covered by approximately carboxyl groups (cooh) for covalent coupling of capture reagents (tang and stratton, ) . magnetic microspheres (figure ) differ in size and structure through the addition of a magnetite layer (dunbar and li, ; houser, ) . usage of magnetic beads improves washing efficiency as the magnetic separation step enables the elimination of unwanted sample constituents. moreover, magplex-tag microspheres are covalently pre-coupled with unique base pair-(bp)-long anti-tag oligonucleotides that serve as an anchor for target sequences containing the complementary tag sequence. this proprietary tag system (xtag technology) is optimized to have minimum cross-reactivity. an assay can be easily designed by adding a complementary tag sequence into the sequence of the primer or detection probe of interest and hybridization to the anti-tag sequence on the microsphere surface. the analysis of beads is in general performed by two lasers. the red classification laser/led ( nm) excites the inner fluorescent dyes of the microspheres, thus identifying a specific microsphere set according to its spectral signature. if the analyte of interest is present, the green reporter laser/led ( - nm) recognizes the fluorescent reporter bound to the captured analyte on the microsphere surface. there are approximately microspheres from each set present in a single sample. this number represents the range in xmap, in which it is possible to perform determination of quantity according to a calibration curve, similarly to qpcr. however, one must bear in mind that inclusion of a pcr amplification step prior to xmap analysis does not reveal the real number of dna molecules present in the original sample, but can only be used for the approximate estimation of dna quantity. therefore, xmap can provide only semi-quantitative data. the simultaneous reading of both spectra is performed in purpose-designed readers ( table ) . they differ by their mechanisms of fluorescence capture and by the maximum number of samples that can be analyzed. the basic detection instrument, which is called magpix, is compatible only with magnetic microspheres (magplex and magplex-tag). the principle of microsphere analysis in the magpix instrument is based on their immobilization in the monolayer on the magnetic surface (figure ) . unlike flowbased instruments, the fluorescent imager of the magpix system reads all the microspheres at once, while generating data that is comparable with other methods. reading a -well-plate takes about min. the maximal reading capacity of magpix instruments is limited to bead sets. advanced detection instruments -the luminex / (bio-plex ) and flexmap (bio-plex) d -are based on flow cytometry principles. the microspheres with bound analyte are focused into a rapidly flowing fluid stream. each microsphere is then individually detected and digitally processed as the stream passes through the imaging cuvette. flow cytometrybased platforms are convenient for applications with samples of limited size. the reading of a -well-plate is faster than in the magpix system and takes min or less. the capacity of the d platform is further increased by the possibility of analyzing -well plates. the microsphere-based technology can be applied in various assay formats, which can be divided, according to the type of analyte, into microsphere-based multiplex nucleic acid assay formats (mbmna) and microsphere-based multiplex immunoassays (mbmi). in general, xmap-based assay formats are in comparison to other commonly used methods very open and flexible, ensuring the result data within few hours, while requiring only minimal amounts of sample. detection assays based on nucleic acids have a potential for high levels of multiplexing, approaching the levels of sensitivity achieved by target amplification methods like multiplex pcr or taqman chemistry assays, while using the same protocols of dna/rna extraction. multiplex oligonucleotide ligation pcr assay format (mol-pcr) is able to simultaneously perform detection and identification, strain typing, detect antibiotic resistance determination, virulence prediction, etc., thereby surpasses other methods like multiplex ligation-dependent probe amplification (mlpa) or qpcr. the disadvantage of technology is that it is not capable to perform quantitative analysis like qpcr, because providing only semi-quantitative data. xmap immunoassays surpass the common enzyme immunoassays in the ability of multiple simultaneous detection, while requiring smaller amount of sample and lower cost. moreover, these assay formats produce superior dynamic range and sensitivity. xmap technology is applicable in numerous nucleic acid assay formats such as, e.g., gene expression analysis, microrna analysis, single nucleotide polymorphism (snp) analysis or specific sequence detection. basically, nucleic acid assays can be developed by coupling sequence-specific capture oligos to magnetic microspheres or by use of xtag technology (angeloni et al., ) . when performing xmap analysis of nucleic acids it is essential to include pcr amplification to enrich the number of targets in the sample to detectable levels. there are two general strategies for including a pcr step in the detection of pathogens using xmap technology. the main difference between the two lies in which phase the pcr amplification is applied. in direct dna hybridization (ddh), allele-specific primer extension (aspe), single base chain extension (sbce), and oligonucleotide ligation assay (ola) all the target dna sequences are amplified in multiplex pcr prior to hybridization to microspheres. the disadvantage of these methods is that in assays containing large amounts of targets multiplex pcr leads to amplification bias, which is caused by the different lengths of the amplicons (nolan et al., ) . in contrast, in the multiplex oligonucleotide ligation pcr assay (mol-pcr) sequence discrimination by detection probes occurs before the amplification step, which can subsequently be run just in singleplex pcr with universal primers. direct dna hybridization is one of the basic approaches used for the selective identification of sequences of interest from heterogeneous mixtures of dnas (figure ) . it is often used, e.g., for identification of species (defoort et al., ; page and kurtzman, ; righter et al., ; liu y. et al., ) or genotyping of pathogens (letant et al., ; zubach et al., ) . in ddh, the amplification of target sequences is ensured by specific primer pairs, and one primer from each pair is fluorescently labeled at the end, permitting detection of the amplicon (christopher-hennings et al., ) . the subsequent incubation of amplicon with microspheres leads to a direct and specific hybridization between matching capture and target sequences. amplicon sequences should be - bp in length figure | direct dna hybridization (ddh, yellow lines = capture oligonucleotide; orange line = amplified target sequence; green star = fluorescent reporter). target dna sequence is amplified, while one of the primers is fluorescently labeled. amplicons are then specifically hybridized (according to complementarity) to capture oligonucleotides on the microsphere surface. to minimize steric hindrance during hybridization and the capture sequence on microspheres should be - bp in size (dunbar, ) . the specificity of the capture sequences and stringency of hybridization conditions allow discrimination up to snp. if the snp or mutation discrimination is intended, the presumed mismatch should be located at the center of the capture sequence (livshits and mirzabekov, ) . this assay format then requires a unique capture sequence coupled to a specific microsphere set to score each snp allele (kellar and iannone, ) . allele-specific primer extension ( figure ) is an approach usually used for determination of allelic variants of pathogens (page and allele-specific detection probes, differing in one nucleotide on the polymorphic side, hybridize to amplified target sequence. after addition of dna polymerase and dntps (one of which is fluorescently labeled), molecules are extended according to complementarity. products are captured by anti-tags on the specific microsphere set. kurtzman, ; lin et al., ) . the defining characteristic of aspe is the extension of two allele-specific detection probes, which contain a polymorphic site at the end, defining the particular allele variant. in this arrangement, dna polymerase can extend detection probes by incorporation of dntps (one nucleotide is labeled, e.g., biotin-dctp), if the allele is present in the sample. just one probe is extended in the case of a homozygous target; conversely, in heterozygotes both probes are extended. the fluorescence signal is generated by a fluorophore bound to labeled dntps, incorporated within the extended probe. the use and assay format of sbce is similar to the previously described aspe. however, there are slight differences, mainly in the design of detection probes. in the case of sbce (figure ) , probe sequences are terminated one base before the polymorphic site ye et al., ) . due to this design the labeled dideoxyribonucleoside triphosphate (ddntp) terminators serve as a "query" nucleotide and are used for single base probe extension at the same time; this assay requires the setting up of separate reactions for each of the four ddntps (ddc, ddg, dda, and ddt). moreover, pcr products from the previous step of pcr amplification of the target sequence need to be treated with exonuclease i and shrimp alkaline phosphatase (exoi/sap) before use as a template in the sbce reaction (ye et al., ; dunbar, ) to get rid of unincorporated primers and dntps. although sbce has been proven to be highly specific and reliable (chen et al., ; syvanen, ) , it is in the process of being replaced by less laborious methods. oligonucleotide ligation-based formats include a ligation step of two oligonucleotide detection probes, which occurs in the presence of a target sequence of a specific pathogen. these assays are based on the ability of detection probes to hybridize next to each other on a complementary target dna sequence (landegren et al., ) . if there are no mismatches near the junction site and there is a phosphate group at the end of a second probe (necessary for phosphodiester bond formation), annealing occurs; dna ligase then recognizes the nick and forms a covalent bond between adjoining nucleotides while creating a single-stranded dna molecule . the most crucial step during the multiplexing of different ligation assays is the design of suitable probes with similar melting temperatures of between and • c (dunbar, ) . in the ola assay format, the target dna sequence is pcramplified prior to the ligation step of the annealed probes (figure ) . ola is suitable for snp genotyping taylor et al., ; ye et al., ) . the multiplex oligonucleotide ligation pcr assay represents an improved version of the previous ola assay format. one advantage is that ligation is carried out prior to the pcramplification (figure ) (nolan and white, ) . unlike in the ola assay, one of the detection probes consists of a sequence complementary to the target sequence and an extension composed of the tag sequence and primer binding site. the figure | principle of single base chain extension (sbce) (red dot = dideoxynucleotide; green star = fluorescent reporter; red line = anti-tag). specific detection probes are terminated one base before the polymorphic site. utilization of fluorescently labeled dideoxynucleotides necessitates a separate reaction for each nucleotide in focus (minimally two). target dna hybridizes with probes after amplification but only the mix with the proper ddntp leads ultimately to the synthesis of a labeled product, which is captured by anti-tag on the microsphere surface. second probe is the same as the first except for the absence of the tag sequence. each probe pair is specific for a particular target sequence, but all pairs share the same primer sequence. basically, these modular detection probes anneal to a target sequence, ligate into a complex single-stranded dna molecule and only if this occurs does the molecule become a template for singleplex pcr using a universal pair of primers (one is fluorescently figure | principle of oligonucleotide ligation assay (ola) (green star = fluorescent reporter; pho = phosphate group; red line = anti-tag). the target dna sequence is pcr-amplified prior to the ligation step of the annealed probes. one of the detection probes consists of a sequence complementary to the target sequence (polymorphic site at the end if snp identification is needed) and also an additional tag tail sequence. the second detection probe is fully complementary to the target sequence and serves as a reporter due to its fluorescent label at the end. detection probes bind next to each other, dna ligase recognizes the nick and makes a bond. the product is captured by anti-tag on the microsphere surface. labeled). additionally, all the ligation products are very similar in length (approximately bp - bp), so the use of a universal primer pair during pcr makes the simultaneous amplification figure | principle of multiplex oligonucleotide ligation pcr assay (mol-pcr) (orange line = detection probe ; green line = detection probe ; blue lines = universal pcr primers; burgundy line = amplified negative strand; green star = fluorescent reporter; pho = phosphate group; red line = anti-tag). specific detection probes bind next to each other to target sequence via complementary parts, while the parts including the tag sequence and binding sites for pcr primers form tails sticking out into space. dna ligase recognizes the nick and makes a bond. the complex sequence of ligated probes becomes a template for singleplex pcr with universal primers; one of the primers is fluorescently labeled. labeled amplicon hybridizes via its tag sequence to capture anti-tag on the microsphere. of many short fragments highly feasible. all these facts ensure that mol-pcr is not susceptible to the amplification bias that is characteristic of multiplex pcr or previously mentioned formats. only a minimal amount of target/sample is required. the mol-pcr upgrade has the potential to have widespread impact on genomic assays, because not only is sequence detection and snp identification possible, but the detection of indels (insertion/deletion), screening tests for pathogens (virus, bacteria, fungi) from various matrices or determination of antibiotic resistances is also feasible thierry et al., ; wuyts et al., ) . mol-pcr could replace, e.g., mlpa or qpcr in certain applications in routine diagnostics . microsphere-based multiplex immunoassay (mbmis) are typically biochemical tests that allow the detection or measuring of the concentration of an analyte (protein) in a solution through the use of an antibody or immunoglobulin (angeloni et al., ) . single-analyte elisa cannot support simultaneous detection of multiple specific antibody responses within a single serum sample (bokken et al., ) , and has further disadvantages, such as the requirement for a relatively large amount of sample, negligible non-specific binding or increased background. mbmis represent an alternative for commonly used indirect tests like elisa. the conversion of an elisa assay to the mbmi format is uncomplicated, efficient, cost-saving and produces an assay with superior dynamic range and sensitivity (baker et al., ) . mbmis are often used in the diagnostics of various pathogens including multicellular organisms, such as e.g., parasites, in tests where the current methods are not sensitive enough. the methods of choice are usually capture sandwich (cs) and indirect serological assay (isa) (figure ) . however, the problems typical for methods based on serology remain: the need for periodical testing in order to avoid false negative results resulting from a wide and inevitable lag between infection and development of a specific response against a parasite in the form of igg antibodies (sero-positivity) (nockler et al., ) . the cs assay utilizes microspheres covalently coupled with a capture antibody (polyclonal antibodies should be purified and mono-specific) that takes up target molecules from the sample. this complex is recognized by a labeled detection antibody (baker et al., ; angeloni et al., ) . the cs format can be used in cases where, for example, confirmation of pathogen identity within the inflammatory focus or altered tissue is needed. in contrast to cs, in isa a specific antibody against an antigen coupled with a microsphere is captured. if the binding of serum antibody to antigen occurs, a labeled secondary antibody (antiantibody) then provides the visualization. isa is typically used for serological screenings (monitoring and prevention purposes) that are carried out on serum samples (van der wal et al., ) . the xmap technology is used in many different applications. this chapter describes the use of this technology for multiplex detection of viral, bacterial, parasitical and fungal agents using the microsphere-based multiplex nucleic acid-assay formats (mbmna) and microsphere-based multiplex immuno-assay formats (mbmi) described above. viruses are a very diverse group of infectious agents and are divided into groups according to a number of properties, e.g., type of nucleic acid, the presence of the viral envelope, antigenic structure, mode of transmission, pathogenicity, etc. they can be classified also according to the syndromes which they cause and mode of transmission, e.g., respiratory viruses, viruses causing gastroenteritis, tumors, hepatitis, rashes or neuroviruses. to date, the majority of applications that enable multiplex viral detection and identification are based on the capture of viral nucleic acid by adoption of various ddh modifications. respiratory viruses are causative agents of the most common diseases of the human upper and lower respiratory tract, which are often associated with significant patient morbidity and mortality (berry et al., ) , e.g., h n subtype of highly pathogenic influenza a virus (neumann et al., ) . the mbmna method for more effective detection and genotyping of h n viral isolates from clinical samples comprising pharyngeal swabs and tracheal aspirates was developed and its efficiency was compared with rt-pcr and qpcr (zou et al., ) . the results using the mbmna approach showed that this assay is vulnerable to viral mutations although the primers were designed according to conserved sequences. therefore, there is a need to monitor viral mutations in order to reduce false-negative results and add new primers and probes to adapt to the mutations, which is a disadvantage of mbmna. on the other hand, mbmna holds a number of advantages compared to rt-qpcr and qpcr, e.g., allele-specific probes with tag sequences can be recognized by a universal set of primers, thus potentially eliminating the problem with different primer sets (which may be incompatible) used in conventional methods. moreover, amplification is carried out with a single set of universal primers where only one primer is labeled; therefore, the background is low and no post-pcr cleanup is required. another application of the mbmna method was developed for the identification of human adenoviruses (hadvs). conventional serological identification of hadvs serotypes is a time consuming process. target-specific extension (tse), which is a variant of aspe was suggested to accelerate identification through the use of mbmna for simultaneous identification of different serotypes; this is not possible using commercially available neutralization tests, antibody studies, or antigen detection by immunofluorescence or conventional pcr (washington et al., ) . universal primers were used for nonspecific pcr amplification and serotype-specific probes coupled to tags were used for tse. this mbmna procedure is methodically simple, the cost is relatively low, and it enables diagnosis of up to five hadv serotypes in a single reaction. besides the in-house assays described above, commercial kits have also been developed for the detection of respiratory viruses by xmap, e.g., xtag r respiratory viral panel (xtag rvp) (krunic et al., ) . xtag rvp is multiplex nucleic acid test designed for detection of multiple respiratory virus nucleic acids in human nasopharyngeal swabs (selvaraju and selvarangan, ; smith et al., ) . qualitative detection of a panel including respiratory syncytial virus (rsv), influenza a virus (influenza a matrix, h subtype, h subtype, h subtype), influenza b (parainfluenza , , , and ), metapneumovirus (hmpv), hadv, entero-rhinovirus, corona nl , corona hku , corona e, corona oc , and bocavirus is possible. bacteriophage ms and bacteriophage λ dna were used as the internal controls. the detection of respiratory virus targets using the xtag rvp, which detects respiratory viral targets, was compared with individual qpcr nucleic acid amplification tests (nats) (pabbaraju et al., ) . the xtag rvp can detect all the respiratory viral targets included in the in-house nat panel, which is used for detection of influenza a, b viruses (ifva, ifvb), parainfluenza virus types to (piv - ), rsv, hmpv, and respiratory adenovirus types (adv). of the , samples tested, were positive by xtag rvp and by in-house nats for these targets. this gives the xtag rvp a sensitivity of . % and a specificity of . %; in addition, xtag rvp can detect picornaviruses (the in-house assays did not detect picornaviruses) and coronaviruses and can subtype ifva positives simultaneously. the xtag rvp includes all the respiratory viral targets that are tested routinely for the diagnosis of acute respiratory tract infections; further, the technology is flexible and can easily allow for incorporation of other targets (e.g., human bocavirus) in the future. the xtag rvp assay was subsequently modified and was marketed as the xtag rvp fast assay, which has a simpler protocol; the results are obtained in a shorter time and handling of the amplified product is not required (amplified dna is mixed with tag primers specific to each viral target), which could be a potential contamination risk (pabbaraju et al., ) . the respiratory samples were tested for a variety of respiratory viral targets by xtag rvp and xtag rvp fast in parallel. the xtag rvp was more sensitive than xtag rvp fast ( . % versus . %) for all the viral targets; in addition, some targets (influenza b virus, parainfluenza virus type , and human coronavirus e) were not detected using xtag rvp fast and, e.g., the sensitivity for detection of ifvb was very low ( . %). therefore, it is not suitable as the primary assay for the detection of ifvb. in addition to respiratory viral diseases the mbmna was successfully applied also for detection of viral pathogens causing acute viral gastroenteritis. acute viral gastroenteritis is usually caused by four distinct families of viruses: rotaviruses, noroviruses, astroviruses, and adenoviruses (liu y. et al., ) . the authors focused on simultaneous detection of rotavirus a (rva), noroviruses (novs), sapoviruses (sav), human astrovirus (hastv), enteric adenoviruses (eads) and human bocavirus (hbov ). altogether fecal samples were tested using the mbmna and rt-pcr in parallel. the specificity of mbmna was equal to the conventional rt-pcr (> %), but mbmna was faster in terms of detection of different viral pathogens in one tube (liu y. et al., ) . the studies of (hamza et al., ) were also directed to the detection of human enteric viruses (human adenovirus (hadv), human polyomavirus (hpyv), enterovirus (ev), rotavirus (rov), norovirus gi (novgi), and norovirus gii (novgii), but environmental water samples were tested (hamza et al., ) . mbmna provided high specificity and no cross-reactivity, but was not as sensitive as qpcr for the identification of viral contamination in river water samples. in contrast, all wastewater samples that were positive in qpcr were also positive by the mbmna and the detection limit was higher than qpcr; mbmna was as sensitive as qpcr for viral detection in wastewater samples. therefore, mbmna could be a reliable method for the simultaneous detection of viral pathogens, but only in wastewater. for detection of gastrointestinal pathogens xtag r gastrointestinal pathogen panel -gpp is commercially available [multiplex detection of various viral, bacterial and parasitic nucleic acids in human stool samples (beckmann et al., ; perry et al., ; wessels et al., ; zboromyrska et al., ) ]. in comparison to the two previous studies mentioned above only three enteric viruses (norovirus, rotavirus and adenovirus / ) can be identified by the gpp (see chapter . ). viruses such as human papillomaviruses (hpv) are also associated with oncogenesis. hpv belong to those viruses, which require simultaneous detection and typing to identify individual hpv types because the genotype determination is necessary for the investigation of epidemiology and behavior of individual hpv types. therefore, ddh was designed for detection and genotyping of hpv using l consensus (primer systems, which can detect to molecules of hpv targets) resulting in the establishment of a method for simultaneous detection of different hpv genotypes including high-risk hpv and lowrisk hpv genotypes (jiang et al., ) . subsequent analysis of the data showed that the -plex method precisely discriminated all high-risk hpv targets and also low-risk hpv targets. another study focused on genotyping hpv also used specific probes targeting a region of the l gene (zubach et al., ) . ddh was optimized for the detection and genotyping of mucosal hpv types, which are associated with infections of the genital, anal, and oropharyngeal mucosae and the method enables a more comprehensive coverage of hpv types compared with the previously mentioned study, where only types of hpv were genotyped. the ddh was more sensitive than the linear array (a leading commercial genotyping method) in terms of distinguishing positive/negative hpv samples, but less sensitive for detection of multiple hpv types; another limitation was the inability of the pcr system to amplify certain variants of hpv . hpv genotype detection was by combined whole genome amplification and xmap technology showed that this method is highly specific and sensitive (lowe et al., ) . this approach is capable to identify all high risk hpv types with the analytical limit of detection copies plasmid dna. many viruses can cause infections with fatal consequences for human health, e.g., hendra and nipah viruses, which can infect cells of the central nervous system and may cause relapsing encephalitis (clayton et al., ) , ebola virus, which causes lethal hemorrhagic disease in humans (takada and kawaoka, ) or menangle virus, which causes an influenzalike illness with a rash in humans (bowden et al., ) ; these zoonotic viruses are linked to bats. the surveillance of zoonotic viruses in wildlife populations is necessary in order to monitor the risk of emerging infectious disease outbreaks. for the complex detection and genotyping of paramyxoviruses in australian bats two bat virus panel assays (bvpa) for detection of paramyxoviruses in australian bats (bvpa- ) and for paramyxoviruses and filoviruses in non-australian bats (bvpa- ) were introduced (boyd et al., ) . examined rna was extracted from the urine of bats and a total of samples were tested in -plex bvpa- and field -plex bvpa- ; both developed assays were proven to be reliable and accurate. a number of pathogens, including viruses, are implicated in reproductive diseases of swine. (chen et al., ) combined onestep asymmetric multiplex reverse transcription pcr (rt-pcr) with ddh for simultaneous detection of respiratory syndrome virus (prrsv), porcine circovirus type (pcv- ), porcine pseudorabies virus (prv), classical swine fever virus (csfv), and porcine parvovirus (ppv). all strains of these five viruses were accurately identified. the results showed that the combination of rt-pcr with the ddh assay is more accurate and specific than the other methods, e.g., conventional rt-pcr, and could be a useful tool in the diagnostics of swine diseases. mbmnas could become very important for veterinary diagnostic testing and (christopher-hennings et al., ) reported the potential use of mbmnas for detection of different pathogens in pigs using panels for the multiplex detection of swine pathogens (viruses and bacteria) in serum, lung, oral fluids, feces and spleen or liver. although direct diagnosis based on the detection of the nucleic acids of viral pathogens described above prevails, xmap antibody-based tests for the detection and typing of viruses are also available. mbmi was used to develop a competitive immunoassay that measures hpv type , , , and specific neutralizing antibodies (opalka et al., ) ; this was later validated for use in epidemiology studies and clinical vaccine trials (opalka et al., ; dias et al., ) . mbmi was also compared with a western blot assay for the detection of hivspecific antibodies (kong et al., ) . the microspheres were coupled with anti-p monoclonal antibody and with hiv antigens: gp , p , p , p , and p recombinant protein. the results of both methods showed that mbmi sensitivity was . % and western blot assay sensitivity was . %. the mbmi was more efficient and precise for screening several parameters and based on the acquired results it was better in hiv diagnostics than western blots. for the determination of antibodies against hcv in patient serum samples mbmi based on the antigenic properties of four recombinant proteins was designed . only a small number of samples was tested and that is why the specificity and sensitivity were %, but in spite of this the mbmi has the potential to become a viable alternative to standard tests due to its excellent specificity and it may be used for screening of hcv infection. detection of antibodies against several epstein-barr virus (ebv) antigens in nasopharyngeal carcinoma patients (npc) showed the possibility of simultaneous detection of multiple markers using mbmi, which is not possible with elisa, and because of the distinct ebv serology spectrum in individual npc patients, the multiplexed microsphere assay has powerful potential to allow serological diagnosis of npc in the future (gu et al., ) . mbmi showed increased sensitivity and the possibility of quantifying antibodies, antigens, as well as other substances (e.g., hormones, cytokines, tumor markers, etc.), in contrast to conventional elisa tests (dupont et al., ) . the majority of applications for multiplex bacterial diagnostics are based on the detection of dna. the most widely used approaches are based on the ddh, ligation assays or aspe, but multiplex detection of bacteria may be performed as well using mbmi. direct dna hybridization was used for the detection of pathogens causing foodborne diseases such as acute gastroenteritis and diarrhea, which are usually associated with ingestion of contaminated food. ddh was applied for the typing of salmonella isolates using the genes encoding the flagellar antigens h (flic and fljb) (mcquiston et al., ) . allele-specific probes for fifteen h antigens, complex major antigens and complex secondary antigens according to the kauffmann-white serotyping scheme were designed. comparison of ddh with traditional serotyping methods revealed that the ddh cannot completely replace these methods because unfortunately not all flagellar antigen types were detected. a similar ddh assay for the typing of salmonella focused only on the most common six serogroups of salmonella in the united states (b, c , c , d, e, and o ), as well as serotype paratyphi a, using the rfb genes required for o-antigen biosynthesis in salmonella (fitzgerald et al., ) . in contrast with the previous study of mcquiston et al. ( ) , the authors showed that the ddh was more specific than traditionally used methods for typing of salmonella. in the previous sections, it was described how ddh can be used for typing of pathogens; however, in most cases ddh is used only for the detection of pathogens, as described below. attempted simultaneous detection of the enteric pathogens aeromonas, campylobacter jejuni/coli, shigella, enteroinvasive escherichia coli (eiec), vibrio, yersinia and as well as salmonella in fecal samples. however, there were some limitations to the method, which included the limited number of clinically significant pathogens or the inability to detect diarrheagenic e. coli, protozoa, or viruses. the full capacity of the ddh assay was utilized when the panel was expanded to include the most common bacterial/viral enteropathogens found in stool samples, such as salmonella, shigella, vibrio, toxin b producer clostridium difficile, campylobacter, clostridium perfringens, yersinia enterocolitica, aeromonas, escherichia coli o :h , verocytotoxin-producing escherichia coli and adenovirus, group a rotavirus, norovirus gi and gii and astrovirus (onori et al., ) . the results showed that the assay is rapid, sensitive, specific, and reliable for screening and for exploring the etiology of gastrointestinal infections. the sensitivity of mbmna was demonstrated to be greater than the routine methods ( . % versus . %), with the exception of salmonella sp. and toxigenic c. difficile where the adoption of multiplex pcr did not always result in a significant improvement of specificity. the causative agents were not found in of ( %) of the presumed infectious gastroenteritis cases, but this could be due to the limitations of the detection panel, which did not include allelespecific probes for detection of parasitic enteric pathogens or emerging viruses related to gastroenteritis. also, using ddh, detection of pathogenic bacteria occurring in environmental samples and causing acute and often fatal diseases (bacillus anthracis, yersinia pestis, francisella tularensis, and brucella melitensis) was optimized in a multiplexed format to allow the maximum sensitivity and specificity (wilson et al., ) . dna was extracted robotically and in combination with ddh a rapid reliable screening approach was developed. detection limits were from fg to pg starting dna concentration when primer sets were multiplexed; in some cases the limits of detection were higher when primer sets were tested separately (range from fg to pg). besides the in-house assays developed for multiplex detection of bacteria described above, there are also commercial solutions based on xmap technology for detection of the most common gastrointestinal pathogens and toxins. the xtag r gastrointestinal pathogen panel is a multiplex nucleic acid test designed for detection of various bacterial, viral and parasitic nucleic acids in human stool samples (beckmann et al., ; perry et al., ; wessels et al., ; zboromyrska et al., ) . the panel allows qualitative detection of campylobacter sp., clostridium difficile (toxin a/b), escherichia coli o , enterotoxigenic e. coli (etec) lt/st, shiga-like toxin producing e. coli (stec) stx /stx , salmonella sp., shigella sp., vibrio cholerae, yersinia enterocolitica, hadv serotypes and , nov gi and gii, rotavirus a, giardia, cryptosporidium and entamoeba histolytica. the xtag gpp was tested and compared with routine tests, which are used in clinical diagnostic laboratories for screening of kinds of enteropathogens, e.g., qrt-pcr kit for detection of viruses, culture methods for detection of bacteria or microscopic examination for detection of parasites (deng et al., ) . samples with discordant results between the routine tests and xtag gpp were tested by singleplex pcr and sequencing. the overall sensitivity of xtag gpp was . % and specificity was . %. the sensitivity of xtag gpp was % for all enteropathogens except salmonella sp. ( . %) and c. difficile toxin b ( . %). the specificity was % for all targets except salmonella sp. ( . %), shigella sp. ( . %), c. difficile toxin b ( . %), and norovirus gii ( . %). xtag gpp is also capable of detecting coinfections; coinfections were detected using xtag gpp, which is more than by the routine tests. however, the authors also reported some disadvantages as xtag gpp failed to detect some important diarrheal pathogens (aeromonas, plesiomonas shigelloides) often detected by routine diagnostic tests; further, the detection of salmonella exhibited low sensitivity ( . %). ligation assays are also often used for multiplex detection of pathogenic bacteria. the main advantage over direct hybridization methods is the ability to simultaneously detect diverse signatures such as unique sequences, snps, indels and repeats (song et al., ) . mol-pcr was initially optimized for the detection of the biothreat agents bacillus anthracis, yersinia pestis, and francisella tularensis . the pathogen-specific sets of moligo pair probes were designed and their specificity and sensitivity were tested using similar species of bacillus anthracis, yersinia pestis, and francisella tularensis and dilutions of isolated dna, respectively. moligo pairs, which showed the highest specificity and sensitivity, were selected for compilation of a final probe panel, which was validated on extracted dna from infected rodent liver and spleen, human blood or pleural fluid spiked with pathogen dna. nine from unknown samples were successfully identified using the final probe panel. the results also showed the ability of this method to simultaneously detect multiple different signatures (snps, indels and repeats). the versatility of mol-pcr was utilized when simultaneous detection of bacillus anthracis, yersinia pestis, and francisella tularensis was supplemented by characterization of antibiotic resistance (ciprofloxacin and doxycycline) of these bacteria based on snp analysis (song et al., ) . the allelespecific probes for detection and characterization of all the known resistance determinants performed well when tested individually, but multiplex use did not provide satisfactory results. due to the ability to simultaneously detect diverse signatures such as unique sequences, snps, indels, and repeats, mol-pcr can be used as a genotyping method as described below. a mol-pcr-based plex snp typing method for mycobacterium tuberculosis complex (mtbc) based on two phylogenetically equivalent sets of snp markers that are specific for the six main human-associated lineages of mtbc was introduced (stucki et al., ) . mol-pcr was compared with taqman qpcr and the obtained results showed that the sensitivity and specificity of both methods were similar (specificity %, sensitivity . % mol-pcr, . % taqman) and that both methods were of comparative cost. mol-pcr was ideal for classification of unknown isolates, while taqman qpcr was faster for confirmation of unknown isolates. mol-pcr was also successfully used for genotyping of bacillus anthracis in a -plex assay to score phylogenetically lineagespecific canonical snps within the genome of bacillus anthracis (thierry et al., ) . allele-specific primer extension was applied for identification of bacteria (lin et al., ) even though it is more commonly used for snp genotyping. aspe was used for the identification of acinetobacter sp. and antimicrobial susceptibilities of the clinical acinetobacter species isolates were also determined (lin et al., ) . the s- s rrna gene intergenic spacer (its) regions of distinct acinetobacter species were amplified and then multiplex aspe was performed. it was shown that this multiplex identification of acinetobacter sp. is applicable also for determination of antibiotic resistance of the clinical acinetobacter isolates. aspe was compared with sbce for identification of bacterial samples (ye et al., ) and both methods provided similar results as they managed to correctly classify bacterial species into groups. in addition to mbmna also mbmi can be used for the direct multiplex detection of bacteria and their products (dunbar et al., ) . in mbmi direct fluorescence (detection antibody that incorporates a fluorescent label) is used for detection of reaction or of emerging product in contrast to elisa and, in addition, mbmi enables measurement of multiple analytes simultaneously. for this reason, mbmi is preferred because time for detection is reduced and also test sensitivity is increased (jun et al., ) . capture sandwich immunoassays (cs) were successfully applied for detection of organism-specific antibodies using microspheres coupled with antibodies for salmonella, campylobacter, escherichia coli, and listeria and it has been demonstrated that mbmi is a suitable method for multiplex detection of bacteria occurring in foodstuffs or for detection of brucella sp. from milk using capturesensitive monoclonal antibodies for the lipopolysaccharide (lps) o-antigen of brucella sp. (silbereisen et al., ) . mbmi was also applied to test bacterial contamination of foods through the detection of staphylococcal enterotoxin b (seb) , staphylococcal toxin a (sea), and toxic shock syndrome toxin (tsst) produced by various strains of staphylococcus aureus (simonova et al., ) using sandwich immunoassays in which microspheres were conjugated with specific antibodies. a similar approach was used for the detection of pneumococcal serotype-specific polysaccharide and c-polysaccharide (c-ps) antigens from urine samples (sheppard et al., ) . for the detection, mbmi was combined with the binax now streptococcus pneumoniae antigen detection kit. the specificity of mbmi was determined by testing serotypes of s. pneumoniae and other strains of streptococci; of the non-pneumococcal serotypes gave c-p positive results, which showed that mbmi could be used for diagnosis of infection caused by s. pneumoniae only in combination with the binax now assay. parasitic zoonoses are recorded worldwide and some of them have endemic character. parasitic agents may pass from animals to humans in several ways, e.g., by direct contact, vector, consumption of raw or undercooked foodstuffs containing the infective stages or by infective stages released into environment (hubalek, ) . in the context of animal health and human food consumption, a list of the top ten parasites has been defined by the un's food and agriculture organization (fao) and world health organization (who) ( table ) . although in the last decades a number of novel diagnostic methodological approaches has been developed, the current diagnosis of some parasitic diseases is still based only on a combination of clinical signs, anamnesis, and direct visual identification of parasitological objects (anderson et al., ) . the most common conventional diagnostic methods, such as microscopic examination, biochemical assays or elisa, are available, but they are laborious, time-consuming and in many cases not reliable (navidad et al., ) . improvements in this field are represented by molecular methods, including also routine pcr diagnostics, increasingly used for detection mainly of intestinal parasites, which are easy to recover from fecal specimens (taniuchi et al., ) or potentially useful for other parasites found in secretions. with regard to the fact that parasites might exhibit very strictly confined localization within the host's bodyintracellular/extracellular or tissue/organ, sampling can be very problematic and it often leads to a false negative results. outbreaks of diarrheal diseases are caused by a wide range of pathogens, including parasites. stool microscopy (detection of eggs, parts of bodies etc.) is the gold standard in the diagnostics of intestinal parasites. however, the presence of parasites in stool may vary and could be naturally low, requiring multiple sampling. in fact, up to % of all cases of diarrhea remain without confirmed etiology (vernacchio et al., ) . therefore, there is space for the development of more sensitive diagnostic assays (taniuchi et al., ) , which should provide more precise determination. among the modern molecular diagnostic methods qpcr assays are most frequently used for determination of intestinal parasites. in areas where co-infections are common (up to % of cases are caused by two or more pathogens) (jansen et al., ; friesema et al., ) , the application of multiplex assays is of great benefit. several pioneering works have been published in relation to this topic. to date, in parasitology, improved multiplex qpcr assays were adapted to ddh, which enables parallel diagnosis of seven intestinal parasites (taniuchi et al., ) ; separate reactions were optimized - plex for protozoa (cryptosporidium sp., giardia intestinalis, and entamoeba histolytica) and -plex for helminths (ancylostoma duodenale, ascaris lumbricoides, necator americanus, and strongyloides stercoralis). the final calculated sensitivity was % and specificity was %. the results of both ddh assays were equivalent or better in comparison to the parent multiplex qpcr. moreover, this approach has been developed as a commercial diagnostic xtag gpp tool-a -plex assay, which enables inter alia detection of the protozoa g. intestinalis, e. histolytica and cryptosporidium sp. the overall performance of xtag gpp compared with conventional methods (standard culture, microscopic examination, immunochromatographic tests, qpcr) showed a sensitivity of . % (range to %) and a specificity of % (range , % to , %) (claas et al., ; mengelle et al., ; navidad et al., ) . if multiplexing more than targets, the limit of detection might be reduced for individual targets when compared to single-target detection (navidad et al., ) . however, the identification of multiple pathogens revealed that very often (in up to % of samples), the physicians do not request testing for the proper pathogen (claas et al., ) . therefore, multiplexing refines the diagnosis and contributes to the selection of a suitable treatment. it was mentioned above that microsphere-based assays can be arranged also as multiplex indirect immunoassays, although the conventional singleplex elisa still represents the gold standard in serodiagnostics for screening of individual human/animal or higher numbers of samples at a population level (ruitenberg et al., ; nockler et al., ; dubey et al., ) . recently, some studies have been done in order to improve the potential of this serological method and to upgrade it to the multiplex level. these studies are mostly focused on parasites with the ability to migrate through the tissues of the host's body -where pcr based detection would not be reliable. in this context, the most studied group of parasites are representatives from the phylum nematoda, including also the important human pathogens, the trichinella sp. the larvae may infect humans during the ingestion of raw or undercooked meat, mainly pork (domestic pig, wild boar) and can induce disease, whose consequences can be fatal (dupouy-camet, ; pozio and murrell, ) . inspection of meat for the most important species, trichinella spiralis, is mandatory at slaughter (anonymous, ) , but currently used methods like artificial digestion and microscopic examination of pooled meat samples (nockler et al., ) are archaic and usually do not properly reflect the real infection. therefore, serodiagnostic methods are considered as a possible alternative and xmap technology in the form of isa, using excretory/secretory (e/s) products, was also developed and tested. the effectivity of isa was tested with t. spiralis-positive pig meat samples. the system was developed as a duplex assay (with toxoplasma gondii), using goat anti-swine secondary antibodies against specific antibodies. the results of this study corresponded to the infection status of the animals with an assay sensitivity of % and specificity of % (bokken et al., ) . when the immunoglobulin binding protein a/g (generic ig-binding protein), which can be used in multiple species in contrast with goat anti-swine secondary antibody, was included, the results showed a similar specificity of %, but an increase in sensitivity from % for anti-swine antibody to % with protein a/g. the xmap technology-isa exhibited % sensitivity and % specificity in comparison with the commercial pourquier elisa, and % sensitivity and % specificity in comparison with the safepath elisa (van der wal et al., ) . with the rising popularity of mbmis, isa was also developed for other members of nematodes, such as representatives from the genus toxocara (anderson et al., ) . the infection by these parasites is typically peroral at areas contaminated by embryonated roundworm eggs, e.g., sand from childrens' playgrounds. the recombinant t.canis and t. cati e/s antigens tc-ctl- and tc-tes- were used to detect toxocara-specific antibodies in sera from humans pre-diagnosed as positive for visceral and ocular larval migrans (vlm, olm). the specificity of isa was % for both sets of samples, but there were differences in the sensitivity, which was % for vlm and % for olm samples. it was recorded that a combination of recombinant antigens improves sensitivity in comparison with conventional immunoassays (e.g., western blot, elisa), which employ native e/s antigens isolated from larvae (limited availability) that also exhibit cross-reactivity with antibodies from other helminthic infections so reducing its usefulness in regions with poly-parasitism. within the unicellular parasitic protozoa isa was tested in representatives from the genus toxoplasma. unlike t. spiralis, no such regulations for meat control exist for t. gondii, even though its prevalence is higher and health complications can be very severe. recombinant tachyzoite surface protein (sag- ) was used for simultaneous serological detection in a set with t. spiralis e/s (bokken et al., ) . similarly to t. spiralis, the results exactly reflected the load of infection; sensitivity was % and specificity was % for t. gondii. the obtained results repeatedly underline the potential of these assays for further implementation in routine diagnostic screening of a wide range of parasites. as we have descibed, the isa represents an improved methodological alternative to current serological diagnostics, enabling multiplex detection of pathogenic agents with higher sensitivity. traditional diagnostic methods for the identifications of fungal pathogens are mostly based on phenotype analysis of fungal cultures or detection of antigens (polysaccharides), but these approaches are time-consuming and not very accurate (diaz and fell, ; bovers et al., ; landlinger et al., ; babady et al., ) . rapid and correct identification methods are important for efficient therapy (diaz and fell, ) , however, available qpcr assays have various levels of sensitivity and specificity and often have a limited range, targeting only a few yeasts or mold species (landlinger et al., ; babady et al., ) . the need for rapid and correct identifications of fungal pathogens was addressed by development of xmap technology based detection methods (diaz and fell, ; page and kurtzman, ; das et al., ; bovers et al., ; babady et al., ; balada-llasat et al., ; farooqi et al., ; landlinger et al., ) . majority of xmap applications for the multiplex detection and identification of fungal pathogens are based on the capture of fungal nucleic acid by ddh assays. to perform rapid and accurate identifications of fungal pathogens in immunocompromised individuals, the ddh was designed detect a wide range of the most commonly occurring clinically relevant fungal pathogens including species of the genera aspergillus and candida and other important pathogens such as cryptococcus, fusarium, trichosporon, mucor, rhizopus, penicillium, absidia, and acremonium (landlinger et al., ). the ddh was used mainly for identifications of fungi due to its ability to detect coinfections with multiple fungal species in patients and may contribute to improved diagnosis of invasive fungal infections. studies employing xmap technology were developed and successfully used to identify individual fungal species within candida sp. (page and kurtzman, ; farooqi et al., ) , or trichosporon sp. (diaz and fell, ) . in these studies, ddh assays for fast and accurate detection and identification of important fungal pathogens were developed. in another study, the xmap technology was used for genotyping of human pathogenic fusarium sp. (o'donnell et al., ) . fusaria were genotyped also by sequence analysis. the independent comparison of the results obtained via xmap technology with results obtained via sequencing showed the xmap incorrectly identified some of fusarium isolates. besides the in-house assays described above, commercial kits have also been developed for the detection of fungal pathogens by xmap, e.g., xtag r fungal analyte-specific reagents (asr) assay and the sensitivity and specificity of the assay were tested within identification of fungal isolates and positive blood culture bottles (babady et al., ) . the candida -plex assay was tested within of candida strains and bacterial strains with no-cross-reaction with any of the bacterial strains. the sensitivity and specificity were %. using -plex assay were tested mold species and the assay correctly identified all species of aspergillus, with % specificity and sensitivity except a. niger ( / isolates). other molds were identify also with % specificity and sensitivity except mucor ( / isolates) and rhizopus ( / isolates). besides the testing of fungal isolates also positive blood culture bottles were tested for the presence of candida species using candida -plex assay. the sensitivity and specificity of the assay was % for each species. the mold plex assay did not detect one rhizopus species and the a. niger strains, so the results were similar as the previous mentioned results in the course of identification of fungal isolates. in addition, asr for identification of candida species do not distinguish between members of candida complexes, e.g., phenotypically indistinguishable groups ii and iii of c. parapsilosis (group i), which have been renamed candida orthopsilosis and c. metapsilosis. similarly, asr for identification of a. fumigatus were unable to distinguish between members of the a. fumigatus complex. the results showed that xtag r fungal asr assay could be used as an adjunct to culture. the mold -plex assay has been developed specifically for the detection of specific species of mold, which may be reason why rhizopus, mucor, and a. niger have not been identified. due to the equal treatment of infections caused by genera mucor and rhizopus, it would be better to design a panel to detect the most common genera of fungi, and not to focus on the detection of particular species. the results showed that the xtag r fungal asr assay is an attractive alternative to reference methods, due to its speed and ability to simultaneously identify multiple fungal species (balada-llasat et al., ) . ddh assay is able to not only identify the fungal pathogens, but it can be used for a genotyping of fungal pathogens. it was applied for identification of closely related pathogenic yeasts cryptococcus neoformans and cryptococcus gattii that may cause meningoencephalitis in immunocompromised individuals (bovers et al., ) . six haploid genotypic groups within these pathogens can be distinguished by several molecular methods e.g. pcr fingerprinting or intergenic spacer genotyping. besides these haploid groups, hybrids have been described as well. ad hybrids are hybrids between the two varieties of c. neoformans and also hybrids between c. neoformans var. neoformans and c. gattii have been described. the ddh has been adapted for the detection of the genotypes within cryptococcus neoformans and cryptococcus gattii. the detection limit was calculated from × to × cells for the various specific probes for each of the six haploid genotypic groups. the results showed that ddh is highly specific method and it is possible not only identify cryptococcal isolates at the species and genotype levels but also allows identification of hybrid isolates that have two alleles of the specific probes region and also able to identify cryptococci in cerebrospinal fluid. however, the optimization of dna extraction methods is needed before routine use in clinical laboratories. detection and identification of pathogens, as well as an understanding of pathogen variation, the pathogenesis of the diseases they cause, and timelines of infection and antimicrobial resistance, are all required in order to obtain the full picture of disease progression and to select effective cures for infected individuals or populations. as the amount of input data required for such decisions increases, so too does the number of tests that are required during laboratory examinations. the multiplex assays for the detection and typing of pathogens using xmap technology are tools of choice as they are capable of providing all of the important information within a reasonable timeframe, and without excessive labor or costs. the major improvement of xmap assays is that they add another dimension to the simple detection, which is represented by the simultaneous analysis of many targets within a single sample, and they therefore represent complementary tools to procedures for the detection and quantification of pathogens such as qpcr, culture, or elisa assays. the significance of such a complex approach for the multiplex detection has grown in recent years, which is documented by the increase in published data and of application of the commercial assays in routine diagnostics. development of a luminex bead based assay for diagnosis of toxocariasis using recombinant antigens tc-ctl- and tc-tes- xmap cookbook: a collection of methods and protocols for developing multiplex assays with xmap technology multicriteria-based ranking for risk management of food-borne parasites: microbiological risk assessment series (mra) . rome: fao regulation (eu) / , laying down specific rules on official controls for trichinella in meat evaluation of luminex xtag fungal analyte-specific reagents for rapid identification of clinically relevant fungi conversion of a capture elisa to a luminex xmap assay using a multiplex antibody screening method detection of yeasts in blood cultures by the luminex xtag fungal assay gastrointestinal pathogens detected by multiplex nucleic acid amplification testing in stools of pediatric patients and patients returning from the tropics identification of new respiratory viruses in the new millennium. viruses basel a novel beadbased assay to detect specific antibody responses against toxoplasma gondii and trichinella spiralis simultaneously in sera of experimentally infected swine identification of genotypically diverse cryptococcus neoformans and cryptococcus gattii isolates by luminex xmap technology menangle virus, a pteropid bat paramyxovirus infectious for pigs and humans, exhibits tropism for secondary lymphoid organs and intestinal epithelium in weaned pigs development of multiplexed bead arrays for the simultaneous detection of nucleic acid from multiple viruses in bat samples absolute quantification of mrna using real-time reverse transcription polymerase chain reaction assays a microsphere-based assay for multiplexed single nucleotide polymorphism analysis using single base chain extension beadbased suspension array for simultaneous differential detection of five major swine viruses fluorescence polarization in homogeneous nucleic acid analysis opportunities for bead-based multiplex assays in veterinary diagnostic laboratories performance of the xtag (r) gastrointestinal pathogen panel, a multiplex molecular assay for simultaneous detection of bacterial, viral, and parasitic causes of infectious gastroenteritis henipaviruses: an updated review focusing on the pteropid reservoir and features of transmission dna probes for the rapid identification of medically important candida species using a multianalyte profiling system simultaneous detection of multiplex-amplified human immunodeficiency virus type rna, hepatitis c virus rna, and hepatitis b virus dna using a flow cytometer microsphere-based hybridization assay a comparison of luminex xtag (r) gastrointestinal pathogen panel (xtag gpp) and routine tests for the detection of enteropathogens circulating in southern china a rapid multiplex assay for nucleic acid-based diagnostics optimization and validation of a multiplexed luminex assay to quantify antibodies to neutralizing epitopes on human papillomaviruses , , , and high-throughput detection of pathogenic yeasts of the genus trichosporon prevalence of viable toxoplasma gondii in beef, chicken, and pork from retail meat stores in the united states: risk assessment to consumers introduction to luminex r xmap r technology and applications for biological analysis in china applications of luminex (r) xmap (tm) technology for rapid, high-throughput multiplexed nucleic acid detection quantitative, multiplexed detection of bacterial pathogens: dna and protein applications of the luminex labmap (tm) system validation and comparison of luminex multiplex cytokine analysis kits with elisa: determinations of a panel of nine cytokines in clinical sample culture supernatants trichinellosis: a worldwide zoonosis enzyme-linked immunosorbent assay (elisa) quantitative assay of immunoglobulin-g species identification of invasive yeasts including candida in pakistan: limitations of phenotypic methods multiplex, bead-based suspension array for molecular determination of common salmonella serogroups development of a multiplex bead-based assay for detection of hepatitis c virus aetiology of acute gastroenteritis in adults requiring hospitalization in the netherlands antibodies against epstein-barr virus gp antigen: a novel marker for serological diagnosis of nasopharyngeal carcinoma detected by xmap technology development of a luminex assay for the simultaneous detection of human enteric viruses in sewage and river water bio-rad'sbio-plex (r) suspension array system, xmap technology overview emerging human infectious diseases: anthroponoses, zoonoses,and sapronoses multiplexed single nucleotide polymorphism genotyping by oligonucleotide ligation and flow cytometry aetiology of community-acquired, acute gastroenteritis in hospitalised adults: a prospective cohort study genotyping of human papillomavirus in cervical lesions by l consensus pcr and the luminex xmap system fluorescence-based multiplex protein detection using optically encoded microbeads multiplexed microsphere-based flow cytometric assays multiplexed magnetic microsphere immunoassays for detection of pathogens in foods luminex xmap combined with western blot improves hiv diagnostic sensitivity advances in the diagnosis of respiratory tract infections: role of the luminex xtag respiratory viral panel a ligase-mediated gene detection technique species-specific identification of a wide range of clinically relevant fungal pathogens by use of luminex xmap technology multiplexed reverse transcriptase pcr assay for identification of viral respiratory pathogens at the point of care application of a microsphere-based array for rapid identification of acinetobacter spp. with distinct antimicrobial susceptibilities simultaneous detection of six diarrhea-causing bacterial pathogens with an inhouse pcr-luminex assay simultaneous detection of seven enteric viruses associated with acute gastroenteritis by a multiplexed luminex-based assay theoretical analysis of the kinetics of dna hybridization with gel-immobilized oligonucleotides hpv genotype detection using hybrid capture sample preparation combined with whole genome amplification and multiplex detection with luminex xmap molecular determination of h antigens of salmonella by use of a microsphere-based liquid array simultaneous detection of gastrointestinal pathogens with a multiplex luminex-based molecular assay in stool samples from diarrhoeic patients evaluation of luminex xtag gastrointestinal pathogen analyte-specific reagents for high-throughput, simultaneous detection of bacteria, viruses, and parasites of clinical and public health importance h n influenza viruses: outbreaks and biological properties detection of trichinella infection in food animals indirect elisa for the diagnosis of trichinosis in living pigs nucleic acid sequence detection using multiplexed oligonucleotide pcr dna polymorphism identity determination using flow cytometry phylogenetic diversity and microsphere array-based genotyping of human pathogenic fusaria, including isolates from the multistate contact lens-associated us keratitis outbreaks of evaluation of a multiplex pcr assay for simultaneous detection of bacterial and viral enteropathogens in stool samples of paediatric patients simultaneous quantitation of antibodies to neutralizing epitopes on virus-like particles for human papillomavirus types , , , and by a multiplexed luminex assay comparison of the luminex xtag respiratory viral panel with in-house nucleic acid amplification tests for diagnosis of respiratory virus infections comparison of the luminex xtag respiratory viral panel with xtag respiratory viral panel fast for diagnosis of respiratory virus infections rapid identification of candida species and other clinically important yeast species by flow cytometry evaluation of the luminex xtag gastrointestinal pathogen panel and the savyon diagnostics gastrointestinal infection panel for the detection of enteric pathogens in clinical samples systematics and epidemiology of trichinella development of a bead-based multiplex pcr assay for the simultaneous detection of multiple mycoplasma species control iii surveillance in swine by immunodiagnostic methods evaluation of xtag respiratory viral panel fast and xtag human parainfluenza virus analyte-specific reagents for detection of human parainfluenza viruses in respiratory specimens development of a sensitive, multiplexed immunoassay using xmap beads for detection of serotype-specific streptococcus pneumoniae antigen in urine samples development of a bead-based luminex assay using lipopolysaccharide specific monoclonal antibodies to detect biological threats from brucella species xmap-based analysis of three most prevalent staphylococcal toxins in staphylococcus aureus cultures semi-quantitative analysis of influenza samples using the luminex xtag (r) respiratory viral panel kit simultaneous pathogen detection and antibiotic resistance characterization using snp-based multiplexed oligonucleotide ligation-pcr (mol-pcr) two new rapid snp-typing methods for classifying mycobacterium tuberculosis complex into the main phylogenetic lineages minisequencing" primer extension for analysis of point mutations and single nucleotide polymorphisms the pathogenesis of ebola hemorrhagic fever advanced techniques in diagnostic microbiology high throughput multiplex pcr and probe-based detection with luminex beads for seven intestinal parasites flow cytometric platform for high-throughput single nucleotide polymorphism analysis a multiplex bead-based suspension array assay for interrogation of phylogenetically informative single nucleotide polymorphisms for bacillus anthracis a beadbased suspension array for the 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influenza a virus (h n ) detection by a novel multiplex pcr typing method novel microsphere-based method for detection and typing of mucosal human papillomavirus types conception of the review: pk; design of the work: vm, nr, pk; writing this review: nr and vm (these authors contributed to this work equally); revision of the manuscript: mk, pm, pk; all authors approved the version to be published in frontiers in microbiology and agreed to be accountable for all aspects of the work. the authors would like to thank neysan donnelly (max-planck-institute of biochemistry, germany) for grammatical corrections of the manuscript. the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © reslova, michna, kasny, mikel and kralik. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- -eq gujk authors: sato, hironori; yokoyama, masaru; toh, hiroyuki title: genomics and computational science for virus research date: - - journal: front microbiol doi: . /fmicb. . sha: doc_id: cord_uid: eq gujk nan rna viruses are highly mutable, yet changes in genomes and proteins would be restricted by the functional and structural constraints inherent in the survival strategies of viruses in nature. rapidly evolving technologies in genomics and computational science are now opening up a new avenue for elucidating the real picture of diversity of the organism in nature and for studying the principles underlying the maintenance and change of structures, interactions, and functions of biomolecules. the information is essential for understanding the evolutionary dynamics of virus-host interactions in virological, immunological, and epidemiological phenomena and for rationally developing methods to control rna viruses. in this research topic, we present timely articles, consisting of reviews, mini reviews, original researches, hypothesis & theory, and perspective, all of which underscore the challenges and increasing importance of incorporating the new technologies to study rna viruses and their impacts on hosts. beerenwinkel et al. ( ) reviewed the challenges and opportunities in inferring the diversity of intra-host virus populations using next-generation sequencing technologies. they discuss the wisdom of reducing artificial errors during sample preparation, existing approaches inferring local and global diversity from sequence data, and successful applications on basic and biomedical studies. tan gana et al. ( ) reviewed the latest articles describing cellular and viral micrornas involved in hiv- infection. they describe recent advances in understanding of the biogenesis and functions of the micrornas in the virus-cell battles and point out roles of the genomics and computational science in obtaining and integrating the information. prabakaran et al. ( ) reported on the antibodyomes of healthy individuals obtained by pyrosequencing and bioinformatics analyses. they showed genetic evidence that the antibody subsets with distinct diversity and related to the already-known neutralizing antibodies against the hiv- , sars coronavirus, and henipaviruses exist in human igm repertoires of uninfected individuals. zhu et al. ( ) reported an antibodyome of an hiv- -infected individual who produced broadly neutralizing antibodies. using pyrosequencing, bioinformatics, and functional analyses, they suggested a role of somatic maturation in generating heavy-and light-chain sequences with varied neutralization phenotypes against hiv- . ode et al. ( ) reviewed the results of molecular dynamic simulations to learn the structural dynamics of proteins in solution. they highlight studies on the structure and function of viral enzymes, virion structures, mechanisms of viral resistance against host immunities and anti-viral drugs, and the development of anti-viral agents. franzosa et al. ( ) reviewed structural systems biology of interactomes in the host-pathogen relationships. they present existing experimental datasets of the host-pathogen interactome and discuss approaches to obtain structural interactome by integrating the biophysical, functional, and evolutionary information. miki and katayama ( ) presented a viewpoint on the in silico -d structural analysis in virus research. they describe importance of incorporating in silico modeling techniques into experimental studies to solve structural problems in their neutralization study of the norovirus. bozek et al. ( ) provided in silico structural models of capsid proteins of hiv- and siv, which revealed marked differences in the electrostatic potential on the interaction surface and suggested a potential role of electrostatic interactions in the evasion of siv from the rhesus restriction factor trim α. daiyasu et al. ( ) reported a new application of information theory to the study of the divergent evolution of function of chemokine receptors and their homologs, such as decoy and viral receptors, in which both sequence and structural information are used to identify amino acid positions that might be responsible for evolving their distinct functions. rusu et al. ( ) provided in silico structural models of the rift valley fever virus glycoproteins gn and gc. the models with the cryo-electron microscopy data allowed the authors to identify four possible arrangements of the glycoproteins in the virion envelope and to indicate how these proteins assemble to form the capsomer base and intercapsomer connections. yokoyama et al. ( ) provided in silico structural models of sapovirus protease docked to its substrate peptides; these models described how this enzyme realizes the functional binding of cleavage sites with distinct sequences and allowed rational identification of the sapovirus protease inhibitors in combination with experimental approaches. iwami et al. ( ) reported a mathematical model to quantitatively characterize the viral replication in cell cultures. in their www.frontiersin.org march | volume | article | study, the data from two time-course experiments of infections with a cell-free virus stock are used to estimate the half-life of infected cells, viral production rate of an infected cell, and the basic reproductive number. takemura and murakami ( ) reviewed structure and function of hiv- capsid proteins. they describe the capsid structure in relation to their abilities to form a conical core in a virion or to interact with various cellular proteins that promote or suppress viral replication. nomura and matano ( ) reviewed the critical roles of host hla/mhc-i genotypes in disease progression in primate lentivirus infections. they highlighted studies showing the association of the hla/mhc-i genotypes with rapid or slow aids progression during hiv/siv persistent infections. kuroki et al. ( ) reviewed the structural biology of the immunologically intriguing cell surface receptors termed paired receptors. by referencing recent studies of two major structural superfamilies, the immunoglobulin-like and the c-type lectin-like receptors, they described how these receptors discriminate self and non-self ligands to maintain homeostasis in the immune system. shiino ( ) reviewed phylodynamic analyses of viral infections, transmission, and evolution in host populations. the author highlighted recent viral researches inferring epidemiologically important parameters, such as infectious clusters, transmission connections, basic reproductive number, and fluctuation of the viral population size. sasaki et al. ( ) proposed a mathematical model that describes the temporal dynamics of the spread of poliovirus avirulent and virulent strains in a human population; this model was used to estimate the risk of outbreak following the cessation of administration of oral polio vaccine and to predict conditions that significantly increased the outbreak risk. in summary, these articles present overviews and examples of "frontiers in virology" to elucidate viral diversity, protein -d structures, virus-host interactions, and evolution of rna viruses using forefront technologies in genomics and computational science. challenges and opportunities in estimating viral genetic diversity from next-generation sequencing data electrostatic potential of human immunodeficiency virus type and rhesus macaque simian immunodeficiency virus capsid proteins evolutionary analysis of functional divergence among chemokine receptors, decoy receptors, and viral receptors toward a three-dimensional view of protein networks between species identifying viral parameters from in vitro cell cultures molecular recognition of paired receptors in the immune system in silico d structure analysis accelerates the solution of a real viral structure and antibodies docking mechanism association of mhc-i genotypes with disease progression in hiv/siv infections molecular dynamics simulation in virus research origin, diversity, and maturation of human antiviral antibodies analyzed by high-throughput sequencing an assembly model of rift valley fever virus estimating the risk of re-emergence after stopping polio vaccination phylodynamic analysis of a viral infection network functional constraints on hiv- capsid: their impacts on the viral immune escape potency micrornas in hiv- infection: an integration of viral and cellular interaction at the genomic level structural basis for specific recognition of substrates by sapovirus protease somatic populations of pgt - hiv- -neutralizing antibodies identified by pyrosequencing and bioinformatics key: cord- -bojfc q authors: han, yelin; du, jiang; su, haoxiang; zhang, junpeng; zhu, guangjian; zhang, shuyi; wu, zhiqiang; jin, qi title: identification of diverse bat alphacoronaviruses and betacoronaviruses in china provides new insights into the evolution and origin of coronavirus-related diseases date: - - journal: front microbiol doi: . /fmicb. . sha: doc_id: cord_uid: bojfc q outbreaks of severe acute respiratory syndrome (sars) in , middle east respiratory syndrome in and fatal swine acute diarrhea syndrome in caused serious infectious diseases in humans and in livestock, resulting in serious public health threats and huge economic losses. all such coronaviruses (covs) were confirmed to originate from bats. to continuously monitor the epidemic-related covs in bats, virome analysis was used to classify covs from bats of species in yunnan, guangxi, and sichuan provinces between august and may . we identified cov strains from individual samples of four bat species. identification of four alpha-covs from scotophilus kuhlii in guangxi, which was closely related to a previously reported bat cov and porcine epidemic diarrhea virus (pedv), revealed a bat-swine lineage under the genus alphacoronavirus. a recombinant cov showed that the pedv probably originated from the cov of s. kuhlii. another alpha-cov, α-yn , from rhinolophus affinis in yunnan, suggested that this alpha-cov lineage had multiple host origins, and α-yn had recombined with covs of other bat species over time. we identified five sars-related covs (sarsr-covs) in rhinolophus bats from sichuan and yunnan and confirmed that angiotensin-converting enzyme usable sarsr-covs were continuously circulating in rhinolophus spp. in yunnan. the other beta-cov, strain β-gx , found in cynopterus sphinx of guangxi, represented an independently evolved lineage different from known covs of rousettus and eonycteris bats. the identification of diverse covs here provides new genetic data for understanding the distribution and source of pathogenic covs in china. coronaviruses (covs) are a group of enveloped viruses with a large positive single-stranded rna genome (∼ - kb in length) of the subfamily coronavirinae under the family coronaviridae. the complete genome of cov contains five major open reading frames (orfs) that encode replicase polyproteins (orf ab), spike glycoprotein (s), envelope protein (e), membrane protein (m), and nucleocapsid protein (n) flanked by a -untranslated region (utr) and a -utr. currently, members of the subfamily coronavirinae are classified into four genera, alphacoronavirus, betacoronavirus, gammacoronavirus, and deltacoronavirus (fehr and perlman, ; su et al., ) . covs can cause upper and lower respiratory diseases, gastroenteritis, and central nervous system infections in a wide variety of avian and mammalian hosts. some covs are human pathogens that cause mild to severe disease, including nl and e of the genus alphacoronavirus, and severe acute respiratory syndrome cov (sars-cov), middle east respiratory syndrome cov (mers-cov), oc , and hku of the genus betacoronavirus (dijkman and van der hoek, ; desforges et al., ; van den brand et al., ; lim et al., ) . the sars pandemic originated in guangdong province in china between and , and spread to countries, resulting in nearly , cases and deaths worldwide (donnelly et al., ) . since being discovered in middle eastern countries in , mers-cov has infected , people with a current fatality rate of . % (mackay and arden, ; van den brand et al., ; de wit et al., ) . with the discovery of diverse wildlife-borne covs in different regions of the world, previous studies have indicated that bats are the main and original natural reservoirs of alphacoronavirus and betacoronavirus (calisher et al., ; woo et al., ) . the special metabolic and immune systems allow bats to tolerate diverse viruses, and the social roosting behavior of many bat species lets them serve as ideal incubators for the occurrence of frequent co-infection, recombination, and intraspecies transmission of covs o'shea et al., ; hu et al., ; lu et al., ; sola et al., ) . although years have passed without a recurrence of the sars outbreak, the consistent discovery of sars-related covs (sarsr-covs) in rhinolophus bats indicates an ongoing risk of sars reemergence balboni et al., ; yang et al., ; wu et al., b; hu et al., ) . recently, a cov of rhinolophus bat origin, swine acute diarrhea syndrome cov (sads-cov), was found to be responsible for the death of , piglets across four farms in guangdong province (zhou et al., ) . besides similar clinical signs, the outbreak of sads coincided with infection by another swine enteric cov, porcine epidemic diarrhea virus (pedv; zhou et al., ) . it is worth mentioning that a pedv-related bat cov, btcov/ , was previously identified in scotophilus kuhlii bats (tang et al., ; wang et al., ; gerdts and zakhartchouk, ; zhou et al., ) . all of these findings emphasize the importance of continuous monitoring for ecological diversity of covs in bats to minimize the impact of potential cov-related diseases on public health and economic growth. as the original site of the sars epidemic in humans and sads outbreak in pigs (heymann et al., ) , guangdong province is also the primary region in china in which wildlife is being consumed. since the supply of wildlife for consumption in guangdong mainly comes from yunnan and guangxi provinces (wu et al., b) , understanding the presence and dynamic changes in sars-or sads-related bat covs within and between these provinces is important for predicting and tracing covrelated diseases. sichuan is next to yunnan province, and has high ecological diversity of animal resources, and it is the largest pig-farming province in china. although pedv-related pig diseases were reported previously (cai et al., ) , no systematic survey of bat-borne covs has been conducted in sichuan to establish if there are sars-or sads-related covs. in this study, a survey of bat-borne covs was performed in bats of guangxi, yunnan, and sichuan provinces between and . samples from rhinolophus bats in sichuan and yunnan provinces, and s. kuhlii and cynopterus sphinx in guangxi province were found to be cov-positive, and a large number of sequencing reads classified into diverse alpha-and beta-covs were obtained using virome analysis. the characterization of covs in bats from different regions provides clues for the evolutionary relationships of pedv, sars-cov, and other batassociated covs. bats were treated according to the guidelines of regulations for the administration of laboratory animals (decree no. of the state science and technology commission of the people's republic of china, ) . the sampling was approved by the ethics committee of institute of pathogen biology, chinese academy of medical sciences & peking union medical college (approval number: ipb ec ). the bat species were initially determined morphologically and subsequently confirmed by sequence analysis of mitochondrial cytochrome b dna (tang et al., ; du et al., ) . anal swab samples from captured bats were immersed in virus sampling tubes (yocon, china) containing maintenance medium and temporarily stored at − • c. the samples were then transported to the laboratory and stored at − • c. the accurate sampling locations were recorded by place name, latitude, and longitude. the samples of each species were pooled by adding ml from each maintenance medium sample into one new sample tube. the pooled samples, classified by species, were processed with a virusparticle-protected, nucleic acid purification method as described in our previous studies (wu et al., (wu et al., , a (wu et al., , . the samples were homogenized and subsequently filtered through a . µm polyvinylidene difluoride filter (millipore, germany). the filtered samples were then centrifuged at , × g for h at • c. to remove naked dna and rna, the pellet was digested in a cocktail of dnase and rnase enzymes. the viral dna and rna were simultaneously isolated using a qiamp minelute virus spin kit (qiagen, united states). first-strand viral cdna was synthesized using the primer k- n and a superscript iii system (invitrogen, united states). the cdna was converted into dsdna by klenow fragment (neb, united states). sequenceindependent pcr amplification was conducted using primer k. the pcr products were analyzed by agarose gel electrophoresis. all dna smears larger than bp were extracted by a minelute gel extraction kit (qiagen). the extracted nucleic acid libraries were then analyzed using an illumina hiseq sequencer, for a single read of bp. the sequence reads were filtered using previously described criteria (wu et al., (wu et al., , a (wu et al., , . reads with no call sites, reads with similarity to the sequencing adaptor and the primer k sequence, duplicate reads, and low-complexity reads were removed. sequence-similarity-based taxonomic assignments were conducted as described in our previous study (wu et al., ) . valid sequence reads were aligned to sequences in the ncbi non-redundant nucleotide database and non-redundant protein database using blastn and blastx, respectively. the taxonomies of the aligned reads with the best blast scores (e score < − ) were parsed by the megan -metagenome analyzer. cov screening of individual samples was performed by amplifying a -bp fragment of the rna-dependent rna polymerase (rdrp) gene of covs using conserved primers ( -ggttgggactatcctaagtgtga- and -ccatc atcagatagaatcatcata- ), as described previously (du et al., ) . sequence reads classified into the same virus genus were extracted. the accurate locations of the reads were determined based on the alignment results exported with megan . specific primers were designed from the located sequence reads. fragments between reads were amplified with nested specific primers and then sequenced. the remaining genomic sequences were determined using -and -rapid amplification of cdna ends. mega . was used to align nucleotide sequences and deduced amino acid sequences using the muscle package and default parameters. the best substitution model was then evaluated by the model selection package. finally, we constructed a maximum-likelihood method using an appropriate model to process the phylogenetic analyses with , bootstrap replicates. recombination among covs was detected with simplot software. all analyses were performed with a kimura model, a window size of bp, and a step size of bp. all genome sequences were submitted to genbank. the accession numbers for the five bat alpha-covs were mk to mk . the accession numbers for the six bat beta-covs were mk to mk . anal swabs from bats of species were collected from yunnan, guangxi, and sichuan provinces in china between august and may . all anal swab samples were classified by species and then combined into pools and subjected to virome analysis. cov-related sequencing reads were detected in ten of the sample pools. these pools related to rhinolophus spp. samples from sichuan, rhinolophus affinis samples from yunnan, s. kuhlii samples from guangxi, and c. sphinx samples from guangxi. pan cov screening revealed that individual samples were cov-positive (table and figure ) . a total of , , reads of bp in length showed the best matches with coronavirinae viral proteins in the ncbi non-redundant database, and these reads showed - % amino acid (aa) identity with known alphaor beta-covs. eleven viruses of representative positive samples were selected for genomic sequencing as quasi-species. there were five viruses in the genus alphacoronavirus; four from s. kuhlii in guangxi province were named as btsk-alphacov/gx a (α-gx a), btsk-alphacov/gx b (α-gx b), btsk-alphacov/gx c (α-gx c), and btsk-alphacov/gx d (α-gx d). the other virus from r. sinicus in yunnan province was named as btrs-alphacov/yn (α-yn ). there were six covs in the genus betacoronavirus. the virus identified from rhinolophus spp. in sichuan province was named as btrl-betacov/sc (β-sc ); four covs from r. sinicus in yunnan province were named as btrs-betacov/yn a (β-yn a), btrs-betacov/yn b (β-yn b), btrs-betacov/yn c (β-yn c), and btrs-betacov/yn d (β-yn d). one cov from c. sphinx in guangxi province was named as btcs-betacov/gx (β-gx ). as shown in table and figure , with the typical genome organization, the full genome sizes of covs ranged from , (α-gx c) to , bases (β-yn b) and their g + c contents ranged from . to . . there were several accessory orfs in each cov strain. these accessory orfs were mainly present in the regions between s and e (named orfs-e), m and n (named orfm-n), or downstream of n (named orfn). the accessory orfs of α-gx (a-d), α-yn , and β-gx were mainly present in orfse and orfm-n regions; the small orfs of β-sc and btsk-alphacov/gx c figure | numbers of bat samples from sichuan, yunnan, and guangxi provinces. the size of the pie chart is in proportion with the total number of specimens collected from each province. red color represents samples positive for cov, and blue color represents negative samples. β-yn (a-d) were mainly present in orfs-e and orfmn regions. we named these accessory orfs according to previous studies. as shown in table , the core sequences of the leader transcriptional regulatory sequence (trs; -cuaaac- ) were identified in the untranslated sequences of α-gx (a-d) and α-yn , which is unique to alphacoronavirus (madhugiri et al., ) . the trs motifs of s, orf , e, and m genes in α-gx (a-d) differed from the core sequences of the leader trs (supplementary table ). the trs motif of s was identified as -ccaaau- or -ccaaac- ; the trs motif of orf was identified as -cauuac- ; the trs motif of e was identified as -cuagac- ; the trs motif of m was identified as -auaaac- . an alternative trs motif ( -guaaac- ) of α-yn was discovered preceding orf , which differed by nucleotide (nt) from those of all other genes in α-yn . in addition to the e of β-gx , the trs motifs of six betacoronaviruses all conformed to the consensus motif -acgaac- , which is unique to betacoronavirus (woo et al., ). an alternative trs motif ( -ucgaac- ) was discovered preceding the e in β-gx . four alpha-covs of s. kuhlii in guangxi province four alpha-covs were identified from s. kuhlii. the full-length genomes of strains α-gx a, b, c, and d were sequenced. the genome sequence similarity among these four covs, pedv, and btcov/ was examined by simplot analysis (figure a ) and pairwise alignment (supplementary table ). the α-gx a, b, and c were closely related to a known cov, btcov/ , identified in s. kuhlii of hainan province with . - % aa identity for orf b, orf , e, m, and n. the differences mainly occurred in s ( . - . % aa identity) and orfx ( . - . % aa identity). there were also significant differences between α-gx a and b and btcov/ at the front end of orf a ( - nt) with . - % nt identity. in addition, although the α-gx d was closely related to that of btcov/ with . - . % aa identity for orf a, orf b, orf , and m, it was unexpected that the aa identity of α-gx d and btcov/ in s region was . %, which was lower than that of α-gx d and pedv ( . % aa identity). although the aa identity of α-gx d and pedv in s region was only . %, we have not found any known viruses with higher identity than this. a novel alpha-cov (α-yn ) was identified in r. affinis of yunnan province. the genome sequence similarity among α-yn , btrf-alphacov/hub (α-hub ), hipposideros bat cov hku (hi-batcov hku ), rousettus bat cov hku (ro-batcov hku ), and cardioderma bat coronavirus/kenya/ky / (ca-batcov/kenya) was examined by simplot analysis (figure b ) and pairwise alignment (supplementary table ) . this virus showed low sequence similarity with any known alpha-covs. α-yn showed the highest sequence similarity to α-hub of rhinolophus ferrumequinum with . - . % aa identity in orf a, orf b, and n; to hi-batcov hku with - . % aa identity in s, m, and orf b; to ca-batcov/kenya in orf a with . % aa identity; and to ro-batcov hku in e with . % aa identity. the sequences of orf a from α-yn showed < % aa identity with those of the afore-mentioned known covs. in addition, we did not find any sequences that were similar to orf b and orf c of α-yn from known viruses. according to the ictv criteria, covs that share < % aa sequence identity in the conserved replicase domains are considered to belong to different species; thus, α-yn could be considered a new species under the genus alphacoronavirus. we obtained five beta-covs from rhinolophus spp. from yunnan and sichuan provinces, which were named as β-yn a, β-yn b, β-yn c, β-yn d, and β-sc . all of a, these four representative bat covs are α-gx d, α-yn , β-yn b and β-gx , respectively. b, for putative orfs, we aligned the trs that preceded the start codon aug with the leader trs. the core sequence is indicated with underscores in bold type. the start codons of genes are in bold blue. these beta-covs had high sequence identity with sarsr-covs. these five sarsr-covs showed - % nt identity with human sars-cov sz . the genome sequence similarity among the five sarsr-covs and sars-cov sz strain was examined by simplot analysis (figure c) . these five sarsr-covs were highly conserved and shared a uniformly high sequence similarity to sars-cov with . - % aa identity in orf a, orf b, e, m, and n (supplementary table ). there were some differences between the sarsr-covs and sars-cov sz in the coding regions of orf a, orf b, orf a, and orf b. after further analysis, the sars-cov sz showed the highest sequence similarity to β-yn b with - % aa identity in s, orf a, and orf b; and to β-yn a with . - . % aa identity in orf a and orf b. in contrast, considerable genetic diversity was shown in the s ( . - . % aa identity) and orf ( . - . % aa identity) among the five sarsr-covs and sars-cov sz strain. β-yn b shared . % aa identity with sarsr-cov wiv , which was higher than that of the other four sarsr-covs ( . - . % aa identity) (figure c and supplementary table ) . remarkably, wiv and wiv (or rs ) were identified between and , and they could use the human angiotensin converting enzyme ii (ace ) receptor for entry, like sars-cov (ge et al., ; yang et al., ) . orfx was also found in the genomes of β-yn b and β-yn d. orfx of β-yn b and β-yn d both shared . % aa identity with wiv . except for orf b ( . % aa identity), β-yn b was highly conserved and shared a uniformly high sequence similarity to sars-cov wiv in every orf ( . - % aa identity) (supplementary table ). a novel beta-cov, β-gx , was identified from this bat species. the genome sequence similarity among β-gx and four known covs, batcov hku - , batcov hku - - , ro-batcov hku , and batcov philippines/diliman was examined by simplot analysis (figure d ) and pairwise alignment (supplementary table ). the full-length genome sequence of this virus had sequence similarity to that of hku related covs of rousettus bats and gccdc -related covs of eonycteris bats (woo et al., ; huang et al., ) , but the sequence identity between them was low. the sequence similarity between β-gx and hku -and gccdc -related cov s were - . % aa identity in orf a, orf b, s, orf , e, m, and n. some partially sequenced genome segments of a recently reported cov, philippines/diliman g / (batcov philippines/diliman) (watanabe et al., ) , identified in c. brachyotis from the philippines, showed the highest aa sequence identity with the β-gx in m ( % aa identity), n ( % aa identity), orf a ( % aa identity), orf b ( . % aa identity), and orf c ( . % aa identity) (watanabe et al., ) . according to the ictv criteria, β-gx could be considered a new species under the genus betacoronavirus. to determine further the evolutionary relationships between these covs, phylogenetic trees were constructed using the aa sequences of rdrp, s, s , e, m and n (figure ) . as we have speculated before, α-gx a, α-gx b, α-gx c, α-gx d and α-yn clustered with the genus alphacoronavirus, and β-sc , β-yn a, β-yn b, β-yn c, β-yn d, and β-gx clustered with the genus betacoronavirus. the results of the phylogenetic analyses were consistent with those of the sequence identity figure | phylogenetic trees based on aa sequences of rdrp, s, s , e, m, and n. the trees were constructed by the maximum likelihood method using appropriate models (wag + g + i for rdrp and n; lg + g + i for s, s , and e; rtrev + g for m) with bootstrap values determined by replicates. only bootstraps > % are shown. the scale bars represent . (rdrp), . (s and s ), and . (e, m, and n), respectively. analyses, and confirmed that the identified covs could be divided into four lineages. lineage e α-gx a, α-gx b and α-gx c clustered with btcov/ , and their branches were short, reflecting the high sequence similarities. in addition, phylogenetic analysis of the s protein supported our previous analysis that α-gx d represented an independent clade between pedv and btcov/ . further analysis of s revealed that α-gx d represented a separate evolution distant from all other members of lineage e. from the length of the branch, α-gx d had a closer relationship with pedv, which means that α-gx d may have undergone recombination with the ancestors of pedv and btcov/ . α-yn clustered with hi-batcov hku , ro-batcov hku , ca-batcov/kenya, and α-hub . however, α-yn had closer relationships with α-hub of r. ferrumequinum in rdrp and n, and closer relationships with hku of hipposideros and rousettus bats in s and m. this means that recombination may have occurred between α-yn and these three related covs. β-sc , β-yn a, β-yn b, β-yn c, and β-yn d clustered with sars-and sarsr-covs under lineage b. in rdrp, e, m, and n trees, these novel sarsr-covs and sars-covs had short branches, which indicated that they were closely related in these regions. phylogenetic analysis of s and s revealed that β-yn b was closer to sars-cov sz and sars-cov tor as well as wiv and wiv . lineage d β-gx of c. sphinx clustered with hku -related covs of rousettus and gccdc -related covs of eonycteris under lineage d; however, β-gx always represented a separate lineage distinct from other members of lineage d. although batcov philippines/diliman of c. brachyotis had a closer relationship with α-yn in m and n, it was only partially sequenced, so we could not analyze its evolutionary relationship with α-yn in rdrp, s, and e, which hindered further analysis. we found that recombinant events had occurred among α-gx d, other bat covs, and pedv of lineage e. α-gx d showed the highest degree of similarity to btcov/ in the orf ab region, and the highest degree of similarity to pedv in the s region. evolutionary analysis showed that the s of α-gx d may have acted as the common ancestors of btcov/ and pedv. we analyzed possible recombination events in lineage e using simplot software. similarity plot and bootscan results demonstrated that the s-n region of the α-gx d had the highest degree of similarity to pedv, and complicated recombination may have happened between bat cov and pedv in the orf ab region ( figure a) . for covs of lineage f, at least two recombination events had occurred ( figure b) . similarity plot and bootscan analysis showed that the s subunits of the α-yn had the highest degree of similarity to ro-batcov hku , while their s subunits had the highest degree of similarity to hi-batcov hku (fig b) . the complicated recombination history between these alpha-covs suggests frequent gene transfers, especially of s, among different covs, which may be the result of the cross-species transmission of these covs. the identification of sarsr-cov in bats in first established a genetic relationship between bats and human sars-covs (lau et al., ; li et al., ) . after that, in the following years, the constant discovery of bat sarsr-covs suggested the risk of re-emergence of sars-cov originating from bat species. other than that, two cov-related cases, human mers in and sads in , which had a great impact on health and the economy were identified as being of bat origin (omrani et al., ; zhou et al., ) . in our previous study, a largescale virome analysis was conducted between and to understand the ecological diversity of bat viruses and the bat origin of emerging infectious diseases. rhinolophus bats in yunnan and guangxi provinces have provided genetic clues to the origin of sars-cov, and a sads-cov-and hku related cov, btrf-alphacov/yn , has also been found in yunnan province (wu et al., a) . sichuan province has diverse wildlife resources and the largest pig breeding in china, and a mers-related cov was reported in bats in this province in (yang et al., ) . it is necessary for us to conduct continuous surveillance for the prevalence, genetic diversity and geographical distribution of covs in some bat species in these potential high-risk regions. using virome analysis, our study investigated the presence and genetic diversity of covs circulating in bat species from sichuan, yunnan, and guangxi provinces between and . the close relationship between five alpha-covs in s. kuhlii in guangxi province and a previously reported cov in s. kuhlii in hainan province reveals that this bat species from different geographic locations contained the same cov species, but with distinct s proteins. the s region of α-gx d is more closely related to that of pedv than that of any other bat cov, and is phylogenetically located at the root of the lineage of pedv and s. kuhlii covs. this finding suggests the presence of a much closer common ancestor of pedv in bats covs. the recombination analysis supports that the s region of pedv may come from the ancestor of α-gx d. considering that bat species such as s. kuhlii prefers to circulate around farmhouses and pigsties, and other pedv-related sequences have been reported in bats in previous reports (simas et al., ; phan et al., ) , the relationship between bat covs and pedv should be further investigated. the discovery of β-sc provides evidence that rhinolophus in sichuan province could harbor sarsr-covs, and reveals a broader geographical distribution of these viruses. the discovery of β-yn b here, combined with previously reported wiv and wiv (ge et al., ; figure | detection of potential recombination events by bootscan analysis. (a) full-length genome sequence of α-gx d was used as a query sequence and btcov/ , α-gx a, and pedv as reference sequences. (b) full-length genome sequence of α-yn was used as a query sequence and ro-batcov hku , hi-batcov hku , and α-hub as reference sequences. all analyses were performed with a kimura model, a window size of base pairs, and a step size of base pairs. the gene map of query genome sequences is used to position breakpoints. yang et al., ) , revealed that these ace -adaptable sarsr-covs were continuously circulating in r. affinis of yunnan province at least from to . these findings emphasize again the importance of continuous longitudinal monitoring for sarsr-covs in rhinolophus over a wide area. although no sads and hku -related cov were found in rhinolophus bats in our study, the surveillance of this cov type in bats is still needed in the future. in our previous study, by the identification of correlated alpha-covs in different miniopterus species from multiple regions, the characteristics of co-infection, recombination, and hostshifting for alpha-covs have been described. here, the alpha-covs of lineage f found in more diverse bat species (including frugivorous and insectivorous bats) extends the known host range of these viruses. however, the recombination events found between α-yn , ro-batcov hku , and hi-batcov hku , and the multiple origins of the s of α-yn , further indicate that a more distant host-shifting in accordance with the recombination of the s region may have happened in the evolutionary history of these viruses among diverse host species. when we collected s. kuhlii in guangxi province, we found that there were many c. sphinx at the sampling site. the pteropodidae bats (c. sphinx bat belongs to pteropodidae) are natural hosts of several emergent human pathogens such as hendra virus, nipah virus, and ebola virus (leroy et al., ; halpin et al., ; sendow et al., ; mari saez et al., ; paez et al., ). an ebola-related filovirus was found in the chinese pteropodidae bats recently (yang et al., ) , and thus, we collected samples of c. sphinx for virome analysis. the c. sphinx beta-cov, β-gx , represents an evolved independent lineage different from hku of rousettus and gccdc of eonycteris under lineage d. based on the partially sequenced region, cov strain philippines/diliman g / previously identified in c. brachyotis from the philippines, could also be clustered into this separate β-gx clade. by providing full-length sequence data, this finding reveals a new host range of this virus in an unreported location. yh, qj, and zw conceived the experiments, analyzed the results, and wrote the manuscript. yh, jd, hs, and zw conducted the experiments and analyzed the results. jz, gz, and sz collected the specimens. all authors reviewed the manuscript. the sars-like coronaviruses: the role of bats and evolutionary relationships with sars coronavirus molecular detection of porcine astrovirus in sichuan province bats: important reservoir hosts of emerging viruses sars and mers: recent insights into emerging coronaviruses human coronaviruses: 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bats as the source of human severe acute respiratory syndrome coronavirus comparative analysis of rodent and small mammal viromes to better understand the wildlife origin of emerging infectious diseases virome analysis for identification of novel mammalian viruses in bat species from chinese provinces novel sarslike betacoronaviruses in bats, china mers-related betacoronavirus in vespertilio superans bats isolation and characterization of a novel bat coronavirus closely related to the direct progenitor of severe acute respiratory syndrome coronavirus characterization of a filovirus (mengla virus) from rousettus bats in china comparative analysis of bat genomes provides insight into the evolution of flight and immunity fatal swine acute diarrhoea syndrome caused by an hku -related coronavirus of bat origin the supplementary material for this article can be found online at: https://www.frontiersin.org/articles/ . /fmicb. . /full#supplementary-material key: cord- -si qml authors: li, hai-yan; zhang, hong-lei; zhao, fu-jie; wang, shi-qiong; wang, zhi-xiang; wei, zhan-yong title: modulation of gut microbiota, short-chain fatty acid production, and inflammatory cytokine expression in the cecum of porcine deltacoronavirus-infected chicks date: - - journal: front microbiol doi: . /fmicb. . sha: doc_id: cord_uid: si qml porcine deltacoronavirus (pdcov) is a novel swine enteropathogenic coronavirus that causes watery diarrhea and induces proinflammatory cytokine responses in piglets. our previous research showed that the specific-pathogen-free (spf) chicks exhibited mild diarrhea and low fecal viral shedding, along with cecum lesions after pdcov infection. disturbances in the homeostasis of the gut microbiota have been associated with various diseases. we aimed to explore the effects of pdcov infection on chick gut microbiota, short-chain fatty acid (scfas) production, and inflammatory cytokine expression in chicks, and also to investigate the relationship between gut microbiota and scfas or inflammatory cytokine expression of the pdcov-infected chicks. results obtained using s rrna sequencing showed that infection with pdcov strain hnzk- significantly altered the composition of chick gut microbiota, with the reduced abundance of eisenbergiella and anaerotruncus genera at days post-inoculation (dpi) (p < . ), and an increased abundance of alistipes genus at dpi (p < . ). the production of scfas in the cecum of pdcov hnzk- –infected chicks, including acetic acid, propionic acid, and butyric acid, decreased in all cases. the expression of inflammatory cytokines (interferon-γ, tumor necrosis factor-α, and interleukin- ) was increased in the cecum tissue and serum of the pdcov hnzk- –infected chicks when detected by quantitative real-time polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. further analysis showed significant correlation between bacterial genera and scfas or inflammatory cytokines expression in cecum of the pdcov infected chicks. these findings might provide new insight into the pathology and physiology of pdcov in chicks. porcine deltacoronavirus (pdcov) is an enveloped, positivesense single-stranded rna virus, with a genome length of approximately kb (zhang, ) . this virus was originally found in hong kong in (woo et al., ) , and the first outbreak of pdcov-related diarrhea in swine herds was reported in the united states in (wang et al., ) . as an important enteropathogen in pigs, pdcov can cause acute diarrhea, vomiting, and dehydration in neonatal piglets (chen et al., ; hu et al., ) . moreover, the infected pigs are characterized by thin and transparent intestinal walls and accumulation of large amounts of yellow fluid in the intestinal lumen (jung et al., ; . further, pdcov-infected piglets show signs of proinflammatory cytokine responses during acute infection (jung et al., ) . pdcov has caused huge economic losses for the pig industries. apart from infection of gnotobiotic (gn) and conventional pigs, pdcov was also reported to have limited ability to infect gn calves (jung et al., ) . recent studies showed the susceptibility of specific-pathogen-free (spf) chicks to pdcov infection, with the clinical syndrome of mild diarrhea and slight lesions in cecum, which was weaker than that of piglets infected with pdcov (liang et al., ; boley et al., ) . however, the reason why pdcov infection can cause intestinal damage is still unknown. the chicken gastrointestinal tract is an important site for immune cell development, which not only regulates gut microbiota, but also maintains extraintestinal immunity. intestinal commensal microbes play a crucial role in gut homeostasis through mutually beneficial interactions with the host immune system (deriu et al., ) . once the intestinal mucosal barrier and microbiota are destroyed, intestinal inflammation will occur. in turn, the host's inflammatory response will elicit changes in the microbial community structure (deriu et al., ) . the gut microbiota produces various metabolites, including short-chain fatty acids (scfas), which can regulate the host antiviral immune response (budden et al., ) . propionate and butyrate have also been reported to induce differentiation of t-regulatory cells, assisting in control of intestinal inflammation (donohoe et al., ; louis et al., ) . in chicks, recent studies have emphasized the key roles of intestinal microbiota in shaping immunity against viral diseases, including avian influenza, marek's disease, newcastle disease (nd), and infectious bursal disease (ibd). these studies not only reflected connections between gut microbiota and distal organs in regulatory functions, but also emphasized the interaction between intestinal microbiota and viral infections and their impacts on immune regulations (lillehoj and trout, ; balamurugan and kataria, ; li, ; chen et al., ; yitbarek et al., ) . it was reported that different commensal bacteria had their own unique role against viral infection by modulating diverse immune mechanisms (abt et al., ) . in the h n avian influenza virus infected chicks, increased levels of interferon gamma (ifn-γ). tumor necrosis factor alpha (tnfα), and interleukin- a (il- a) led to intestinal microflora dysbiosis (li h. et al., ) . however, there are no studies emphasizing how pdcov affects the gut microbiota in chicks. thus, in the current study we aimed to observe the intestinal microbiota, scfas, and inflammation responses to pdcov infection in chicks and elucidate the potential associations among them, which will provide the theoretical basis for investigation of the etiology and pathogenesis of pdcov. the virulent pdcov strain-hnzk- (genbank accession number mh ) was isolated and identified by our laboratory and propagated in llc-porcine kidney (llc-pk) cells. passage of this strain was used in current study. virus propagation and virus titers were performed as described previously (liang et al., ) . the spf chicks were purchased from jinan sipafas poultry co. ltd. in jinan, china. twenty-four-day-old spf chicks with similar body weight (about g/chick) were randomly divided into two groups, the control (mock) and pdcov hnzk- infected groups. all chicks were housed in the same facility but in different rooms under biosafety conditions and allowed free access to water and feed during the experiment. feed was formulated to meet or exceed the national research council nutrient requirements for chicks (national research council [nrc] , ; table ). each chick was intragastrically inoculated with pdcov strain hnzk- or eagle's minimum essential medium (mem) with µl/chick ( . log ge/chick) (n = /per group). based on our previous study (liang et al., ) , three chicks in each group were selected randomly for necropsy at and dpi, respectively. the cecal contents were collected for analysis of gut microbiota and scfas. the cecum tissue was collected for inflammatory cytokine mrna measurements. the serum was collected for inflammatory cytokine detection by enzyme-linked immunosorbent assay (elisa). viral rna was extracted from cecal content suspensions using the trizol tm method (invitrogen, carlsbad, ca, united states) according to the manufacturer's instructions. the viral rna was further reverse transcribed into cdna. viral rna titers were determined using quantitative real-time polymerase chain reaction (qrt-pcr) as reported previously (liang et al., ) . the detection limit of the qrt-pcr was . log ge/ml. the scfas in cecum were determined using gas chromatography (gc) according to a previously described method (stewart et al., ) . the samples were placed on a db-wax column ( m long × . mm diameter and . µm film thickness) and were separated by using a trace tm gc with flame ionization detector (fid). the temperature program was • c for . min, then raised to • c at • c/min and held for min, then raised to • c at • c/min and held for min. samples were run with a : split ratio and a . ml/min column flow. high-purity hydrogen was used as the carrier gas. the temperatures of the injector and detector were and • c, respectively. the levels of expression of the inflammatory cytokines (ifn-γ, tnf-α, and il- ) in the cecum of spf chicks were quantified by qrt-pcr. cecum tissue samples were homogenized by the tissuelyser- (jingxin, shanghai, china). total rnas were extracted from the supernatant of the cecum tissue lysate by trizol tm reagent kit (invitrogen, carlsbad, ca, united states) and were quantified by nanodrop spectrophotometer (thermo fisher scientific, waltham, ma, united states), then further reverse transcribed into cdna. the total reaction volume of µl contained . µl of dna, . µl of sybr green qpcr mix (takara bio inc., tokyo, japan), µl of h o, and . µl of each specific primer. primers were synthesized according to previous reports (emami et al., ) and are shown in table . reactions were performed under the following conditions: one cycle of preincubated samples at • c for min and cycles of amplification samples at • c for s, • c for s, • c for s. target gene expression of each sample was normalized using glyceraldehyde -phosphate dehydrogenase (gapdh). the relative expression of the target gene for each sample was calculated using the − ct method (livak and schmittgen, ) . the expression levels of the ifn-γ, tnf-α and il- in serum were detected using elisa kit from nanjing jian cheng biological institute (nanjing, china). serum samples were obtained and measured as previously described (huang et al., ) . absorbance was read at nm using a microplate spectrophotometer. the inflammatory cytokine concentrations were calculated according to a best-fit standard curve. raw fastq files were demultiplexed, quality-filtered by trimmomatic, and merged by flash (magoč and salzberg, ) . sequences with ambiguous bases, shorter than bp, and sequences with an average mass less than were removed. the high-quality reads were clustered into operational taxonomic units (otus) with % similarity cutoff using uparse (version . ) and used for further analysis of the venn diagram, alpha diversity indices (shannon and simpson), and richness estimators (ace and chao). beta diversity measures were based on weighted-unifrac distance analysis and shown by principal coordinate analysis (pcoa), which was conducted to assess the relationships among the different groups. lefse was applied to recognize which bacterial taxa contribute to the differences between the two groups (segata et al., ) , and the minimal threshold of linear discriminant analysis (lda) was set at . . the difference between specific taxa was analyzed using one-way analysis of variance (anova) followed by post hoc t-tests. data analysis of inflammatory cytokines and scfas was performed using graphpad prism (version . , la jolla, ca, united states). a p < . or . was considered statistically significant or highly significant, which is indicated as follows: * p < . , * * p < . . spf chicks, intragastrically inoculated with pdcov hnzk- at days old, showed mild diarrhea at dpi. pdcov rna was detected in the intestinal contents by qrt-pcr at and dpi while non-infected control chicks appeared normal throughout the course of the experiments. pdcov hnzk- infection had the operational taxonomic units (otus) were defined at a % similarity level. the coverage percentage (good's) and richness estimators (ace and chao) and diversity indices (shannon and simpson) were calculated using good's method and the mothur program, respectively. in the same column, values with the same superscript letter are not significantly different (p > . ); those with different superscript letters differ significantly (p < . ). figure | venn map. venn map of shared otus based on the sequences with more than % similarity (n = ). the mock group at dpi (mock- d), the pdcov hnzk- group at dpi (hnzk- - d), the mock at dpi (mock- d), and the pdcov hnzk- group at dpi (hnzk- - d). no effect on feed consumption compared to controls during the whole experimental period. a total of cecal concent samples, the control chicks (n = ), and pdcov hnzk- groups (n = ) at and dpi, respectively, were evaluated using s rrna gene sequencing. a total of , sequences with a median read length of bp (range from to bp) were collected. the total number of unique sequences from the four groups was . the sequence and species-level otus of each group are shown in table . the venn diagram showed that otus of the total gut microbial richness ( ) were shared among all the sequenced samples. two hundred and fifty-nine otus were shared between the samples of the mock and pdcovhnzk- groups with a total of at dpi. three hundred and seventeen otus of the total gut microbial richness ( ) were shared between the samples of mock and pdcov hnzk- groups at dpi (figure ). alpha diversity estimators of community are shown in table . gut microbial richness, according to the ace and chao indexes, was significantly increased in the pdcov hnzk- group when compared with that of the mock group at dpi (p < . ), while there was no significant difference between the two groups at dpi (p > . ). in shannon and simpson alpha diversity indexes, the differences between the pdcov hnzk- and the mock groups at and dpi did not reach statistical significance (p > . ). beta diversity analysis showed that there were some similarities in the microbial composition between the mock and the pdcov hnzk- groups at dpi. however, the microbial composition from chicks with pdcov inoculation and the controls could be divided into two different clusters at dpi, indicating that the cecal microbial community structure and composition in the control group were significantly different from those in the pdcov hnzk- group at dpi (figure ) . to further investigate whether pdcov hnzk- infected chicks experienced any significant alteration in gut microbiota, we analyzed the relative abundance of microbiota at the phylum, family, and genus levels. at dpi, genus level analysis showed the taxonomic abundance was altered in the pdcov hnzk- group, which was characterized by lower abundance of eisenbergiella and anaerotruncus (p < . ). there were no significant differences at the levels of phylum and family between the mock and the pdcov hnzk- groups (figure a) . at dpi, the ratio of firmicutes to bacteroidetes in the pdcov hnzk- group was lower than that of the mock group. at the family level, the proportion of rikenellaceac in the chicks inoculated with pdcov hnzk- significantly increased (p < . ). at the genus level, alistipes and the unclassified f-ruminococcaceae were significantly increased (p < . ), whereas the proportion of ruminococcaceae-ugg- was significantly decreased in the pdcov hnzk- group when compared to that of the mock group at dpi (p < . ) (figure b) . the characteristics of bacterial taxonomic abundant in the collected samples were further analyzed using lefse algorithm (figure ) . lefse analysis revealed that there existed main and discriminative features from phylum to genus (lda score > ) between the mock and the pdcov hnzk- -inoculated chicks at and dpi (figures a,b) , respectively. more specifically, eisenbergiella and anaerotruncus were the most differential microbiota in the mock at dpi. moraxellaceae, acinetobacter, pseudomonadates, eubacterium, and norank-f lachnospiraceae in the pdcov hnzk- group increased when compared to that in the mock group. at dpi, we identified microbial biomarkers from the pdcov hnzk- group, including rikenellaceae, bacteroidia, bacteroidetes, bacteroidales, alistipes, unclassified-f-ruminocococcaceae, sphingomonadaceae, novosphingobium, and lachnoclostridium, figure | beta diversity measures in cecal microbiota of chickens (n = ). the scatterplot of the principal coordinate analysis (pcoa) scores shows four different clusters (weighted-unifrac distance), the blue represents the pdcov hnzk- group at dpi (hnzk- - d), the red represents the mock group at dpi (mock- d), the purple represents the pdcov hnzk- group at dpi (hnzk- - d), and the green represents the mock group at dpi (mock- d). frontiers in microbiology | www.frontiersin.org figure | significant differences analysis in cecal microbiota of chickens. (a) statistical comparison of the relative microbial abundance from phylum to genus between the mock and the pdcov hnzk- groups at dpi (n = ). (b) statistical comparison of the relative microbial abundance from phylum to genus between the mock and the pdcov hnzk- groups at dpi (n = ). **p < . ; *p < . . lefse identifies the taxa with the greatest differences in abundance from phylum to genus between the mock and the pdcov hnzk- groups at dpi (n = ). (b) lefse identifies the taxa with the greatest differences in abundance from phylum to genus between the mock and the pdcov hnzk- groups at dpi (n = ). linear discriminant analysis (lda) effect size (lefse) analysis showing those abundance from phylum to genus between groups; only taxa of an lda significant threshold of . are shown. which could be distinguished from the mock group. clades were more abundant in the mock group than that in the pdcov hnzk- group, including firmicutes, ruminocococaceae-ucg- , clostridia, norank-f-clostridiales-vadinbb -group, clostridiales-vadinbb -group, anaerostipe, eisenbergiella, coriobacteriaceae, coriobacteriales, actinobacteria, and actinobacteria. there were bigger differences on microbiota compositions between the early and later stages of pdcov infection (at and dpi, respectively). compared to the control, there was a decreasing trend in the amounts of acetic acid (aa), propionic acid (pa), and butyric acid (ba) in the pdcov hnzk- group at and dpi. furthermore, pa and aa levels in the pdcov hnzk- group were greatly decreased at and dpi, respectively (p < . ) (figure ). as shown in figure , the expression of ifn-γ (p < . ), tnfα (p < . ) and il- (p < . ) was significantly upregulated at dpi compared with that in the mock chicks (figures a-c) . furthermore, ifn-γ expression was significantly upregulated (figure a ) at dpi (p < . ). to evaluate if pdcov infection could affect ifn-γ, tfn-α, and il- secretion in serum, the concentrations of ifn-γ, tfnα, and il- in serum were tested with elisa kits. our results showed that the changes of the inflammatory cytokines in serum were similar with those in the cecum tissue. the levels of ifnγ and tnf-α were approximately two and three times as high as in the pdcov hnzk- group when compared to that of the mock group at dpi (p < . ). the level of il- in the pdcov infection group was also increased at dpi (p < . ). the serum levels of ifn-γ in the pdcov-infected chicks were higher than in the mock group at dpi (p < . ), and no significant differences were observed for the secretion of tnfα and il- between the mock and the pdcov hnzk- groups at dpi (figures a-c) . spearman's correlation analysis was conducted between the top bacterial genera and the environmental factors (scfas, proinflammatory cytokines), and was directly reflected by a heatmap (figure ) . the threshold | r| > . was considered as having a correlation. the results indicated that eisenbergiella was negatively correlated with tnfα (p < . ) and ifn-γ (p < . ) expression levels, and was positively correlated with pa (p < . ) at dpi ( figure a) . the ruminiclostridium_ also showed a positively correlation with the il- expression (p < . ), while norank_f_ ruminococcaceae was negatively correlated with ba (p < . ). at dpi (figure b) , alistipes and unclassified_f_ ruminococcaceae showed an extremely significant positive correlation with ifn-γ secretion (p < . ), but eisenbergiella and norank_f_clostridiales_vadinbb _group were negatively correlated with it (p < . ). the level of pa was associated with the increase in the abundance of ruminiclostridium_ (p < . ). ruminococcus and eisenbergiella were positively correlated with aa (p < . ), while alistipes showed a negative relationship with it (p < . ). eisenbergiella, unclassified_f_lachnospiraceae, ruminococcaceae_ucg- , norank_f_clostridiales_vadinbb _group, and ruminiclostr idium_ were positively correlated with ba. gut microflora play an important role in shaping immunity against viral diseases in host. disruption of microbial homeostasis is associated with a variety of pathological states, which helps the establishment of acute viral infections in chickens (abaidullah et al., ) . in this study, the infection model of pdcov was developed on spf chicks. our results demonstrated similar clinical diseases, and the dynamics of the virus shedding in cecum were observed in all spf chicks inoculated with the pdcov hnzk- strains when compared with our previous experiment (liang et al., ) . the influence of pdcov infection on cecum microbiota of spf chicken was evaluated, pdcov hnzk- infection significantly altered the gut microbiota composition and decreased scfas products in chicks' cecum. simultaneously, the expression levels of inflammatory cytokines (ifn-γ, il- , and tnf-α) in serum and cecum tissue were significantly increased, resulting in the inflammatory response in the chicken. the analysis showed a significant correlation between bacterial genera and scfas or inflammatory cytokines. the diversity of indigenous intestinal microbiota is one of the key factors in resisting the colonization of invading pathogens (keesing et al., ) . several studies have reported that the diarrhea-relating coronaviruses could influence the intestinal microbiota diversity in pigs (liu et al., ; song et al., ; huang et al., ; tan et al., ) . indeed, many viral agents have been shown to alter the intestinal microbiota in chickens, such as aiv (yitbarek et al., ) , marek's disease virus (mdv) (lillehoj and trout, ) , ndv (li, ) , and ibdv (li l. et al., ) . in pdcov-infected chicks, the species richness indices were increased at dpi, and there were no significant changes in the overall α-diversity of cecal microbiota at dpi. it is possible that the reduced immune response may account for the change of microbial diversity. from the pcoa results, microbiota structure in the pdcov infected group was clearly distinguished from that of the mock group at dpi, which also reflected spf chicks' resistance to pdcov. from microbial community profiling, the difference between the groups was analyzed at the phylum, family, and genus level. eisenbergiella and anaerotruncus genera were significantly decreased in the pdcov hnzk- at dpi, indicating that pdcov hnzk- infection had a marked influence on chicks' cecal microbiota. according to previous studies, the decrease of the ratio of firmicutes-to-bacteroidetes was observed in mice with diabetes (wen et al., ) and in some patients with crohn's disease and ulcerative colitis (frank et al., ) . in our results, a decreased ratio of firmicutes-to-bacteroidetes occurred in the pdcov hnzk- group at dpi. more remarkably, the alistipes, considered an opportunistic pathogen (pandit et al., ; zhang et al., ) , was significantly increased in the pdcov hnzk- group at dpi. we used linear discriminant analysis (lda) of effect size (lefse) to confirm the taxa that most likely explains the differences between the pdcov hnzk- and mock groups. these findings provide compelling evidence that pdcov hnzk- induced microbiota imbalance and showed that recovery was difficult in a short time period after pdcov hnzk- infection. production of scfas, as the end products of protein and carbohydrate fermentation (cummings and macfarlane, ) , can easily be affected by the status of the gut microbiota (lloyd-price et al., ) . studies have shown that lactobacillus salivarius and l. agilis could increase propionate and butyrate contents in cecum of chicks (meimandipour et al., ) . the changes in butyrate and propionate can influence intestinal physiology and immune function, while acetate can alter lipid metabolism (macfarlane and macfarlane, ) . our data showed that the changes of gut microbiota composition contributed to the decreased scfas, which reduced this protective effect of scfa in the cecum. however, the interaction of gut microbiota with scfas to respond to pdcov infection needs further studies. inflammatory immune responses in the gut can alter the gut luminal environment in a way that favors dysbiosis (winter et al., ) . chicks with dysbiosis are more prone to acute viral infection (abaidullah et al., ) , which may in turn further affect the pathogen infectivity. eisenbergiella is strongly correlated with increased levels of tnf-α and ifn-γ, which help in modulating the functional activities of the cells of the immune system (liu et al., ) , and to block virus replication and prevent clinical disease (bradley, ; praveena et al., ) . this may be the reason that eisenbergiella was decreased in the pdcov hnzk- group at dpi. moreover, the observation is similar to the increased proinflammatory (tnf-α) cytokine responses of -day-old gn pigs to acute pdcov infection (jung et al., ) . alistipes was strong positively correlated with ifn-γ levels, and the numbers of ruminococcaceae_ucg- were strong negatively correlated with ifn-γ levels, indicating that these bacteria can be tolerated, with ifn-γ responses benefiting for those genera of bacteria. the anti−inflammation activity could be explained in the study regarding to the administration of norank_f_lachnospiraceae in gut, and was related to the il− cytokine (olszak et al., ) . the results showed the change of microbiota in the cecum was related to intestinal inflammation (directly or indirectly, positively or negatively). in conclusion, the present study revealed that obvious changes were found in the microbial community of chicks infected with pdcov hnzk- . pdcov-inoculated chicks showed a decrease in eisenbergiella and anaerotruncus populations at dpi and an increase in pathogenetic alistipes at dpi with a reduction in scfas production. the changes in the microbial community may be related to changes in inflammatory cytokine expression. these results provide new insights into the pathophysiology of chicks infected with pdcov. the obtained raw sequencing reads have been added to the ncbi sra page (accession number: prjna ). other data generated during the study are included in this article. the experimental procedures were approved by the laboratory animals ethics committee at hennan qixiang biological technology co. ltd. (hnqx- - ) , and all husbandry practices and euthanasia were performed with full consideration of animal welfare. hl and hz performed the experiments. hl, fz, and sw performed statistical analyses and wrote the manuscript. z-xw and z-yw designed the study and revised the manuscript. all authors read and approved the final manuscript. current findings on gut microbiota mediated immune modulation against viral diseases in chicken commensal bacteria calibrate the activation threshold of innate antiviral immunity economically important non-oncogenic immunosuppressive viral diseases of chicken-current status porcine deltacoronavirus infection and transmission in poultry, united states tnf-mediated inflammatory disease emerging pathogenic links between microbiota and the gut-lung axis role of the intestinal microbiota in the immunomodulation of influenza virus infection isolation and characterization of porcine epidemic diarrhea viruses associated with the disease outbreak among swine in the 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detection and genetic characterization of deltacoronavirus in pigs innate immunity and intestinal microbiota in the development of type diabetes the dynamics of gut-associated microbial communities during inflammation discovery of seven novel mammalian and avian coronaviruses in the genus deltacoronavirus supports bat coronaviruses as the gene source of alphacoronavirus and betacoronavirus and avian coronaviruses as the gene source of gammacoronavirus and deltacoronavirus gut microbiota-mediated protection against influenza virus subtype h n in chickens is associated with modulation of the innate responses porcine deltacoronavirus: overview of infection dynamics, diagnostic methods, prevalence and genetic evolution the effect of exposure to high altitude and low oxygen on intestinal microbial communities in mice the authors thank dr. hui hu from henan agricultural university for designing the study and revising the manuscript. key: cord- - xemmaqt authors: ferreira, eliane de oliveira; penna, bruno; yates, edwin a. title: should we be worried about clostridioides difficile during the sars-cov pandemic? date: - - journal: front microbiol doi: . /fmicb. . sha: doc_id: cord_uid: xemmaqt nan the outbreak caused by the novel coronavirus sars-cov- and its associated symptoms, termed covid- disease, originally in wuhan, china in , has rapidly become a global pandemic (park, ) . the advent of this contagious virus continues to challenge healthcare systems and has impacted the global economy severely. by th june , it had been reported that at least countries and territories were witnessing cases, with , , confirmed cases and deaths. notably, a significantly higher mortality rate has been observed among patients over years old and the immunocompromised (guo et al., ) . although the sars-cov- seems to be less virulent than other coronavirus family viruses, it still has a high rate of transmissibility and can infect lung cells and enterocytes. in severe cases, the symptoms arise from the immune/inflammatory response, resembling macrophage activation syndrome (mas), and can include diffuse pulmonary intravascular coagulation, resulting in hypoxia. in the majority of patients, elevated levels of cytokines il β, il- , and il- , and tnfα (tay et al., ) have been reported, and can progress to a so-called "cytokine storm, " multiple-organ failure and death tay et al., ) . the spread of sars-cov- resulted in a rapid increase in the admission of patients, requiring artificial respiratory support, often stretching the capacity of healthcare systems and highlighting the urgent need for effective therapeutic strategies. currently, there are no vaccines or treatments that have been developed specifically for sars-cov- infections, although vaccines and a number of drugs, both of existing re-purposed and novel medications, are in clinical trials (chary et al., ; park, ) . in a great number of individuals, the infection will cause a sub-clinical illness or, mild upper respiratory tract disease. however, when a patient progresses to severe disease, clinicians have to consider using one of the following options: (i) trial drugs, such as: remidesivir, ribavirin, favipiravir and others that aim to hinder virus replication indirectly; (ii) corticosteroids to suppress lung inflammation; (iii) the use of biologics targeting some of the cytokines reported to be up-regulated in patients; (iv) the convalescent plasma transfusion therapy; (v) heparin therapy for improving hypoxia, to avoid intravascular coagulation, which may also hinder the invasion of sars-cov- ; (vi) zinc, which controls the proliferation of neutrophils, nk cells, macrophages and lymphocytes and the adverse effect of ros; (vii) antibiotics (azithromycin and doxycycline), commonly used to modulate the inflammation response (mycroft-west et al., ; guo et al., ; rahman and idid, ; zhai et al., ) . to make things worse, several studies have demonstrated, based on laboratory, clinical and epidemiology research, that secondary bacterial infections can be a complication of respiratory viral infections, increasing morbidity and mortality. bacterial co-infections were also reported with mers-cov patients in intensive care units (morris et al., ) . indeed, during the influenza epidemics, the majority of deaths were due to bacterial infections (morens et al., ; gill et al., ; morris et al., ) . one of the reasons that bacterial co-infections occur is that the upper respiratory tract has a diverse microbiota and can harbor opportunistic pathogens including streptococcus pyogenes, s. pneumoniae, haemophilus influenzae, staphylococcus aureus, and pseudomonas aeruginosa. while this problem is likely to be most prevalent among the aged and immunocompromised patients, it is by no means restricted to these groups. for example, pneumonia caused by the rare pathogen, mycoplasma pneumoniae, has been reported in a years old man with ventilatory support during sars-cov- infection in china, by fan et al. ( ) . bengoechea and bamford ( ) , have suggested three scenarios concerning sars-cov- /bacterial co-infections: (a) secondary sars-cov- following bacterial infection or colonization; (b) combined viral/bacteria pneumonia; (c) secondary antimicrobial resistant bacterial (amr) after sars-cov- . the authors also point out that administering high doses of drugs to modulate the immune response, such as glucocorticoids to decrease inflammatory processes, can predispose them to fatal secondary bacterial respiratory infections. although the first two perspectives are of great concern, here, we would like to emphasize the need to consider carefully any increased antibiotic use in combating covid- (bengoechea and bamford, ) . this is particularly relevant in light of the appearance of new hypervirulent bacteria, such as clostridioides difficile, which is still responsible for outbreaks in several countries and remains a global health problem. furthermore, since the likelihood of other viral diseases emerging in the future is high, the development of an integrated and broadly applicable strategy to enable an early assessment of the inter-linked considerations of disease treatment and secondary, bacterial disease, would be desirable. antimicrobial agents have been used for decades and have altered medicine significantly, making a major contribution to the control of infectious diseases (wright, ) . however, following their discovery and their global over-and misuse, they have also contributed to the appearance of novel resistant microbes and so called "super-bugs" in all niches, including the human gastrointestinal tract microbiome, which is a huge reservoir for bacteria, archaea, fungi and viruses. the human gut microbiome plays an important role in health, by fermenting indigestible food components into absorbable metabolites, producing essential vitamins, removing toxic compounds, competing with pathogens and helping to shape the immune system (heintz-buschart and wilmes, ). most of these functions are interconnected and tightly linked with human physiology. equally though, this human gut consortium can be influenced by several factors that include antibiotics (rice o'connor, ) and, its overall composition and diversity can be altered, or become unbalanced. changes in gut microbiota structure and function after antibiotic treatment create a metabolic environment that favors c. difficile germination and colonization, associated with infectious diarrhea, which is debilitating to patients and extremely costly, with symptoms ranging from diarrhea to fulminant colitis, toxic megacolon, and death (farooq et al., ; czepiel et al., ) . nearly every antibiotic has been implicated in the development of cdi, including the drugs metronidazole and vancomycin, which are used for its treatment. the risk for development of cdi is -to -fold higher during, and in the weeks subsequent to, antimicrobial therapy and -fold higher for the next months (hensgens et al., ) . covid- patients receive an empirical antimicrobial therapy with moxifloxacin, cefoperazone, or azithromycin , drugs that are strongly associated with cdi. apart from antibiotic use, there are other factors associated with cdi, including higher age (> years), longer hospitalization, the use of proton pump inhibitors, comorbidities, chemotherapy, chronic kidney disease, and feeding tubes (bagdasarian et al., ) . although some of those risk factors for cdi are also related to higher probability rates of mortality in severe sars-cov- infection, the limited number of cdi cases reported among covid- patients is somewhat surprising. both infections can present similar digestive manifestations including diarrhea, nausea, vomiting and abdominal pain, meaning clinicians need to be even more vigilant for potential co-infections with c. difficile. another problem that covid- may cause relates to fecal microbiota transplantation (fmt) (chiu et al., ; khanna and pardi, ) , which is used for recurrent cases of cdi, accounting for - % of cases (martin and wilcox, ) , in particular, for those patients whose treatment failed, which represent - % of cases (vigvari et al., ) . fmt is based on stools donated by volunteers, which are screened and so are considered healthy donors although fmt seems to reduce the risk for recurrent cdi, the efficacy and safety of this procedure is still under evaluation, especially because the inconsistency found with clinical trials (wilcox et al., ) and deficiency of standards methods for producing fmt (nicco et al., ) . in addition to the possible transmission of opportunistic and amr bacteria, in light of the sars-cov- , all microbiome replacement therapies from now on will require accurate diagnosis to guarantee their safety or, may need to be suspended until the pandemic is over. to the best of our knowledge, only two clinical surveillances studies reviews have been published. the first one in may , reporting cdi with covid- at the medical center in detroit, michigan, usa. patients were screened from th march to th april, , and in cases received antibiotic therapy, presented diarrhea, experienced sars-cov- infection and were co-infected with c. difficile (sandhu et al., ) . the authors emphasized that when cdi is present as a co-infection with covid- and the diarrhea persists, therapy can be difficult because of the sars-cov- infection. another concern is the inappropriate use of antibiotics, particularly among patients with mild covid- , which could increase the long-term threat of amr and new epidemic strains. the second one, is from the saint michael's medical center in newark (new jersey, usa), of a year-old man who tested positive for c. difficile at admission and presented diarrhea for days. he did not use any drugs correlated with cdi development and had no previous contact with any individuals presenting diarrhea. besides that, he tested positive for sars-cov- and was presenting fever, respiratory symptoms and lymphopenia. after he was mechanically ventilated, he received vancomycin and metronidazole, but unfortunately, he died with pneumoniae and septic shock. authors emphasize that doctors should consider cdi in patients with covid- presenting diarrhea (lakkasani et al., ) . the dearth of studies regarding secondary infections, such as clostridioides difficile, in covid- patients makes it difficult to measure the effect of the pandemic on antimicrobial stewardship programs and on long term antimicrobial resistance. while increased awareness regarding personal hygiene and extensive use of protective equipment may lead to reductions of healthcare associated infections, the challenge of strictly isolating and managing covid- patients in many healthcare systems, often in proximity to patients colonized with c. difficile, and the inevitable higher workload imposed on healthcare staff could lead to additional hospital transmissions. the increased use of antibiotics to treat covid- may, inadvertently, have resulted in an under-reporting of c. difficile infection. actually, spigaglia ( ) has published an article expressing her opinion about the covid- and the impact in elderly patients, who will probably become more susceptible to cdi. the author also demonstrates her concern about the low number of bacterial infections cases related to patients with sars-cov- . to ensure appropriate treatment and to improve patient outcome, increased vigilance and improved diagnosis are both necessary. given that future emerging viral diseases are highly likely, we would urge increased awareness of the issue and call for informed debate around how to implement effective measures to meet these challenges. in conclusion, it seems highly likely that cases of cdi are being under-reported among covid- patients and the increased use of antibiotics may, in part, be responsible. to ensure appropriate treatment and improve patient outcome, increased vigilance and improved diagnosis are both 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infection (cdi): possible implications for elderly patients the trinity of covid- : immunity, inflammation and intervention faecal microbiota transplantation in clostridium difficile infections the efficacy and safety of fecal microbiota transplant for recurrent clostridium difficile infection: current understanding and gap analysis the antibiotic resistome: the nexus of chemical and genetic diversity since january elsevier has created a covid- resource centre with free information in english and mandarin on the novel coronavirus covid- . the covid- resource centre is hosted on elsevier connect, the company' s public news and information all authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication. the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © ferreira, penna and yates. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- - ee i authors: kruchten, anne e. title: a curricular bioinformatics approach to teaching undergraduates to analyze metagenomic datasets using r date: - - journal: front microbiol doi: . /fmicb. . sha: doc_id: cord_uid: ee i biologists with bioinformatic skills will be better prepared for the job market, but relatively few biology programs require bioinformatics courses. inclusion in the curriculum may be hindered by several barriers, including lack of faculty expertise, student resistance to computational work, and few examples in the pedagogical literature. an -week wet-lab and in silico research experience for undergraduates was implemented. students performed dna purification and metagenomics analysis to compare the diversity and abundance of microbes in two samples. students sampled snow from sites in northern minnesota and purified genomic dna from the microbes, followed by metagenomic analysis. students used an existing metagenomic dataset to practice analysis skills, including comparing the use of excel versus r for analysis and visualization of a large dataset. upon receipt of the snow data, students applied their recently acquired skills to their new dataset and reported their results via a poster. several outcomes were achieved as a result of this module. first, youtube videos demonstrating hands-on metagenomics and r techniques were used as professional development for faculty, leading to broadened research capabilities and comfort with bioinformatics. second, students were introduced to computational skills in a manner that was intentional, with time for both introduction and reinforcement of skills. finally, the module was effectively included in a biology curriculum because it could function as either a stand-alone course or a module within another course such as microbiology. this module, developed with course-based undergraduate research experience guidelines in mind, introduces students and faculty to bioinformatics in biology research. in , botanist hans winkler coined the term "genome" as a fusion of the words gene and chromosome (winkler, ) . since that time, the "omics" fields have exploded, creating such terms as "pseudome" (the population of pseudogenes), "translatome" (the population of proteins in the cell, weighted by their abundance level), and many others that are increasingly becoming a normal part of the lexicon for biologists . the term "bioinformatics" was defined by luscombe et al. ( ) , as "conceptualizing biology in terms of molecules (in the sense of physical chemistry) and applying "informatics techniques" (derived from disciplines such as applied maths, computer science and statistics) to understand and organize the information associated with these molecules, on a large scale. in short, bioinformatics is a management information system for molecular biology and has many practical applications." undergraduates in biology should be trained in this field to successfully compete in the job market and make vital contributions to the biological sciences as their careers mature. the vision and change: a call to action report of (brewer and smith, ) emphasized that undergraduate biology students should have competence in computational and systems level approaches and the ability to use large databases. only a small fraction of institutions offer a full undergraduate bioinformatics program (mellon, ) , but several offer courses on bioinformatics. in the state of minnesota, % of public and private school biology departments offer a bioinformatics course in their curriculum but none appear to require it for the degree. this may reflect a lack of expertise among faculty to teach the course. in , bachelor's degrees in biology were conferred to graduates in the united states (us-doe, ). among bachelor's degree holders - years old, biology graduates' annual salaries were not significantly different than the median annual income of all degree holders of $ , , but computer and information science degree holders had an annual income of $ , , well above the median income (nces, ) . a slight increase in biology-related computer information jobs is predicted, suggesting that biology majors would be well-served to develop computer information skills to complement their biology degrees (araneo et al., ) . bioinformatics is a broad field that encompasses gene alignment tools, crowdsourcing approaches, metagenomics, and many others. rather than lecturing about bioinformatics, many groups have chosen to incorporate bioinformatics tools into cures (course-based undergraduate research experiences). in cures, students are working in classes on research projects of interest to the broader scientific community (auchincloss et al., ) . on the curenet website , several bioinformatics cures have been shared for faculty adoption and participation, including a crispr-cas project , a study of iron uptake in insects , genome solver: microbial comparative genomics , and the genomics education partnership (gep) . these programs, and many others across the country, teach students a variety of gene-based bioinformatics approaches including using blast, multiple gene alignment, primer design, and many others. students develop strong gene analysis skills while also contributing to active scientific research projects in the process. while (wang et al., ) and crowdsourcing datasets of antibiotic resistance in microbes (freeman et al., ; small-world, ) . students in these courses develop research skills such as bacterial culturing, sterile technique, pcr, and hypothesis building. few projects, however, teach undergraduates the computational skills required to statistically analyze "big data" in biological fields. computational skills are required to analyze and find patterns in big data, which includes the four vs: volume of data, velocity of processing the data, variability of data sources, and veracity of the data quality. applications of big data analysis can be found everywhere, but for biologists especially important applications include genome sequencing, ecological studies (such as of microbiomes), and health care information (li and chen, ) . graduates of biology programs have opportunities for employment in any of these fields but may not have the important computational skills in parallel with wet lab or field biology skills to be successful in big data fields. there seem to be few cures or similar programs published in the literature that provide instructions for how faculty can implement curricular modules to help students develop these big data skills. several groups have outlined a series of bioinformatics competencies for life scientists, including coursesource (the bioinformatics learning framework) (rosenwald et al., ) , the curriculum task force of the international society of computational biology (iscb) education committee (mulder et al., ) , and the network for integrating bioinformatics into life sciences education (niblse) (wilson sayres et al., ) . building on previous work from both coursesource and iscb, niblse surveyed instructors at us institutions and used the data to develop a list of core competencies for undergraduate life scientists. while many of the core competencies focus on genomics-based bioinformatics skills, several of the competencies are addressed by the work in this project. the competencies are listed below (wilson sayres et al., ) , and the bolded ones are addressed by the approach in this project: • c . explain the role of computation and data mining in addressing hypothesis-driven and hypothesis-generating questions within the life sciences. • c . summarize key computational concepts, such as algorithms and relational databases, and their applications in the life sciences. • c . apply statistical concepts used in bioinformatics. • c . use bioinformatics tools to examine complex biological problems in evolution, information flow, and other important areas of biology. • c . find, retrieve, and organize various types of biological data. • c . explore and/or model biological interactions, networks, and data integration using bioinformatics. • c . use command-line bioinformatics tools and write simple computer scripts. • c . describe and manage biological data types, structure, and reproducibility. • c . interpret the ethical, legal, medical, and social implications of biological data. importantly, both the coursesource bioinformatics learning framework and the iscb curriculum task force recognize that there are different levels of users of bioinformatics curriculum, including bioinformatics engineers, bioinformatics scientists, and bioinformatics users. the approach described here is geared toward bioinformatics users, including both faculty who are interested in learning about these tools and students who will be moving forward into a variety of careers in research, medicine, education, and others. this course module is a starting point for introducing students to low level bloom's taxonomy areas such as knowledge and comprehension of bioinformatics. it is hoped that this introduction will spark an interest in students to learn more about the field and become bioinformatics scientists. this approach is also intended to provide an entry point for faculty to begin developing new courses in bioinformatics within their undergraduate biology programs and collaborate with colleagues in computer science fields to pool interests and resources. in response to the need for a big data cure, i have developed an week course that meets for two -hour sessions weekly in which students gain hands-on experience using r and excel to analyze large datasets. to mimic an authentic research experience as closely as possible, the students work as a research group as they discuss the literature, develop hypotheses, and plan experiments. individuals or pairs are responsible for collecting samples and performing the actual sample preparation and experiments. data analysis is completed individually and then discussed and improved in the full research group. while this course was developed as a stand-alone experience, it could easily be incorporated as a module in a broader full length course. the primary student learning outcome for this course was to develop students' data science skills using excel and r. the premise of this research course was to perform a metagenomic analysis of the microbiota in two different snow samples. to accomplish this research project, students perform a literature review, develop hypotheses, collect and prepare samples, perform metagenomic sequencing (through a third party vendor), learn data analysis skills, and present their research findings via a poster presentation. secondary student learning outcomes for this course include those described in the cure network: making discoveries of interest to the broader scientific community, an iterative work experience, communication of their findings, and development of scientific research skills (curenet, ). weeks and : literature review, hypothesis development, and sampling table highlights the main activities completed in the course, beginning with a literature review. because the primary learning outcome for this course is the development of r and excel skills, the instructor can assist in the literature review process by developing the initial research question and providing some preliminary resources to begin the discussion. in this project, i developed the initial research question of "how does the bacterial population vary between two snow samples from different locations on campus?" and provided several primary and secondary articles about microbiomes, microorganisms often found in snow, and bacterial abundance and diversity. students used these resources as jumping off points to find more sources (usually pdfs, websites, and videos) which were collected in a class google folder. students visually mapped these sources into three broad categories on the whiteboard: "snow, " "microbiomes, " and "microbial diversity." after a group discussion, each student was responsible for developing an individual literature review from these and other sources they found. this fast-paced literature review process leads to the development of a research question, hypothesis, and sampling procedure. metagenomic analysis with our vendor takes - weeks, so it was essential to collect and prepare samples right away to allow time for the primary student learning outcome of developing skills in excel and r. to this end, after discussion, most of the class agreed upon the same research question and hypothesis, with slight variations that could be accommodated within the sampling and sample preparation processes. our research question asked if the microbiota of snow samples would differ between an area heavily trafficked by both foot and automobile traffic compared to campus trails primarily traveled by snowshoe. most students hypothesized that the area with both foot and automobile traffic would have more bacteria overall and more diversity of bacteria. students demonstrated their understanding of the field and our research question development by submitting a draft of an introduction for their final poster project (see supplementary material section for teaching materials). sampling and sample purification were relatively simple and inexpensive. students used ml plastic conical tubes (vwr - ) to collect three samples spaced at one meter intervals along a line at each of the two sites for days in a row. to purify microbial dna from the samples, the snow was melted and filtered through a . micron polyester membrane using an aeropress coffee press . the membranes containing the filtered microorganisms were then processed using the qiagen dneasy powerwater kit (qiagen - -nf). after confirming the presence of bacterial dna via pcr with a s primer set (idtdna.com; s rrna for # - - - , s rrna rev - - - ), the samples were sent for metagenomic sequencing off campus. weeks and : introducing metagenomics, big data, and r the first step in teaching students about bioinformatics was to guide them through an understanding of how metagenomic sequencing works and how the dataset was generated. a prerequisite for this course was a one semester foundations in biology course covering the essentials of molecular biology, including central dogma concepts such as dna, rna, base pairing, replication, and transcription. the literature review is initiated by the instructor to save time and is further developed by students. students use their literature review to develop their hypothesis, identify sampling methods, and prepare dna samples, allowing a pairing of wet lab skills with in silico activities. when wet lab resources are unavailable, this step can be completed by the instructor or replaced with an existing publicly available dataset. students build on foundational knowledge of dna from prerequisite courses by viewing video material on pcr and sequencing. instruction in statistics, excel, and r using a combination of video material and in class discussions builds a foundation of data analysis skills. practice data analysis using an existing dataset students use their developing data analysis skills to mimic the instructors' actions using excel and r to analyze an existing dataset. reinforcing data analysis skills with snow dataset students apply the data analysis skills they have learned and practiced to a new dataset from samples collected on campus. students showcase all the skills practiced in the course in a poster containing a research question, background material, a hypothesis, methods, results, and discussion. students complete the semester by recording a video presentation of themselves presenting their poster. if available, students also present their posters at a campus-wide research symposium. the supplementary material contain a list of resources used for reviewing foundational dna and pcr knowledge (supplementary material section ) . with this background in mind, students work to understand the polymerase chain reaction, or pcr. this foundational knowledge is essential, in part because it strips away the complexities of how we typically teach replication with emphases on all of the different enzymes (polymerases i and iii, primase, ligase, helicase, etc.) and focuses on the simple concept of creating a complement sequence of dna to the template. after mastering pcr, students then move on to understanding dna sequencing, beginning with sanger sequencing. to do this, students watch a series of youtube videos on sanger sequencing , the evolution of next-generation sequencing , and finally illumina sequencing used by our vendor (see supplementary material section for more details). after watching the video on illumina sequencing, students usually express a combination of fascination and confusion. to provide further practice in understanding this extremely important process, we break into student pairs and have each pair illustrate the processes of cluster formation on whiteboards using color coding. after performing a similar exercise to better understand base calling, we complete this section of the instruction by discussing how multiple overlapping dna segments from one organism can be used to generate the sequence for the entire s ribosomal rna gene. it is common for biology students in our program to have a fear or aversion to mathematical and other quantitative or computational approaches. % of traditional undergraduate https://www.youtube.com/watch?v=jnk_ maf fk https://www.youtube.com/watch?v=jfcd q qstm&t= s https://www.youtube.com/watch?v=fcd b hraz students enrolled in our college identify as female, % identify as first generation college students, % have family incomes less than $ , , and % come from rural communities and small cities. many students have taken the minimum mathematics courses required by the state graduation guidelines. in a study of life sciences majors conducted by andrews and aikens (andrews and aikens, ) , both females and first generation students exhibited a lower interest in mathematics topics in biology than their counterparts, and females perceived a higher cost associated with doing math in biology than their male counterparts. they also found that students' likelihood of taking a biostatistics class was positively related to their interest and perceived utility of the course. a goal for this course module is to spark future interest in bioinformatics training, so it was important to demonstrate to students the utility of statistical analysis both for the project and their future careers. in recognition of these factors, i began the bioinformatics instruction with a review (or novel instruction) of basic statistical analysis. to accomplish this, students first reviewed major statistical functions such as mean, median, standard deviation, standard error, p-values, and student's t-test using a freely available resource compiled by mit . these concepts were practiced using a very simple assignment completed in pairs during class time examining the statistical significance of simple drug treatment data (see supplementary material section for details). in class discussion helped to sort out problems in understanding before moving on to larger dataset analysis. next, students are introduced to fundamental concepts in data analysis, including data clean up and developing the research question. to facilitate this process, i provided the students with a dataset previously collected in the boundary waters canoe area https://web.mit.edu/$\sim$csvoss/public/usabo/stats_handout.pdf frontiers in microbiology | www.frontiersin.org wilderness (bwcaw). this dataset included triplicate sampling of four different sample sites resulting in columns of data on a spreadsheet. after metagenomic sequencing, , unique bacterial species or otus (operational taxonomic units) were identified in the spreadsheet rows, resulting in , unique cells of data. given that most students' experience of using excel to this point had been in traditional lab courses, this was by far the largest excel file any of them had ever opened. to make the experience less overwhelming for the students, i provide them with a version of the dataset that condensed otus into phyla, resulting in a dataset with sampling columns and rows of identified phyla. my goal was for them to be able to use excel to average the triplicate results from each sample site and make comparisons across the data, either between the four individual sample sites or between phyla. to do this, i created a video of myself using excel to average the sites, perform a t-test comparing the data between sites, and then sort the data by increasing p-value, thus reordering the data so that the most significant p-values were at the top of the list. students then were required to repeat the actions of this video on both the phyla dataset and the otu dataset. in doing this, students gained experience cleaning up and renaming columns, writing formulas, accessing the formula bank, sorting, and visualizing data. after establishing comfort with analyzing data in excel, we moved on to r. r is a freely available statistical computing program (the r-foundation, ) used across many fields for the analysis and visualization of data. for the purposes of this course module, i wanted to introduce students to the pros and cons of using the programming language r versus using excel both for data analysis and for data visualization, particularly for its ability to generate a heat map of large datasets. this includes establishing student knowledge, but not necessarily application, of using a command line and understanding the function of packages, bundles of shareable code created by experts in the field and freely available for use. when students learned coding was involved, there was an immediate sense of anxiety in the room. to alleviate this stress, i returned to an approach with which the students were familiar: learning by watching videos. just as they had learned to use excel functions by watching me perform tasks via video, the basics of r were laid out by watching a series of publicly available youtube videos. many videos are available, but i chose the "r programming for beginners" playlist from the r programming youtube channel (see the supplementary material section for a complete list of videos). in this series, the host, public health specialist greg martin, guides viewers through the whole process of using r, including downloading r and r studio onto their computers, learning basic commands such as identifying variables and manipulating a preloaded dataset of health characteristics of star wars characters, and installing and using r packages. this playlist resonated with the students, both https://www.youtube.com/channel/ucfjyq p k_suqfxvdqieqnw because of the clear instructions and because of the link to public health, a field with which many of the biology students could identify. students watched this series of videos on their own and their sole assignment was to replicate exactly what the host did and turn in a screenshot of their final r studio product. once the students achieved some initial comfort with r, i gave them a fully composed sheet of code to copy and paste into the script window of r. the code was created by modifying freely available code (albert and yoder, ) , including the packages gplots, vegan, and rcolorbrewer to plot data, create the heatmap, and apply a color scheme. i used this approach for three reasons. first, students did not yet have the capability to compose their own code because they didn't have enough knowledge of syntax to do what was needed. second, because r is an open access community, students and instructors can find existing code for many functions on the internet and modify it to fit their needs. third, by providing code that was annotated (with # lines explaining each line of code), students were able to walk through each line of code, understand the function, and run the code to achieve a final product of a heat map demonstrating the diversity and abundance of microbial samples across sampling sites in the bwcaw (figure ; full code in the supplementary material section ). because the purpose of this course module was to introduce bioinformatics users to command line coding, the ability to generate a finished product was important both to increase their level of confidence in using r and in order to demonstrate the analysis capabilities available in r that were not available in excel. at this point in the course, students had participated in a strong introduction to data analysis using both excel and r. they had manipulated a dataset larger than any of them had seen before and reflected on the pros and cons of each tool in analyzing the datasets. each student had observed excel and r being used via video and followed up with practice completing the work themselves. this iterative approach follows best practices in pedagogy where students are offered multiple opportunities to observe, practice, and learn a skill. when the data from the metagenomic analysis of the snow samples was returned to us in week six, students were ready to analyze it. the final project was a standard scientific poster presentation of their background, research question, hypothesis, methods, results, and discussion. to accomplish this task, students had to return to the notes they took for the analysis of the bwcaw dataset and apply these approaches for the snow dataset. this task involved cleaning up the data, and properly labeling sample columns, and changing existing lines of code in r to import the proper.csv file, identifying columns correctly and creating an appropriate visualization. by using this iterative approach of first observing, then practicing, and finally applying, all the students were able to successfully assign the right syntax to the code and create a successful project. as presented, this process allows students to experience both wet bench and in silico research. however, it is important to note that the project could be modified to include only the in silico figure | example heat map and r code. students used r to generate two heat maps in the course, first with a practice set of data from the boundary waters canoe area wilderness which was followed by a heat map of snow sample data to reinforce skills. (a) representative student-generated heat map of the bwcaw data. on the right axis, triplicate samples are boxed with corresponding colors; bacterial species' names are on the bottom axis. r-generated dendrograms are on the left and top axes. (b) a snapshot of the script window of r studio showing the code students used to generate the heat maps. a full copy of the code is available in supplementary material. frontiers in microbiology | www.frontiersin.org september | volume | article experience for students, as was the case in the second iteration of this course in spring due to the covid- pandemic and the closure of college facilities. it would be possible to provide this experience with the many publicly available datasets, but during the college closure i chose to perform the wet bench portion myself prior to the beginning of the course so that students felt they had a more "personal" sample rather than a dataset to which they had no personal attachment. this approach resonated with students as evidenced in their comments in the course evaluations. during the covid- pandemic in spring , the course was delivered using both asynchronous and synchronous (zoom) methods. the course meeting schedule was altered to limit zoom fatigue by meeting synchronously on tuesdays and working asynchronously on course materials during the remainder of the week. thursday meeting sessions were reserved for open office hours, an approach that well was received by students and widely used. tuesday synchronous meetings were initially used for discussions of the overall project, research design, and sequencing videos. breakout rooms in zoom were used extensively to facilitate small group discussions of research questions and to build comprehension of the sequencing videos. beginning in week , the course took on essentially a "flipped" format. students viewed and practiced skills introduced in the videos and synchronous class time was used for troubleshooting, comprehension checks, and setting up the "next steps." students in this virtual course were still able to successfully use r for statistical analysis and visualization of their data. one of the most important take home messages of this work is that we should take advantage of technology both to continue skill development as faculty and to teach resourcefulness to students. many faculty who teach undergraduate students completed their dissertations before the age of bioinformatics or in an area that did not focus on quantitative skills. these faculty may not currently possess the skills to incorporate a bioinformatics module into a course. youtube affords faculty the opportunity to learn new skills in a step-bystep manner when the technology and approaches may be wholly new to them. this is a very inexpensive and efficient way to acquire professional development that can serve to enhance both classroom teaching and potential new areas of research. as part of the course evaluation, students were asked to answer a series of confidence questions about skills developed in the course ( table ) . on a scale of - , with being high confidence, all students rated themselves as a ten when asked about confidence in pipetting a variety of liquids with micropipettors, reflecting the skills developed in the wet lab portion of the course. when asked about explaining sanger sequencing and next generation sequencing to another scientist, the class averages were . and . out of , respectively, for these new skills learned in the course. the course successfully introduced students to basic knowledge about r, as reflected in an . average score to "i can copy, perform, and run a simple code in r." as expected from an week introductory module, the students did not feel confident enough to create and run their own r code (average score . ). students were also asked, "after completing this course, how has your interest in biological research changed?" all of their free response answers are below: • i am still interested in it, and now realize the importance of being able to effectively use r and excel to convey my data. • my interest in research has stayed quite high after taking this course. i am planning on working in the more biochemical side, but this was still very interesting and helped me make sure that a career in research is where i belong. • i feel like i have a better understanding of how questions are being asked in the biological community. • my interest in biological research has grown even stronger. i knew before that i love research, but every time i continue to do it, my passions grow stronger. • it greatly raised my interest in biological research. it was cool to see how the experiments we performed gave us numbers, that we could find relationships between. • i was always curious about how scientists made the figures they did. after using r, examining larger datasets is a lot less frightening. • i have a greater understanding of the importance of microbiomes and am interested in my own microbiome! • i was very hesitant about research before this course because i had a few bad experiences, but this class changed my outlook on it. i am definitely more interested and would like to do more. • my interest has greatly increased in biological research, specifically, on human microbiomes like the gut microbiota. also, conducting my own biological research and experiencing the challenges of creating a poster has made me appreciate all the hard work scientist do to give us informative papers. • i am once again excited now about the medical applications of molecular biology and studies! i'm excited to skim new articles and have a better toolbox to understand them after learning about r and how microbiome data can be represented. while this course had a very small sample size (n = ), these responses suggest that this approach to using r was positively received by students. moreover, the students saw a utility in learning r, which research shows may lead to continued interest in participating in mathematical biology experiences (andrews and aikens, ) . in a short time frame, the course introduced students to bioinformatics and provided an opportunity for further practice. because of the students' ability to effectively visualize the dataset with r, they were able to think critically about the data and consider future research questions. from the r-generated heat map, the students realized that their initial hypothesis was incorrect. the heavy foot and automobile traffic sample site did have a higher abundance of bacteria but the diversity of bacteria was much lower than the sample site with light traffic. several students continued their analysis of the data even after the course ended and proposed a new research question for the next offering of the course. several outcomes were achieved as a result of this module. first, faculty expertise was enhanced in a time efficient manner using youtube training videos, leading to broadened research capabilities and comfort. second, students were introduced to computational skills in a manner that was effective and intentional, with time for both introduction and reinforcement of skills. finally, the module was effectively included in a biology curriculum because it could function as either a stand-alone course or a module within another course such as microbiology, leading to flexibility in the curriculum. this module, developed with cure guidelines in mind, is an effective and easily implementable way to introduce a broad group of students to bioinformatics in biology research, and also serves as a springboard for interested students to pursue further training and research in bioinformatics. the original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author. the author confirms being the sole contributor of this work and has approved it for publication. making heatmaps with r for microbiome analysis. the molecular ecologist life science majors' math-biology task values relate to student characteristics and predict the likelihood of taking quantitative biology courses advising biology majors about career choices: resources & information for biology instructors assessment of course-based undergraduate research experiences: a meeting report vision and change in undergraduate biology education: a call to action what is a cure crowdsourced data indicate widespread multidrug resistance in skin flora of healthy young adults † big biological data: challenges and opportunities what is bioinformatics? a proposed definition and overview of the field list of educational programs in computational biology the development and application of bioinformatics core competencies to improve bioinformatics training and education chapter employment outcomes of bachelor's degree holders the coursesource bioinformatics learning framework small world initiative: crowdsourcing antibiotic discovery the r project for statistical computing. vienna: the r foundation digest of education statistics do you kiss your mother with that mouth? an authentic large-scale undergraduate research experience in mapping the human oral microbiome † bioinformatics core competencies for undergraduate life sciences education verbreitung und ursache der parthenogenesis im pflanzen-und tierreiche special thanks to the students in biol for enthusiastically and successfully attempting a task outside their comfort zone. the supplementary material for this article can be found online at: https://www.frontiersin.org/articles/ . /fmicb. . /full#supplementary-material conflict of interest: the author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © kruchten. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- - og umiq authors: xin, shuyu; du, shujuan; liu, lingzhi; xie, yan; zuo, lielian; yang, jing; hu, jingjin; yue, wenxing; zhang, jing; cao, pengfei; zhu, fanxiu; lu, jianhong title: epstein-barr virus nuclear antigen recruits cyclophilin a to facilitate the replication of viral dna genome date: - - journal: front microbiol doi: . /fmicb. . sha: doc_id: cord_uid: og umiq epstein-barr virus (ebv) nuclear antigen (ebna )-mediated dna episomal genome replication and persistence are essential for the viral pathogenesis. cyclophilin a (cypa) is upregulated in ebv-associated nasopharyngeal carcinoma (npc) with unknown roles. in the present approach, cytosolic cypa was found to be bound with ebna into the nucleus. the amino acid - of the ebna domain was important for the binding. cypa depletion attenuated and ectopic cypa expression improved ebna expression in ebv-positive cells. the loss of viral copy number was also accelerated by cypa consumption in daughter cells during culture passages. mechanistically, cypa mediated the connection of ebna with orip (origin of ebv dna replication) and subsequent orip transcription, which is a key step for the initiation of ebv genome replication. moreover, cypa overexpression markedly antagonized the connection of ebna to ubiquitin-specific protease (usp ), which is a strong host barrier with a role of inhibiting ebv genome replication. the ppiase activity of cypa was required for the promotion of orip transcription and antagonism with usp . the results revealed a strategy that ebv recruited a host factor to counteract the host defense, thus facilitating its own latent genome replication. this study provides a new insight into ebv pathogenesis and potential virus-targeted therapeutics in ebv-associated npc, in which cypa is upregulated at all stages. epstein-barr virus (ebv) is a member of the gamma herpesviruses and was the first confirmed human tumor virus (ding et al., ; lu et al., ) . ebv ubiquitously infects more than % of the global population and is closely associated with the development of several malignancies, including burkitt's lymphoma and nasopharyngeal carcinoma (npc) (shair et al., ; yu et al., ; young, ; young and dawson, ; liu et al., ) . npc, which is primarily of epithelial origin, is a type of metastatic head-and-neck neoplasm that is highly prevalent in southern china and some other areas in east asia and africa (tang et al., ; tu et al., ; jiang et al., ) . ebv is able to establish life-long persistence in the human host (young and murray, ; . it contains a large genome approximately kb in size and has two phases in its life cycle (the latent and lytic stages) (yu et al., ; hammerschmidt and sugden, ) . ebv mainly spreads through the saliva of human host, infects b cells through the oral mucosal epithelium, and then transforms b lymphocytes into resting memory b cells through a series of viral latent transcription programs, thus establishing a lifelong latent infection pattern (thorley-lawson et al., ) . the majority of ebv infections in vivo are latent (thorley-lawson, ) . during ebv latency, the ebv genome exists in the form of episome dna, few viral genes are expressed, and no virion is produced. ebv nuclear antigen (ebna ) is the only viral protein that is expressed in all types of ebvassociated tumors (lu et al., ; tao et al., ) . determining how ebv is able to maintain its stable latent status in host cells is a topic of interest, because it may provide understanding about the pathogenesis of ebv and new targets to inhibit the persistence of ebv genome in the therapy of ebv-associated cancers. ebv replication is under the control of some host and viral factors that are not fully understood. ebna plays a key role in the replication and mitotic segregation of ebv dna episomes to daughter cells (yates and guan, ; frappier, b) . ebna -mediated s-phase episome replication depends on binding of ebna to the ebv origin of genome replication (orip) (reisman et al., ) . viruses are obligate intracellular parasites, and their replication cycles depend on some host cell factors. for example, some studies suggested that cellular origin recognition complex (orc) and minichromosome maintenance (mcm) complex are related to the ds element of orip, implicating them in the initiation of ebv dna replication (frappier, a; capone et al., ) . these host factors may also be potential targets for antiviral therapy. cyclophilin a (cypa) is a protein with multiple functions as a typical member of the cellular peptidyl-prolyl cis-trans isomerase (ppiase) family (braaten et al., ; bahmed et al., ) . cypa was discovered initially as an intracellular receptor of the immunosuppressive drug cyclosporin a (csa) (braaten et al., ; bahmed et al., ) . studies have shown that cypa can use il- to induce cell signal conversion, activate tyrosine phosphorylation and nuclear transport of transcription factor , and can bind and activate nf-κb . cypa is involved in the life cycles of multiple viruses and plays a critical role in their successful infectivity and replication, including human immunodeficiency virus type (hiv- ), hepatitis c virus (hcv), hepatitis b virus (hbv), vesicular stomatitis virus (vsv), vaccinia virus (vv), coronaviruses (covs), and feline coronavirus (bose et al., ; naoumov, ; jyothi et al., ; phillips et al., ) . the interaction between cypa and hiv protein promotes the replication and infection of hiv particles; cd is the main signal receptor of cypa, and the two interact to regulate the early steps of hiv replication (ciesek et al., ; tang et al., ) . conversely, cypa suppresses the replication of some viruses, such as rotavirus, infectious bursal disease virus and influenza virus (xu et al., ; liu et al., ) . however, the role and mechanism of cypa in the function of ebv remain unknown. our laboratory previously performed a proteomics study using npc tissues and found that cypa was upregulated from the early stages of npc (atypical hyperplasia and stage i) to the malignancy stages (yang et al., ) . since ebv infection also occurs during the early stage of npc (morrison et al., ) , we speculated that a potential relationship might exist between cypa and ebv, which initiated the present approach. recently, we have reported that the exosomal cypa level in npc is positively related that of exosomal latent membrane protein , which is another latent protein of ebv. the result suggests a relationship between cypa and ebv (liu et al., ) . ubiquitin-specific protease (usp ) is a type of deubiquitinase that is also known as herpesvirus-associated ubiquitin specific protease (hausp) and has been documented as a host factor that inhibits ebv replication (holowaty et al., a) . the usp -ebna interaction was selectively disrupted by deletion of ebna residues - just upstream of the dna binding domain, and the resulting ebna mutant exhibited significantly increased dna replication activity (holowaty et al., b) . usp also plays a role in the replication inhibition of kaposi's sarcoma-associated herpesvirus (kshv) by interacting with the latency-associated nuclear antigen (lana), which is the homology of ebna (jager et al., ) . as it is known, kshv also persists in infected cells mainly in a latent state, and lana, is expressed in all latently kshv-infected cells (rainbow et al., ) . however, the mechanism by which ebv overcomes host suppression to maintain its own pathogenesis remains to be investigated. here, we demonstrate that ebna binds to and recruits cypa to the nucleus to support the function of ebna in the replication of the viral latent genome. cypa overexpression could antagonize the host barrier, usp . the results revealed a strategy that ebv recruited one host factor to counteract another, thus favoring the viral dna replication and persistence in latent infection. the study provides novel insights into understanding ebv pathogenesis in epithelial cells. the p plasmid (maxi-ebv), which contains the complete ebv genome of the b - strain, was kindly provided by dr. w hammerschmidt (delecluse et al., ) . the human embryonic kidney hek cell line (hek ) was originally obtained from atcc and was used to establish latent infection of the whole ebv genome (p ) by using hygromycin for the screening, resulting in the c cell line that was previously described by our group (zuo et al., ) . subsequently, the cells were divided into groups of shrna (using prnat-u . -shrna-cypa treated cells), the nc shrna group (prnat-u . -shrna treated cells cypa nc), liposomal transfected cells are based on lipofectamine transfection reagent (invitrogen-thermo fisher; united states). the day after transfection, cells were treated with g was added (sigma-merck; germany) for cell selection. cells were subcultured every days. the cells were grown in dulbecco's modified eagle's medium (dmem; sigma) supplemented with % fcs. the c - cell line is an ebv-positive npc cell line (zuo l.l. et al., ) . all recombinant plasmids used for bimolecular fluorescence complementation (bimc) were constructed using standard cloning techniques according to the schematic diagram (supplementary figure s ) by using the pcaggs vector, which is a gift from dr. harty (lu et al., ) . cypa and cypb were amplified from cells by rt-pcr. cypa or cypb gene was fused with the n-terminal fragment of the yellow fluorescent protein (yfp), constructing the plasmids of pcaggs-flag-cypa-ny (cypa-ny) and pcaggs-flag-cypb-ny (cypb-ny), respectively. the ebna protein was expressed as a fusion protein with the c-terminal fragment of (yfp), constructing the plasmid pcaggs-myc-ebna -cy (ebna -cy). the expression plasmids pcaggs-flag-cypa and pcaggs-flag-cypb were also constructed respectively. the myc tag sequence was added to the n-terminal of ebna . myc-tagged ebna was generated by cloning into the sphi and nhei sites of the pcaggs vector to produce the pcaggs-myc-ebna plasmid. the truncations ebna (nt, - ) and ebna ( - ) were generated by pcr based on the plasmid pcaggs-myc-ebna using the primers listed in supplementary table s . the ebna deletion mutants ebna (nt, - ), ebna ( - ) and ebna ( - ) were generated by inverse pcr. for example, primer complementary to - bp (anti-sense) and - bp (sense) of ebna were used for amplification, and the resulting pcr. product was gel-purified and self-ligated, resulting in ebna ( aa - ). then a sphi-nhei fragment from ebna ( aa - ) was inserted into the sphi and nhei sites of the pcaggs-myc-ebna to produce the pcaggs-myc-ebna ( aa - ). the orip-sv -luc expression vector was obtained by introducing the flanking sequence of orip amplified from the pc -orip plasmid, which was a gift from prof. frappier (sivachandran et al., ) . the orip sequence was cloned into the saci and xhoi sites of the luciferase reporter vector, pgl -enhancer. the rt-qpcr was performed as previously described (yu et al., ) . briefly, total rna was isolated from cells using the trizol reagent (invitrogen). first, trizol was added to lyse the cells, then / volume of chloroform was added to the lysate to separate the dna, protein and rna, followed by the isopropanol and % absolute ethanol to precipitate the rna. for rt, µg of rna was reversely transcribed into cdna using the one-step gdna removal and cdna synthesis supermix kit (transgen biotech, beijing, china). real-time quantitative pcr (rt-qpcr) was then performed by using the kit of transstart r top green qpcr supermix (transgen biotech, beijing, china) according to the manufacturer's instructions. the following program was used for the rt reaction: • c for min, ice for min, followed by • c for min and then • c for s. the cfx multicolor detection system (bio-rad) was employed for the detection. primers for rna detection by qrt-pcr were designed based on rna sequences, and bate-actin was used as an endogenous control. the following procedure was used for the rt-qpcr: • c for min, followed by cycles of • c for s and • c for s. data obtained by the conventional method of calculating comparative method (− ct) . repeated three times for each sample in parallel in each experiment, and the results were expressed as the mean of three independent experiments. the sequences of the qrt-pcr primers are provided in supplementary table s . western blotting (wb) was performed using a standard protocol as described previously (zuo l.l. et al., ) . cells was lysed in ripa ( mm tris, ph . , mm nacl, % np- , . % sodium deoxycholate, . % sds, sodium orthovanadate, sodium fluoride, edta, leupeptin) followed by incubation on ice for min, protein quantification according to the specification of bca kit (beyotime, shanghai, china). the cells lysates were centrifuged at × g for min at • c. supernatant fractions were used for detection. samples with equivalent amounts of denatured protein were separated using % sds polyacrylamide gels (epizyme, shanghai, china). after electrophoretic separation, the proteins were transferred to a polyvinylidene difluoride (pvdf) membrane (millipore, danvers, ma, united states). the membrane was blocked with % skimmed milk for h at room temperature, followed by an overnight incubation with the primary antibody at • c. after washing three times, the membrane was incubated with a secondary antibody for h at • c. finally, the proteins of interest were detected using the luminata crescendo hrp substrate (millipore) and viewed with the chemidoc xrs + molecular imager (bio-rad). the results were analyzed using the image lab software (bio-rad). the following antibodies were used for the immune detection: anti-ebna (santa cruz biotech, de, united states, sc- ), anti-cypa (proteintech, chicago, il, united states, - -ap), anti-c-myc (sigma, st. louis, mo, united states, c ), and anti-flag (sigma, h ). gapdh (proteintech, chicago, il, united states, -i-ap) and bate-actin (proteintech, chicago, il, united states, -i-ig) were used as the loading controls. the secondary antibodies used for wb and if were as follows: hrp-conjugated anti-rabbit (cst, chicago, il, united states, # p ), hrp-conjugated antimouse (ge healthcare, united kingdom, na v) and alexa fluor donkey anti-rabbit igg (life technologies, united states, ). c cells were plated in a -well plate at a density of × cells per well. the next day, different csa concentrations as indicated were applied for treatment. at , and h posttreatment, the cell counting kit- (cck ) was used to test cell viability according to the manufacturer's instructions. the absorbance was detected at the nm wavelength using a microplate reader. cyclosporin a (dalian meilun, china) is a type of immunosuppressive preparation that targets cypa (chatterji et al., ) . the drug was dissolved in dimethyl sulfoxide (dmso). ebv-positive cells were cultured in medium containing µm of csa. after h of treatment, the cellular proteins and total rna were extracted and subjected to detection. bimolecular fluorescence complementation assay was performed as described previously (liu et al., ; lu et al., lu et al., , . the schematic diagram of bimc assay is shown in supplementary figure s . in brief, human hek t cells were grown on coverslips in six-well plates. the pcaggs-myc-ebna -cy plasmid or the mutant constructs and the pcaggs-flag-cypb-ny plasmid were cotransfected into the cells using the lipofectamine transfection reagent (invitrogen) according to the manufacturer's instructions. approximately . µg of each plasmid was used for the transfection. transfection of a single plasmid for each used in bimc assay was performed as a negative control. after h, the cells were gently washed for three times with pbs and fixed with cold % paraformaldehyde for min at room temperature. then, the cells were washed and subsequently stained with hoechst (sigma) for min at room temperature. finally, the cells were washed again, and the slides were observed for the specific yfp signal under a fluorescence microscope. a typical co-ip procedure was performed as described (liu et al., ; lu et al., lu et al., , . cell lysates were harvested directly or at h post-transfection as indicated. the lysed sample was centrifuged at , rpm for min at • c. the supernatant lysates of about mg were incubated with a primary antibody, such as an anti-flag monoclonal antibody (mab) (h , sigma, : ) or anti-mouse igg antibody (control) (sc- , santa cruz, : ), for h at • c. after centrifugation, the supernatant was transferred to a new tube containing µl of protein g beads (transgen biotech, beijing, china) and incubated for h at • c. after extensive washes with cold lysis buffer, the immunoprecipitated proteins were eluted in sds sample loading buffer (aurigene biotech, changsha, china), separated by sds-page, transferred onto polyvinylidene difluoride membranes (millipore), and detected by wb (zheng et al., ) . three small interfering rnas (sirnas) targeting cypa (genbank accession number: nm ) were designed and synthesized by guangzhou ribobio company. to evaluate the knock-down efficiency of the sirnas, pmol of each sirna was transfected into hek cells. the cells were lysed with ripa as described by wb, and lysates were used for western blotting with anti-cypa and anti-bate-actin antibodies. the sirna with the best knock-down efficiency was chosen for the subsequent experiments. a scramble sirna-nc was used as the control. hek t and vero cells grown on coverslips in six-well plates for h were washed gently three times with pbs, fixed with % formaldehyde in pbs for min, and then permeabilized with . % triton x- in pbs for min. subsequently, the cells were blocked with fresh % goat serum. the cells were incubated with an anti-cypa rabbit polyclonal antibody and anti-flag mouse mab overnight at • c, respectively. after washing five times with pbs, the secondary antibody was added for h of incubation at • c. then, hoechst was applied for nuclear staining for min at room temperature. the coverslips were finally mounted onto slides and observed under a florescent microscope (bx , olympus, japan). the chip assay was performed to detect the binding of ebna -orip according to previous reports (shen et al., ) . the immunoprecipitation kit (millipore) was used for the assay. hek cells were transfected with lipofectamine (invitrogen) according to the manufacturer's protocol. cells were collected after h and lysed with lysis buffer (beyotime, shanghai, china) [ mm tris (ph . ), mm nacl, % triton x- , sodium pyrophosphate, β-glycerophosphate, edta, na vo , leupeptin]. the immunoprecipitated nucleoprotein complexes were eluted by incubation twice for min at • c with µl of elution buffer ( % sds and mm nahco ), and the crosslinks were reversed by incubation at • c for h. the dna was extracted with phenol/chloroform and precipitated with ethanol. then orip dna was amplified by qrt-pcr using the primers listed in supplementary table s . the experimental procedure for the detection of orip transcription activation mediated by ebna was carried out according to previous description from other group . cells were seeded into -well plates h prior to transfection. the following day, ng of the orip-sv -luc reporter plasmid was transfected into the cells using lipofectamine . the sv -luc reporter plasmid was transfected as a control. cell lysates were collected at h post-transfection and assayed for luciferase activity using a luciferase assay kit (promega, madison, wi, united states) on the panomics luminometer. the renilla luciferase activity was also measured using an enzyme assay kit (promega). the results were normalized to the renilla activity. three parallel repeats were performed for each sample in each experiment, and the results were expressed as the mean of three independent experiments. the detection of ebv copy number was performed as described previously (zuo et al., ) . dna was extracted from the c -shnc and c -shcypa cells using the general allgen frontiers in microbiology | www.frontiersin.org kit (cwbio, hunan, china) and quantified. the relative ebv copy number was determined by rt-qpcr using the ebv dna quantitative fluorescence diagnostic kit (sansure biotech, hunan, china) according to the manufacturer's instruction. in this product, bamhi-w fragment in ebv genome is designed as the specific primers and probes. the ebv copy number concentration (copies/cell) of the samples was calculated according to the level of internal reference. the experiment was repeated for three times. the statistical analyses were performed using graphpad prism (graphpad software, ca, united states). differences between groups were determined using student's t-test or one-way analysis of variance (anova). the data are expressed as the means ± standard deviations (sds). single, double and triple asterisks indicate statistical significance ( * p < . , * * p < . and * * * p < . ). by immunofluorescence (if) assay, cypa was detected to be mainly localized in the cytoplasm in vero and hek t cells (supplementary figure s ) . to study the potential interaction between ebna and cypa, bimc assay was performed. the bimc assay is a useful tool for detection of protein-protein interactions in living cells, and the results can be visualized under a fluorescence microscope (hudry et al., ; yang et al., ) . a diagram of the basic principle of the bimc assay is shown in supplementary figure s . the ebna protein was expressed as a fusion protein with the c-terminal fragment of the yfp (pcaggs-myc-ebna -cy), and cypa was fused with the n-terminal fragment of yfp (pcaggs-flag-cypa-ny). these two plasmids were co-transfected into hek t cells, with the single pcaggs-flag-cypa-ny plasmid transfection as a negative control. the results showed that ebna and cypa interacted with each other in the nucleus (figure a , top). when the nuclear localization signal (nls) sequence of ebna was deleted, the proteins interacted in the cytoplasm ( figure a) . this translocation showed the specificity of the interaction mediated by ebna with an nls. cyclophilin b (cypb) is another member of the cyclophilin family, but we did not detect any binding signal for cypb-ebna in the bimc assay ( figure a) . there was no fluorescence signal in the cells transfected with only single plasmid which was a negative control (figure a , lower). ebna and cypa/b expression in the bimc assay was detected by wb ( figure b) . we further evaluated ebna and cypa intracellular localization in ebvpositive cells by if assays (figure c) . the result showed that cypa expressed in both cytoplasm and nucleus in ebv-positive c cells, while mainly in the cytoplasm of the ebv-negative hek cells (figure c) . the co-immunoprecipitation (co-ip) assay was used to verify the interaction of ebna with cypa. these results showed that both endogenous and exogenous cypa interacted with ebna (figures d-f) . the plasmids pcaggs-myc-ebna and pcaggs-flag-cypa or pcaggs-flag-cypb were transfected in hek cells, myc-ebna was immuneprecipitated with anti-flag antibody. as shown in figure g , the result of co-ip assay further validated that ebna interacted with cypa but not cypb. to identify the contribution of the functional domains of ebna to the cypa-ebna interaction, we constructed five deletion mutants of myc-tagged ebna named ebna - , ebna - , ebna - , ebna - , and ebna - . the plasmid structures are illustrated in figure a . the bimc assay revealed that yfp was reconstructed in hek t cells using wild-type ebna and four of the mutants (ebna - -cy, ebna - -cy, ebna - -cy and ebna - -cy) but not the ebna - -cy mutant (supplementary figure s a) . each negative control with single plasmid transfection is shown in supplementary figure s c . subsequently, we co-transfected pcaggs-myc-ebna or a pcaggs-myc-ebna mutant plasmid with pcaggs -flag-cypa into hek cells. a co-ip assay using a flag-tag antibody for the pulldown showed the same result (i.e., cypa did not interact with ebna - ) ( figure b ). this domain, which is also the region containing the usp binding domain in ebna (figure a) , was thus critical for the binding of ebna to cypa. co-expression of ebna - -cy (only this one domain of ebna ) and cypa-ny recovered their binding function based on the bimc assay results (supplementary figure s b) . the nls (amino acids - ) is also included in the domain containing amino acids - , and thus yfp was detected in the nucleus (supplementary figure s b) . these results indicated that amino acids - of ebna , which contain the usp -binding domain, were essential for the interaction between ebna and cypa. we designed three sirnas to observe their efficiency on cypa protein expression in hek cells ( figure a) . the results showed that the knock-down of sicypa- was best, and this sirna was used in the subsequent experiments, whereas a scrambled control sirna (sinc) had no effect on cypa expression. as shown in figures b,c , ebna protein and mrna expression decreased in response to sicypa in ebvpositive npc c - and c cells. conversely, ectopic cypa overexpression in the ebv-positive cell lines resulted in an increase in the ebna expression levels detected by wb and qrt-pcr (figures d,e) . as ebv episomal genome is easy to be lost with culture passages in vitro (frappier, b) , we investigated whether cypa depletion might expedite this process in consecutive passages. then we collected gdna from different generations figure | detection of cypa-ebna binding by the bimc and co-ip assays. (a) detection of the ebna -cypa interaction by the bimc assay. hek t cells were transfected with the indicated plasmids, including cypa-ny, ebna -cy, ebna nls-cy, and cypb-ny following by bimc analysis, and fluorescence was observed. transfection of a single cypa-ny plasmid did not lead to the production of fluorescence. scale bar, µm. (b) the proteins expressed from the plasmids used in the bimc assay were detected by wb. (c) the detection of cypa and ebna in ebv-negative and ebv-positive hek cells by if assay. scale bar, µm. (d) endogenous cypa interacts with ebna in hek cells. the plasmid pcaggs-myc-ebna was transfected into cells. an anti-myc antibody was used for the pull-down the cypa, and the wb assay was carried out for detection. (e) endogenous cypa interacts with ebna in c cells. ebna was immune-precipitated with anti-cypa. ebna was detected by wb (f) exogenous cypa interacts with ebna . hek cells were transiently transfected with flag-cypa alone or with myc-ebna . flag-cypa was immune-precipitated with anti-myc antibody. igg was used as a negative control for the pull-down in the co-ip assay. flag-cypa was detected by wb. (g) co-ip assay for comparison of the interactions of cypa and cypb with ebna . pcaggs-myc-ebna and pcaggs-flag-cypa or pcaggs-flag-cypb were transfected in hek cells. myc-ebna was immune-precipitated with anti-flag antibody. figure | identification of the ebna domain required for binding to cypa. (a) diagram of the ebna deletion mutants. the start and end amino acid residues for each fragment are indicated according to a previous report (young and murray, ) . (b) validation of the interaction between each mutant ebna and cypa by the co-ip assay. pcaggs-myc-ebna , pcaggs-myc-ebna mutants, and pcaggs-flag-cypa were transfected into hek cells. myc-ebna was immune-precipitated with anti-flag antibody. flag-cypa and myc-ebna were detected by wb. of cells and detected the copy number of each cell. as it shown in figure f , cypa depletion significantly facilitated loss of ebv copy numbers. ebna -orip binding has been validated to be necessary for the replication initiation of ebv genome (shen et al., ) . here, we investigated the effect of cypa knockdown on the orip luciferase activity in cells transfected with an orip luciferase reporter in c cells. ebv-positive c and ebv-negative hek cells were stably transfected with a lentivirus expressing a short hairpin (sh) of cypa and a scramble control (shnc) (figure a) , and the results showed that cypa was successfully knocked down. ebna protein expression was restored in the c -shcypa cells transfected with the wild-type cypa expression plasmid ( figure b ). in these cells, an orip luciferase reporter, orip-sv -luc was transiently transfected. after h transfection, luciferase activities were detected. the ebna activated orip luciferase was decreased by . -fold in the luciferase assay compared to that in the control cells (c -shnc) due to cypa depletion. in contrast, there was no effect on the transfection of an sv -luc reporter plasmid with cypa depletion (p < . , figure c ), ebna mrna was detected by qrt-pcr. when ebna was lacked, cypa knockdown did not affect luciferase activity of orip-sv -luc ( figure c) . the results indicated that cypa enhanced the ebna activation of orip transcription. the chip assay was used to further validate that cypa played a role in ebna -orip binding. pcaggs-myc-ebna and orip-sv -luc were co-transfected into hek -shnc and hek -shcypa cells. as shown in figure d , cypa interference (shcypa) reduced the ebna binding to orip dna by approximately % compared to that of the control (shnc) in hek cells (p < . ). cypa is an intracellular receptor of csa, which in turn inhibits the ppiase activity of cypa through binding to the hydrophobic pocket of cypa (chatterji et al., ). based on above data about cypa regulation in ebv genome transcription initiation, we further investigated the possibility of csa against ebv replication. the working concentration of csa was considered as µm according to its inhibition effectiveness and low cytotoxicity by the cell counting kit- assay ( figure a) . rescue experiments showed that ebna protein levels were restored compared to the csa treatment group (figure b ). ebna protein expression was evaluated by wb after treated or untreated with csa ( figure c, left) . rt-qpcr analysis showed that there was a significant reduction for the ebna following the decrease of cypa caused by csa (figure c, right) . subsequently, c cells were transiently transfected with the cypa expression plasmid together with orip-sv -luc. luciferase activities were increased at h post-transfection compared with that of no cypa overexpression, but there was no change for sv -luc (without orip), and ebna -orip-luc activity increased in cypa overexpression ( figure d) . the protein level of cypa and the mrna level of ebna are detected ( figure d) . we further investigate the role of csa in ebna -mediated orip transcription. after transfection of orip-sv -luc, the cells were treated with csa. the result showed that csa decreased ebna-orip-luc activity compared to the untreated group, with a decrease in cypa and ebna ( figure e) . however, overexpression of cypa and treatment of csa in hek cells did not affect the activity of orip-sv -luc ( figure f ). these data indicated that csa inhibited the ebna -mediated orip activation. chip assay followed by quantitative pcr (chip-qpcr) was used to evaluate the the effect of cypa depletion on loss of ebv copy numbers during passages. ebv-positive c was used for shrna stable transfection and selection. cell lines stably transfected with shrna-cypa or shrna-nc were established for the experiment. cell dispersal for each passage was performed at a ratio : . the copy number of the c -shcypa cells was decreased compared with the c -shnc, with passage th of c -shcypa set to . * p < . , * * p < . , * * * p < . . effect of cypa overexpression and csa treatment on ebna binding to orip in hek cells. myc antibody (for myc-ebna ) efficiently pulled down orip-dna from the above samples, and the results showed that csa treatment eliminates ebna -orip binding, while cypa overexpression increases its binding ( figure g) . the results suggested that the ppiase activity of cypa was required for the role of cypa in ebv latent replication. restoration of ebna protein expression in c -shcypa cells transfected with wild-type cypa expression plasmids. cypa and ebna proteins were detected by wb. (c) effect of cypa knockdown on ebna -orip-mediated transcription activity in the luciferase reporter assay. the ebna mrna was detected by rt-qpcr. (d) effect of cypa knockdown on binding of ebna to orip in the chip assay. cypa was depleted from hek with shrna. antibodies against myc and igg control were respectively used for the pulldown in chip assayshek . the precipitation of orip dna was quantitated by rt-qpcr. cypa and ebna proteins were detected by wb. * p < . , * * p < . , * * * p < . . ubiquitin-specific protease has been implicated in strong inhibition of ebv replication through its tight connection with ebna (holowaty et al., b) . in this study, as the above results demonstrated, the cypa binding domain was located within the domain containing amino acids - , which spanned the usp -binding domain in ebna . because cypa played an opposite role in regulating ebna function compared with usp , hek -shnc and hek -shcypa cells were transiently transfected with pcaggs-flag-cypa alone or with pcaggs-myc-ebna , a co-ip assay was carried out. the results showed that usp -ebna binding was strong in the hek -shcypa and hek -shnc cells. however, when cypa was overexpressed, the amount of usp bound with ebna was decreased remarkably (figure a) . in order to study the ppiase activity of cypa in this mechanism in ebv replication, a co-ip assay was performed to using the treatment of csa. the result verified that csa eliminated the antagonism of cypa on ebna -usp interaction ( figure b) . to investigate the ability of cypa to influence the ebna -orip connection through antagonizing usp , in hek cells, orip-sv -luc was transfected with pcaggs -myc-ebna , pcaggs-flag-cypa or pcaggs-myc-ebna mutant, a chip assay was designed based on ectopic cypa expression. as shown in figure c , the interaction of orip and the ebna mutant with deletion of amino acids was significantly enhanced. in contrast, cypa overexpression increased ebna -orip binding to a high level. the result demonstrated that the deletion of the binding site for both usp and cypa exhibited the enhancement of ebna -orip binding ( figure c ). this was a priority effect of the release of usp inhibition but not cypa facilitation. only when cypa was overexpressed, could the effect of usp inhibition be reversed. the results further showed that cypa overexpression was essential to overcome the usp suppression in regulating ebna replication function. ebv latent infection is an important causative factor in the development of related cancers such as npc (dittmer et al., ; zheng et al., zheng et al., , , although the mechanism is largely unclear. viral replication and genome maintenance in host cells are important for the pathogenesis. cypa was found to be highly expressed in npc in the previous work from our laboratory (yang et al., ; liu et al., ) and played an unknown role related to ebv. herein, we reveal that cypa, especially when increasingly expressed, contributes to the replication function of ebna . cypa first was recruited by ebna into the nucleus and then mediated ebna -orip binding and replication activity. the mrna expression of ebna measured by rt-qpcr. ebv-positive c cell lines were used for the test. (d) c cells were transfected with orip-sv -luc reporter plasmid and cypa expression plasmids. overexpressed cypa significantly increased ebna -orip-dependent luciferase activity, but had no effect on sv promoter dependent luciferase activity (left). ebna mrna was measured by rt-qpcr (right) . (e) orip-sv -luc reporter plasmid was transfected into c cells, following the csa treatment. csa treatment greatly reduced ebna -orip luciferase activity compared with untreatment. (f) csa and elevated cypa on ebna -orip-mediated transcription activity in the luciferase reporter assay in hek cells. (g) chip-qpcr was used to determine ebna -orip binding. csa treatment reduced ebna -orip binding while elevated cypa increased binding. cypa and ebna proteins were analyzed by wb. * p < . , * * p < . , * * * p < . . (c) effect of cypa overexpression and ebna - mutation on the ebna -orip binding detected by chip assay. * p < . , * * * p < . . figure | schematic for the mechanism of cypa in supporting the replication function of ebna . ebv genomic dna exists within the host genome in the form of extrachromosomal episomes. cytoplasmic cypa can be hijacked by ebna into the nucleus. overexpressed cypa can overcome the suppression of usp in binding to ebna . nuclear cypa mediates ebna -orip transcription, and thus contributing to the viral genome replication and maintenance. on the other hand, when cypa was upregulated, the usp suppression in ebna -mediated replication could be reversed. this interaction is a type of quantity-driven winning for cypa, because its rival usp is too powerful. it was reported that the affinity of usp -ebna was -fold higher than usp -p , implying the strong binding of usp -ebna (saridakis et al., ) . the csa treatment demonstrated that the ppiase activity of cypa was required for this function. the schematic working model is shown as in figure . cyclophilin a has been implicated in the life cycles of several viruses and plays a critical role in the successful infectivity and replication of these viruses, including some tumor viruses, such as hbv and hcv (bose et al., ; naoumov, ; jyothi et al., ; phillips et al., ) . it has not been reported for the relationship between cypa and kshv, another tumor virus in the same family of gamma herpesvirus as ebv. though cypa is involved in the regulation of several viruses, its function mode is different from that in other viruses depending on the different mode of viral infection and replication. for example, the interaction between cypa and hiv gag was proposed to facilitate disassembly of the viral rna containing core following virus entry and thus supports the efficient reverse transcription of the hiv- genome (luban et al., ; ott, ) cypa also enhances virus attachment to the host cell membrane through interactions with heparans (saphire et al., ) and after membrane fusion through interaction with cd , thereby promoting viral infection. in the present study, for the first time, we showed that cypa was also utilized by ebv in the modulation of viral replication function in epithelial cells. thus, cypa is involved in the maintenance of the virus during its latency in host cells. ebna mediates dna episome replication from orip (frappier, a) . our results revealed that cypa was recruited to influence ebna -orip-mediated transcription. cypa depletion significantly facilitated loss of ebv copy numbers (figure f) , suggesting that impairment of ebna-orip binding further weakened successful replication and maintenance of the ebv genome. epstein-barr virus can naturally infected b cells and latently maintained in resting b cells (thorley-lawson et al., ) . as cypa is a multi-functional protein, which can be expressed in all kinds of cells, and ebna is the only expressed protein of ebv in all latency types. therefore, we think that both cypa and ebna may be expressed in resting b cells. how cypa plays a role in the function of ebv in b cells remains to be further investigated. the deubiquitinase usp is also known as a hausp and has been found to be associated with several herpesviruses, including hsv- , ebv, and kshv (holowaty et al., b; jager et al., ; hammerschmidt and sugden, ) . a previous study demonstrated that usp suppressed ebv replication (holowaty et al., b) . here, we showed that ebv hijacked the host factor of elevated cypa to counteract usp , thereby adding to the definition of hausp. additionally, our data showed that deletion of the binding domain in ebna for both usp and cypa resulted in significantly increased ebna -orip binding activity (figure c) , mainly due to release of usp inhibition. the result was consistent with that of previous report (holowaty et al., a) , demonstrating that the suppressive role of usp was strong enough to greatly overshadow the improved role of cypa. in summary, the study reveals that cypa is a novel critical host factor utilized by ebna in the viral dna replication in epithelial cells. elevated cypa levels remarkably antagonize usp in the interaction with ebna . the results revealed a strategy that ebv recruited a host factor to counteract the host defense, thus facilitating its own latent genome replication and efficient persistence. our findings implied that ebv has evolved sophisticatedly. this study provides a new insight into ebv pathogenesis and potential virus-targeted therapeutics in ebvassociated npc, in which cypa is upregulated. the data used to support the findings of this study are available from the corresponding author upon request. we thank dr. wolfgang hammerschmidt (gsf-national research center for environment and health, germany) for kindly providing us the maxi-ebv system, which was used in the establishment of c cell line. we thank dr. lori frappier (university of toronto, canada) for the kind gift, plasmid pc -orip. we also thank dr. ronald n. harty (university of pennsylvania, united states) for providing us the pcaggs vector. the supplementary material for this article can be found online at: https://www.frontiersin.org/articles/ . /fmicb. . /full#supplementary-material figure s | the schematic diagram of the bimc assay. figure s | cypa expression was detected by if assay in vero and hek t cells. scale bar, µm. after transfected the pcaggs-flag-cypa plasmid for h in vero cells, the flag antibody was incubated overnight in • c, followed by green fluorescent secondary antibody in • c for h, hoechst stained nucleus. hek t cells, cypa antibody incubated overnight, followed by red fluorescent secondary antibody, stained nucleus. extracellular cyclophilin-a stimulates erk / phosphorylation in a cell-dependent manner but broadly stimulates nuclear factor kappa b requirement for cyclophilin a for the replication of vesicular stomatitis virus new jersey serotype cyclophilin a is required for an early step in the life cycle of human immunodeficiency virus type before the initiation of reverse transcription ebvassociated cancer and autoimmunity: searching for therapies the isomerase active site of cyclophilin a is critical for hepatitis c virus replication nucleolin is important for epstein-barr virus nuclear antigen -mediated episome binding, maintenance, and transcription cyclosporine a inhibits hepatitis c virus nonstructural protein through cyclophilin a propagation and recovery of intact, infectious epstein-barr virus from prokaryotic to human cells proteomics analysis of gastric epithelial ags cells infected with epstein-barr virus. asian pac multiple pathways for epstein-barr virus episome loss from nasopharyngeal carcinoma ebna and host factors in epstein-barr virus latent dna replication the epstein-barr virus ebna protein replication of epstein-barr viral dna protein interaction domains of the ubiquitin-specific protease. usp /hausp protein profiling with epstein-barr nuclear antigen- reveals an interaction with the herpesvirus-associated ubiquitin-specific protease hausp/usp hox proteins display a common and ancestral ability to diversify their interaction mode with the pbc class cofactors the ubiquitin-specific protease usp modulates the replication of kaposi's sarcoma-associated herpesvirus latent episomal dna evaluation of circulating ebv microrna bart - p in facilitating early detection and screening of nasopharyngeal carcinoma liver-targeted cyclosporine a-encapsulated poly (lactic-co-glycolic) acid nanoparticles inhibit hepatitis c virus replication extracellular vesicles: novel vehicles in herpesvirus infection exosomal cyclophilin a as a novel noninvasive biomarker for epstein-barr virus associated nasopharyngeal carcinoma cyclophilin a restricts influenza a virus replication through degradation of the m protein characterization of filovirus proteinprotein interactions in mammalian cells using bimolecular complementation a 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replication of plasmids derived from epstein-barr virus is composed of two cis-acting components human immunodeficiency virus type hijacks host cyclophilin a for its attachment to target cells structure of the p binding domain of hausp/usp bound to epstein-barr nuclear antigen implications for ebv-mediated immortalization ebv latent membrane protein effects on plakoglobin, cell growth, and migration ribosome protein l is essential for epstein-barr virus nuclear antigen function epstein-barr virus nuclear antigen replication and segregation functions in nasopharyngeal carcinoma cell lines cypa-cd -erk / -cyclin d signaling pathway is upregulated during rat left ventricular hypertrophy global trends in incidence and mortality of nasopharyngeal carcinoma novel roles and therapeutic targets of epstein-barr virus-encoded latent membrane protein -induced oncogenesis in nasopharyngeal carcinoma ebv persistence-introducing the virus the pathogenesis of epstein-barr virus persistent infection genome-wide analysis of epstein-barr viruses isolated from primary nasopharyngeal carcinoma biopsy specimens chicken cyclophilin a is an inhibitory factor to influenza virus replication identification of candidate biomarkers for the early detection of nasopharyngeal carcinoma by quantitative proteomic analysis epstein-barr virus-derived plasmids replicate only once per cell cycle and are not amplified after entry into cells epstein-barr virus at -future perspectives epstein-barr virus and nasopharyngeal carcinoma epstein-barr virus and oncogenesis: from latent genes to tumours epstein-barr virus downregulates microrna through the oncoprotein latent membrane protein : a contribution to increased tumor incidence in epithelial cells a precise excision of the complete epstein-barr virus genome in a plasmid based on a bacterial artificial chromosome epstein-barr virus microrna mir-bart - p inhibits p expression lactoferrin suppresses the epstein-barr virus-induced inflammatory response by interfering with pattern recognition of tlr and tlr inhibition of epstein-barr virus infection by lactoferrin the copy number of epstein-barr virus latent genome correlates with the oncogenicity by the activation level of lmp and nf-kappab an update: epstein-barr virus and immune evasion via microrna regulation cadherin is activated by epstein-barr virus lmp to mediate emt and metastasis as an interplay node of multiple pathways in nasopharyngeal carcinoma key: cord- -slmlhqnb authors: yap, sally s. l.; nguyen-khuong, terry; rudd, pauline m.; alonso, sylvie title: dengue virus glycosylation: what do we know? date: - - journal: front microbiol doi: . /fmicb. . sha: doc_id: cord_uid: slmlhqnb in many infectious diseases caused by either viruses or bacteria, pathogen glycoproteins play important roles during the infection cycle, ranging from entry to successful intracellular replication and host immune evasion. dengue is no exception. dengue virus glycoproteins, envelope protein (e) and non-structural protein (ns ) are two popular sub-unit vaccine candidates. e protein on the virion surface is the major target of neutralizing antibodies. ns which is secreted during denv infection has been shown to induce a variety of host responses through its binding to several host factors. however, despite their critical role in disease and protection, the glycosylated variants of these two proteins and their biological importance have remained understudied. in this review, we seek to provide a comprehensive summary of the current knowledge on protein glycosylation in denv, and its role in virus biogenesis, host cell receptor interaction and disease pathogenesis. most denv infections are asymptomatic or remain as mild febrile illness. a classical den fever is diagnosed when the patient shows self-limiting high fever, headache, and muscle/joint pain - days after a mosquito bite. a small proportion of den patients may develop den hemorrhagic fever and/or den shock syndrome (dhf/dss) which are life-threatening. the clinical manifestations of dhf/dss include hemorrhagic fever, vascular permeability and plasma leakage, thrombocytopenia and circulatory failure in dss. to date, there is no specific treatment for den and no licensed anti-denv drug is available. for severe den cases, clinical complications are managed by supportive therapy to avoid mortality. the progression to severe den (dhf/dss) has been linked to a phenomenon known as ade of infection (halstead, ) . the ade hypothesis postulates that during a secondary heterologous denv infection, preexisting anti-denv antibodies bind to but fail to neutralize the virus, and promote increased uptake of sub-neutralized virions by fcgamma-receptor bearing cells such as dc, macrophages, and monocytes (kliks et al., ; boonnak et al., ) . in addition, ligation of fc receptor stimulates production of interleukin (il)- which in turn suppresses the cellular anti-viral response (suhrbier and la linn, ) . these events lead to increased viral loads which are believed to correlate with disease severity (vaughn et al., ) . to reduce den morbidity and eventually eliminate the disease, an effective vaccine is urgently needed. however, the development of den vaccine has been greatly hampered by the potential risk of ade. the only licensed den vaccine (cyd-tdv) available is a tetravalent, recombinant, live attenuated den vaccine developed by sanofi pasteur (guy et al., ) . the vaccine has shown varied efficacy against different serotypes and in different age groups, with safety issues in children below years of age (capeding et al., ; villar et al., ) . in addition, large scale efficacy studies have suggested that this vaccine works best in people with pre-existing denv immunity (capeding et al., ) . thus, who recommendations have limited the use of the cyd-tdv vaccine in geographical settings with high den burden and in age group - years old (who, ) . clearly, while this first-in-human tetravalent den vaccine will certainly provide a wealth of knowledge and improve our understanding of immune correlates of protection, a better vaccine is needed to protect the . billion people that are at risk of den infection. several promising vaccine candidates are currently under development; some have entered the clinical pipeline [reviewed in (schmitz et al., )] . it is hoped that they will address the shortcomings of the cyd-tdv vaccine. denv belongs to the family flaviviridae of which the members are well known as human pathogens, including wnv, zika virus, yellow fever virus, tick-borne encephalitis virus, jev, and hepatitis c virus (hvc). they are enveloped viruses with positive sense, single stranded rna and many of them are arthropod-borne viruses. among all flaviviruses, denv has the highest impact on global disease burden. the virus particle is about nm in size and the rna genome (∼ . kb) is encapsulated by a protein shell which consists of three structural proteins, namely capsid (c), envelope (e), and (pre)membrane protein (prm/m) (kuhn et al., ) . in order to establish infection, denv first binds to the host cell receptors via e proteins on the cell surface. the ligand-receptor interaction initiates uptake of the virion through receptor-mediated endocytosis (acosta et al., ) . inside the acidic late endosome, membrane fusion occurs as the virion envelope fuses with the endosomal membrane (allison et al., ; modis et al., ) , followed by uncoating of the nucleocapsid and then release of the viral rna into the cytoplasm. the rna genome of denv is translated into a single polyprotein by host ribosomes and is made of three structural (c, e, prm/m) and seven non-structural (ns) (ns , ns a/b, ns , ns a/b, ns ) proteins. the polyprotein is then cleaved by host and viral proteases to release individual viral proteins (acosta et al., ) . the viral genome replication process within the host cell is mainly driven by the ns proteins. ns anchors the replication complex to the er membrane and interacts physically with ns b (youn et al., ; muller and young, ) . ns a is responsible for viral rna synthesis and virion assembly (xie et al., ) . ns functions as a serine protease, rna helicase and nucleotide triphosphatase/rna triphosphatase, and its protease activity is dependent on the cofactor ns b (falgout et al., ) . ns a has been reported to induce membrane rearrangement within the host cell, thereby assisting the formation of replication vesicles (miller et al., ) . ns is a multifunctional enzyme with a methyltransferase (mtase superfamily) domain and a rnadependent rna polymerase domain (acosta et al., ) . during virion assembly, the newly synthesized viral rna interacts with c proteins to form the nucleocapsid. a spiky immature virion is formed when e and prm proteins encounter the nucleocapsid acosta et al., ) . the maturation process takes place in the trans-golgi network where prm is cleaved by host furin to generate a smooth surfaced mature virion (stadler et al., ; yu et al., ) , which is released into the extracellular environment through the secretory pathway. glycosylation is the post-translational modification of biomolecules such as proteins or lipids through the enzymatic attachment of complex oligosaccharide structures to the peptide backbone or lipid anchor (varki et al., ) . over % of the eukaryotic proteome is glycosylated (dell et al., ) . the range of complexity of these structures is reflected by their covalent attachment to the protein, the monosaccharide composition of the glycan and combinations of anomeric ring linkages between these monosaccharides. this complexity affects the branching, antennae and topology of the glycan structures, which translates to the overall tertiary and quaternary structure of the glycoprotein. there are two types of protein glycosylation distinguished by their site of attachment on the protein backbone; n-linked glycosylation where the glycan is covalently attached to the asparagine (n) (consensus motif; nxs/t except where x is a proline); and o-linked glycosylation where the glycan is linked to the oxygen from some serine (s) or threonine (t) residues of the protein backbone (chandler et al., ; varki et al., ) . n-linked glycans share a common chitobiose core structure. furthermore, n-linked structures fall under three different classes: ( ) high mannose, where the non-reducing composition of the glycans are dominated by mannose sugars that extend from the core, ( ) complex, where the branching and extension of the glycans from the core is initiated by n-acetyl glucosaminyl transferases; and ( ) hybrid structures where the core is extended by both high mannose arm and a complex structure on the other arm (chandler et al., ; varki et al., ) (figure ) . such complexity can be found with the isomerism/anomercity of a sugar. whilst two glycans may have the same composition, the differences of their isomeric linkages can affect the selectivity of their host receptors and thus biology. for example, in the context of avian influenza, the haemagglutinin specifically and exclusively recognizes , -linked sialic acids that are found in the avian host. this is in contrast to , linked sialic acid normally found in the human host receptors with very low , -linked sialic acid expressed in the lower respiratory tract. cross reaction between avian h n and such sialic acids caused the recent human epidemics (shinya et al., ; walther et al., ) . glycosylation is a highly organized process that involves a network of glycotransferases and glycosidases in the er-golgi complex that enzymatically synthesize the glycan as well as trim down the structures so as to achieve the refined structure. in mammalian cells, n-glycosylation takes place predominantly within the er. briefly, the initial stages of glycosylation involve the enzymatic synthesis of dolicholphosphate-nacglcnac man glc which usually takes place across the membrane of the er. this involves firstly the enzymatic synthesis of dolichol-p-p-glcnac man , before this is "flipped" across the membrane into the er lumen where further monosaccharides are added (shi and jarvis, ; dell et al., ) . the nacglcnac man glc is transferred to the appropriate nxs/t motif of the protein via the oligosaccharyltransferase as the incipient protein is being translated. as the n-glycans transition through the er-golgi complex, a series of glycosidases trim down the mannose residues before glycotransferases present in the golgi extend the antennae of the glycans to produce larger hybrid or complex structures (varki et al., ) . in contrast to n-glycosylation, o-glycosylation occurs entirely in the golgi apparatus. it does not involve any glyco-lipid intermediates and no glycosidases appear to be involved in their synthesis and processing. within the insect kingdom, glycosylation is far simpler yet interestingly insect are able to produce elaborate protein glycosylation in a restricted fashion compared to glycosylation of higher eukaryotes (rendić et al., ) . most our knowledge in insect glycosylation was derived from studies performed on drosophila melanogaster and baculoviral-insect systems. in fact, the bulk of mammalian glycoproteins expressed and/or purified in insect cells have used cell lines from spodoptera frugiperda (sf , sf ) or trichoplusia ni (high five) (rendić et al., ) . glycoproteins derived from these insect cell systems display glycans that contain predominantly high mannose type structures. however, the long belief that these high mannose and paucimannosidic n-linked structures are the dominant forms in insect-cell derived glycoproteins has been recently challenged by the advent of high resolution glyco-analytical tools which were able to identify glyco-epitopes such as [alpha] - fucosylation (hsu et al., ; takahashi et al., ; rudd et al., ) and double core fucosylated structures ([alpha] - and [alpha] - fucosylation on the core glcnac) (staudacher et al., ; rendic et al., ) . there were also reports of the extension of the [alpha] , -arm of the chitobiose core as opposed to the [alpha] - arm extension found in most mammalian cell lines (kubelka et al., ) . higher complex n-glycans can be found in many insectderived n-linked glycoproteins, however, if grown in serum free media, lysed cell extracts from sf and high five cells do lack the nucleotide donors for sialic acid (cmp-neuac) (rendić et al., ) . further to this, the presence of a truncated trimannosyl n-glycan with an [alpha] , -linked fucose was reported (shi and jarvis, ; rendić et al., ) . extensive work has shown that such structures are the result of the action of an endogenous hexosaminidase specific for the nacglcnac[beta] , -man structure rather than low activity of the [beta]- , nac glcnac transferase ii responsible for the extension of the mannose arms of complex structures (kubelka et al., ) . studies performed with mosquito cell lines a. albopictus and a. aegypti showed that glycoproteins produced in these cell lines display predominantly high mannose and pauci-mannosidic structures (hsieh and robbins, ) . interestingly, within these initial experiments, the presence of mannosidase-resistant structures was observed (rhomberg et al., ) . glycomics as a field has experienced a significant maturation from low to high resolution analysis. in the past decades, scientists have mostly relied on digestive enzymes (johnson et al., ; mondotte et al., ; hacker et al., ) , chromatography (johnson et al., ) , lectin-binding assay (johnson et al., ; hacker et al., ) and radioactive labeling (smith and wright, ) to study the glycan structure on glycoproteins. enzymatic digestion by endo h and peptide:n-glycosidase (pngase f) remains the most popular method due to its simplicity and allows the investigator to determine whether the glycan structure is asparagine linked (n-linked). in this approach, purified glycoprotein is subjected to specific enzymatic digestion prior to separation on sds-page. after cleavage of the attached oligosaccharide chains, the digested protein migrates ahead of the undigested form due to a lower molecular weight. enzymatic digestion of glycoproteins reveals, however, relatively little information on the glycan structure. over the recent decade, glycan analysis has dramatically improved through developments in fluorescent labeling, lc and mass spectrometry. there is an abundance of techniques such as lc, ce and mass spectrometry which are available for glycomic analysis. for lc and ce, the field has benefited from labels such as -ab, -aa (lc separation) and -aminopyrene- , , -trisulfonic acid (ce separation), whereby glycans are tagged at a glycan's reducing end and the identity of these glycans is assigned based on their retention time behavior across a hilic or ce, respectively (rudd and dwek, ; callewaert et al., ) . approaches coupling fluorescence with mass spectrometry (flr-ms) have helped increase the efficiency of glycomic approaches. in such a platform, the retention time and fluorescence are usually coupled with mass detection to add further confirmation (houel et al., ; zhang et al., ) . the use of exoglycosidase arrays adds further confidence to a glycan's structure elucidation and can help to identify co-eluting glycan species (marino et al., ) . the evolving development of glycan labeling has meant that newer, faster labeling and highly sensitive labels such as procainamide or rapidfluor mass spectrometry (rfms) label can increase the throughput and efficiency of a glycomic analysis. various modes of mass spectrometry have been applied to the analysis of released glycans. the most common are maldi and electrospray ionization. maldi is a straight-forward method that often requires methods such as permethylation and esterification to increase signal intensity and stabilize labile glycans that contain sialic acids. electrospray ionization involves a milder desolvation technique and coupled with lc methods and further fragmentation of the molecule, provides high resolution techniques to qualitatively characterize a glycome (nguyen-khuong et al., ) . detection of glycan fragments which result from fragmentation along the glycosidic bonds (detected in positive mode) and cross-ring (detected in negative mode) (harvey et al., ; everest-dass et al., ) help to understand the composition and topology of the glycan without the need to adulterate the glycan through derivatization. glycoproteomics allows investigators to understand the degree of glycosylation on various sites of the glycoprotein. the platform is adapted from proteomics and as such relies heavily upon mass spectrometry and substantial data analysis. whilst most glycoproteomic methodologies follow similar approaches to proteomics such as trypsin digestion and analysis, key to any glycoproteomics method is the enrichment of glycopeptides (mysling et al., ; kolarich et al., ) . this is important to reduce the ion suppression from peptide mass spectrometry signals without enrichment. this can be performed via hilic chromatography, in which the enrichment is centered upon exploiting a glycan's hydrophilicity. fragmentation data is vital to glycopeptide identification and data analysis must be able to exploit the information which can come from fragmentation modes such as collisionally induced dissociation (cid), high collisional dissociation or electron transfer dissociation/electron transfer high collisional dissociation (etd/ethcd) available to the investigator (scott et al., ; thaysen-andersen et al., ; stavenhagen et al., ) . depending on the strength of fragmentation, information such as glycan composition, site of attachment and peptide backbone are all able to be divulged from a single spectrum (yang et al., ) . glycans either directly or indirectly have diverse biological functions which span but are not limited to inflammation, immunology, infectious diseases, metabolism, embryogenesis, cancer biology and neurodegeneration. at the protein level, glycosylation is responsible for correct protein folding/structure, protein trafficking and stability, receptor/ligand recognition as well as increasing its half-life in the blood stream (park et al., ; bork et al., ; sumer-bayraktar et al., ; hart, ; palmisano et al., ) . at the cellular level, complex sugar structures modulate receptor functions and thus are integral to regulate normal cell-cell, cell-substrate communication and adhesion (vercoutter-edouart et al., ; varki et al., ) . glycosylation disorders can adversely affect immunity and cancer development. from an immunological perspective, all living cells are covered by a dense glycocalyx and indeed, pathogens and foreign objects must deal with this complex forest of cell surface glycoconjugates upon entering the host (pickles et al., ; rodrigues et al., ) . viruses do not possess their own glycosylation machinery and by virtue of their opportunistic nature, are heavily dependent upon the glycosylation machinery of the host cell to glycosylate their proteins. hiv, influenza virus, hendra virus, severe acute respiratory syndrome coronavirus (sars-cov), hepatitis viruses and wnv are examples of viruses for which glycosylation was shown to be critical to their stability, infectivity and antigenicity (mir-shekari et al., ; vigerust and shepherd, ; medina et al., ; doores, ) . firstly, glycosylation can be involved in receptor binding. this is exemplified by hiv and denv which rely on high mannose type glycosylation to bind to their mrs or dc-sign that are present on host immune cells (cambi and figdor, ; cambi et al., ) . furthermore, glycosylation is required to facilitate proper protein folding and trafficking of the viral membranes using the host chaperones such as calnexin and/or calreticulin proteins (meunier et al., ; land and braakman, ; slater-handshy et al., ) . importantly, glycosylation is a means to evade immune recognition within the host by changing glycan sites (medina et al., ) , which in turn can increase the diversity of the glycosylation on the virus. in addition, the glycan structure has been reported to mask particular antigenic sites from recognition by neutralizing antibodies (doores, ; walls et al., ) . the external protein shell of den virion consists of copies of e ( - kda) and prm glycoproteins whereby only e proteins are exposed on the surface (kuhn et al., ) . extensive research over the years has revealed multiple functions of e protein in host receptor attachment, cellular uptake of virion and membrane fusion. e protein forms dimers on the virion surface (kuhn et al., ) . the ectodomain of each e monomer without the transmembrane domains and membrane-associated "stem" region displays an elongated structure under cryo-em, which is further defined into three distinct domains (domains i, ii, and iii) (rey et al., ) . the central n-terminal di separates the dimerization dii from the c-terminal diii. diii has been proposed to be the receptorbinding domain (kuhn et al., ) whereby neutralizing monoclonal antibodies against diii most efficiently block virus initial attachment to mammalian cells (crill and roehrig, ) . the fusion peptide located at the tip of dii is essential for endosomal membrane fusion and is essential for virus entry (allison et al., ; kuhn et al., ; huang et al., ) . dimerization of e proteins at neutral ph positions the fusion loop into a hydrophobic pocket formed by di and diii of the adjacent e monomer. this helps to prevent premature exposure of the fusion loop before endocytosis of the virion by a new host cell. di forms part of the flexible hinge region which facilitates structural rearrangement of e protein during virion maturation and fusion process (zhang et al., ) . inside the acidic endosome, the ph-dependent hinge at the di-dii interface (allison et al., ; modis et al., ) allows e dimer to dissociate and rearrange into a trimeric form which serves as a pre-fusion intermediate promoting membrane fusion (modis et al., ) . in the er lumen of the host cell, membrane-associated e protein is generated after co-translational processing of the viral precursor polypeptide by host signalase (acosta et al., ) . the newly synthesized e protein rapidly heterodimerizes with prm (lorenz et al., ) and three prm-e heterodimers further oligomerize to form a total of sixty heterotrimeric prm-e spikes per subviral particle (konishi and mason, ; zhang et al., ) . this higher-order oligomer has been proposed to represent the preassembly complex (wang et al., ) . translocation of this complex from er to golgi is critical as the transition from immature to mature virion is completed only in the trans-golgi network, where the spiky prm-e trimers are rearranged into flat dimers in a head-to-tail orientation on the virion surface (kuhn et al., ; zhang et al., ) . in the er, denv e protein undergoes n-linked glycosylation at two asparagines, n and n located in dii and di, respectively (chambers et al., ; johnson et al., ; hacker et al., ) . the n glycosylation site is unique to denv and has been proposed to interact directly with dc-sign, one of the host cell receptors (pokidysheva et al., ) (see virus attachment to cell surface and cell entry process). in contrast, n (n in other flaviviruses) represents the conserved glycosylation site in the family flaviviridae. high resolution crystal structure of tick-borne encephalitis virus e dimer shows the n -oligosaccharide chain projected overhead of the hydrophobic groove where the fusion loop fits in, suggesting that it functions as an "epitope shield" over the fusion loop to stabilize the dimer contacts (rey et al., ) . consistently, denv and denv mutant viruses lacking n glycans due to a single point mutation within the glycosylation motif displayed elevated fusion ph threshold compared to their parental counterpart (guirakhoo et al., ; lee et al., ) . the authors proposed that the altered fusion activity of these mutants was likely due to instability of the e dimers. the glycan structure on denv e protein has been studied using digestive enzymes (johnson et al., ; mondotte et al., ; hacker et al., ) , chromatography (johnson et al., ) , lectin-binding assay (johnson et al., ; hacker et al., ) and radioactive labeling (smith and wright, ) . endo hand pngase f-enzymatic digestion revealed the presence of n-glycosylation in denv e protein. no o-linked glycan has been detected to date (johnson et al., ) . in mosquito cell-derived virions, the n-glycans attached to e protein display heterogeneity in structure and sugar composition where high mannose and paucimannose with terminal mannose residues are the dominant glycoforms (figure a ) (smith and wright, ; johnson et al., ; hacker et al., ) . recently mass spectroscopy has been applied to denv glycoprotein studies to provide a comprehensive and detailed profile of the glycan moieties (dubayle et al., ; lei et al., ) . using an integrated mass spectroscopy strategy consisting of lectin microarray and maldi-time of flight mass spectrometry (maldi tof-ms), lei et al. ( ) have successfully determined the detailed composition of n-glycans attached to the e protein from mosquito cell derived mature denv . among the distinct n-glycans detected, contain terminal galactosylation while the remaining glycans were identified as high mannose type, complex type, fucosylated and sialylated n-glycans. in a separate study, the n-glycans from denv - (vaccine cyd-tdv) produced in mammalian vero cells have been reported to consist of high mannose, complex and hybrid glycans with complex glycans as the major glycan species (figure a ) (dubayle et al., ) . by performing in-gel proteolysis of e-protein, site specific n-glycans have been determined. sialylated complex glycans and high mannose ( - residues) glycans were detected at n in all denv except for denv . besides, most of the complex or hybrid glycans at n were found fucosylated. interestingly, fucosylated glycans were detected only at n but not at n across all four denv serotypes. since high mannose binding dc-sign interacts only with n glycans on the viral surface (pokidysheva et al., ) and n -glycan is dispensable for virus production in mosquito and mammalian cells (bryant et al., ) , this suggests that n glycans may serve a distinct function from n glycans in den pathogenesis possibly via interaction with an unknown fucose binder or act as a viral glycan shield. for n specific glycans, denv was reported to have a different sugar composition from the other three denv serotypes (dubayle et al., ) whereby a higher content of complex or hybrid glycans was found in denv . high mannose glycans were detected as the main glycan species for denv , and . in addition, sialylated n-glycan was detected only in denv at this site. the differential glycosylation pattern between denv and denv , , may impact on various aspects of dengue pathogenesis including virus tropism, virus fitness, and induction of host responses (see role of glycosylation in denv life cycle). non-structural protein (ns ) was first identified as a nonhemagglutinating, soluble complement-fixing antigen in the brain and serum from denv -infected mice (brandt et al., ; smith and wright, ) . ns has a molecular weight range of - kda depending on its glycosylation status. it is a multifunctional glycoprotein which presents in different oligomeric forms and locates at various cellular compartments (westaway and goodman, ; flamand et al., ) . non-structural protein monomer consists of three structural domains namely a β-roll dimerization domain, a wing domain and a β-ladder domain (akey et al., ) . the monomer structure is stabilized by six intramolecular disulfide bonds and no intermolecular disulfide bond has been identified in dimeric ns (winkler et al., ) . however, any one of the three cysteine residues at the c-terminal has been reported to be important for dimer formation (pryor and wright, ) . the β-roll domain and part of the extended wing domain form a hydrophobic protrusion surface that acts as the er membrane and replication complex (ns b) interacting site, which is critical for viral rna replication (youn et al., ; akey et al., ) . ns dimer is formed when two β-roll domains dimerize at the center and these dimers tend to trimerize resulting in hexameric ns (flamand et al., ; gutsche et al., ; muller et al., ) . the ns hexamer crystal structure revealed a barrel-shaped oligomer with a central open channel. three dimers are arranged symmetrically in a way such that the β-roll domains are entirely facing inwards and the channel interior is lined by the hydrophobic protrusion surface contributed by each dimeric component (akey et al., ) . the hydrophobic lining allows the ns hexamer to be secreted as a lipoprotein whereby the lipid cargo is loaded into the central channel (gutsche et al., ) . in contrast to the β-roll domains, glycosylation sites and most of the linear epitopes of ns identified are facing outward, representing the most accessible parts of ns hexamer by host antibodies (akey et al., ) . intracellular ns is predominantly in dimeric form whereas secreted ns is mainly in hexameric form ( figure b ) (flamand et al., ) . during protein synthesis, ns is cleaved from the viral polypeptide and translocated into the er lumen. in er, newly synthesized e protein is glycosylated and heterodimerizes with prm protein to form a higher order oligomeric preassembly complex. the immature virus particle with prm-e spikes is formed when the nucleocapsid associates with prm-e-rich membranes which buds into the er lumen ( ). the glycans remain of high mannose type on the immature virus particle as it is translocated to golgi apparatus along the secretory pathway ( ). the conformational rearrangement of prm-e spikes and cleavage of prm by host protease furin occurs in the golgi to produce a mature, smooth virus particle. in mammalian cells, the glycans are further processed and modified into complex glycans before the virus particle is released to the extracellular milieu (route a). in mosquito cells, majority of the glycans are high mannose or galactosylated due to the different glycosylation enzymes expressed in insect cells (route b). high mannose glycan on the e protein, particularly the n -glycan facilitates dc-sign(+) cell infection and virus propagation. the function of complex glycan on e protein is currently unknown. the glycosylated pr peptides are bound to e protein after furin cleavage and only dissociate at neutral ph in the extracellular milieu. (b) monomeric ns protein is glycosylated with high mannose glycans at n and n . the monomer rapidly dimerizes in the er and membrane-associated ns dimers ( ) are involved in virus rna replication. three ns dimers form a soluble hexameric ns but the exact location of hexamer formation remains unknown ( ). in mammalian cells, the n glycans are modified into complex glycans before the soluble ns hexamer is secreted out of the cells (route a). in mosquito cells, generation of complex glycans doesn't happen and the lack of complex glycans (n ) on ns hexamer affects hexamer stability and greatly reduces its secretion (route b). a subset of dimeric ns are found on the infected cell surface but the trafficking pathway has yet to be determined (route c). high-mannose glycan at n stabilizes ns dimer. the soluble monomer undergoes dimerization to gain partial hydrophobicity (flamand et al., ) , allowing membrane association of ns dimer in the absence of a transmembrane domain (winkler et al., ) . the exact mechanism of ns hexamer formation remains unclear and two possible locations have been proposed including along the golgi secretory pathway, or immediately after dimerization at the er (muller and young, ) . the functions of ns are closely associated to its cellular location throughout the virus replication cycle. er membraneassociated dimeric ns has been found to co-localize with viral dsrna (mackenzie et al., ) . circulating hexameric ns is able to bind to the plasma membrane of mammalian cells via the interaction between its n-glycans and cell surface glycosaminoglycans, heparin sulfate and chondroitin sulfate e (avirutnan et al., ) . recently, it has been reported that hexameric ns contributes to disease pathogenesis of severe den (beatty et al., ; modhiran et al., ) . the soluble protein acts as a viral toxin that induces pro-inflammatory cytokine response and vascular leakage via toll-like receptor expressed on immune cells and endothelial cells (modhiran et al., ) . beatty et al. ( ) showed that ns vaccination protects mice from ns -induced vascular leakage which was independent of complement components. glycosylation of denv ns occurs right after its cleavage in the er (winkler et al., ) at two asparagines, n and n (putnak et al., ; winkler et al., ; flamand et al., ) . these two n-glycosylation sites are conserved in the family flaviviridae. recently, a less conserved glycosylation site at n has been reported in wnv, st. louis encephalitis virus and murray valley encephalitis virus but is absent in all four serotypes of denv (akey et al., ) . intracellular and extracellular denv ns display different types of n-glycans as the oligosaccharides undergo modification during the maturation process (winkler et al., ; pryor and wright, ; flamand et al., ) . intracellular dimeric ns n-glycans are of high mannose composition regardless of the host cell type (mammalian or mosquito cell) (mason, ) . on the other hand, in extracellular hexameric ns , the n -glycans consist of complex oligosaccharides whereas the n -glycans are made of high mannose type sugar chains (mason, ; pryor and wright, ; flamand et al., ) . as dimeric ns passes through the golgi apparatus, two n -glycans are further modified into the endo h-resistant, multi-branched complex type before the protein is released (winkler et al., ) . the differential modification at these two sites is due to the inaccessibility of n -glycan by golgi-resident enzymes after the dimerization of ns (flamand et al., ) . in the denv replication cycle, prm interacts with e protein and acts as a chaperone to ensure proper e protein folding (lorenz et al., ) and to prevent premature fusion of the virus particle along the secretory pathway by concealing the e fusion loop yu et al., ) . glycosylation of the prm/m glycoprotein in denv has not been extensively studied. the protein is glycosylated at n (table ) with circumstantial evidence for n-linked glycosylation at sites , , and (courageot et al., ) . it was found that α-glucosidase inhibitor reduced the amount of prm-e heterodimer, suggesting the n-glycans are required for productive folding pathway of these glycoproteins (courageot et al., ) . triglucosylated n-glycan at n of denv affects the folding of prm by causing a delayed formation of prm-e heterodimer (courageot et al., ) . n-glycosylation on both e and ns proteins has been shown to play important roles throughout the denv infection cycle from virion attachment, entry, maturation, assembly to secretion. carbohydrate chains on the denv e proteins play a critical role in host cell infection at the early step of host receptor binding. indeed, virus attachment and penetration into mammalian and mosquito cells were blocked by pre-incubation of virus with concanavalin a, a plant lectin that binds to alpha-linked terminal mannose of high mannose or hybrid glycans (hung, ) . lectins are a group of proteins that recognize carbohydrates through a carbohydrate recognition domain [reviewed in ±the n-glycosylation site is located at residue to depending on the serotype. (zelensky and gready, ) ]. to date, various lectin families such as c-type, p-type, l-type, galectin and calnexin have been shown to interact with viral components . c-type lectins are particularly important for denv infection as they have been shown to be involved in host cell attachment and disease pathogenesis (see disease pathogenesis). the cell membrane-anchored c-type lectin dc-sign has been identified as host cell receptor for many viruses , among which denv infects dc and monocyte via dc-sign (navarro-sanchez et al., ; tassaneetrithep et al., ) . the interaction between dc-sign and denv can be inhibited by the addition of mannose and mannan and has been further examined at the molecular level by structural analysis. cryo-em data of denv/dc-sign complexes reveals that the carbohydrate recognition domain of dc-sign interacts directly with the n -glycan of e dimers (pokidysheva et al., ) . consistently, lectin (hha)-resistant denv which lacks both n-glycosylation sites on e protein failed to infect dc-sign(+) dc, in contrast to productive infection and replication in dc-sign(−) and carbohydrate-independent cells such as vero, huh , c / and baby hamster kidney fibroblasts (bhk- ) (alen et al., ) . the presence of n -glycan on e protein also allows denv to infect endothelial cells in liver and lymph node via dc-sign-related proteins known as dc-signr and l-sign, the close homologues of dc-sign (tassaneetrithep et al., ; alen et al., ) . in addition to dc-sign, mr has been identified as another c-type lectin utilized by all four serotypes of denv to infect macrophages and dc (miller et al., ) . both mr and dc-sign bind to denv e protein with high affinity (k d in the sub-nanomolar range) (lo et al., ) , despite a different ligand specificity for these two host receptors (miller et al., ) . mr shows a preferential binding to terminal mannose, fucose and n-acetyl glucosamine while dc-sign binds to high-mannose oligosaccharides (miller et al., ) . as dc-sign and mr have been proposed to be the primary host receptors for denv during infection (lo et al., ) , the engagement to these c-type lectin receptors with diverse glycoforms of e protein may allow denv to infect a wide range of host cells. in contrast and interestingly, n deglycosylated (n − ) denv and denv were found to display enhanced infectivity in mosquito cells (c / ) compared to wild type (wt) (ishak et al., ; lee et al., ; alen et al., ) . for mosquito cells, the entry mode employed by flavivirus (denv and jev) was shown to involve membrane fusion instead of receptormediated endocytosis (hase et al., a,b) . hence, it is possible that absence of the n -glycans from the virion surface reduces steric hindrance and therefore promotes cell membrane attachment and membrane fusion. finally, n deglycosylated (n − ) denv mutant displayed reduced infectivity ( -fold lower) in both mammalian and mosquito cells compared to wt, possibly due to impaired virus entry process (lee et al., ; hacker et al., ) , whereby loss of the n -glycan affected the conformational stability of e proteins and led to premature exposure of the fusion peptide (yoshii et al., ) . early studies on denv e protein showed that n-glycosylation is not essential for virus replication in mosquito cells (bryant et al., ; mondotte et al., ) . instead, loss of the n glycosylation site through site directed mutagenesis (n q) in e protein was sufficient to render denv (strain ) growth defective in bhk- cells, a dc-sign(−) cell line (bryant et al., ) . direct transfection of n q mutant rna into bhk- cells neither produced intracellular viral antigen nor released new virus progeny. the lack of virion release may be due to impaired virion secretion along the er-golgi secretory pathway in the absence of n -glycan tag on e protein. however, the same mutant replicated and grew comparably to wt counterpart in c / mosquito cells in vitro and in a. aegypti mosquito in vivo (bryant et al., ) , thus supporting that n-glycosylation of e protein at position n is essential for productive infection in mammalian cells only, consistent with earlier studies (bryant et al., ; mondotte et al., ) . further investigation on the importance of the n-glycosylation motif was done by lee et al. ( ) through extensive point mutation within the conserved n-x-t/s motif of denv (strains puo- and ngc), whereby the conserved residue t was replaced with residues of different side chain propensity. replacement of t by a larger and more hydrophobic residue (leucine and valine) either by molecular cloning (lee et al., ) or by passaging the virus under selection pressure (alen et al., ) generated viable virus that retained efficient growth in bhk- and vero cells in the absence of n glycan. in addition, n q/d mutant virus generated in strain puo- propagated in mammalian cells at reduced growth rate, which is inconsistent with previous studies carried out with denv strain (bryant et al., ; mondotte et al., ) . the differential and virus strain-dependent outcome led to the hypothesis that in the absence of n glycosylation, the aminoacid composition of the dii region determines virus survival in mammalian cells (lee et al., ) . multiple sequence alignment showed indeed that strain differed from strains puo- and ngc at two positions, arginine (r) in dii and t in di (figure ) . uncharged polar threonine replaces charged r and hydrophobic isoleucine (i) replaces t in strains puo- and ngc. it is possible that the presence of hydrophobic residues facilitates protein folding even without the glycan tag for chaperone-assisted folding and followed by productive protein secretion. nevertheless, structural comparison of the wt strains and their respective mutants needs to be carried out to confirm this hypothesis. in contrast to the varying outcomes obtained with n mutant viruses and their ability to grow in mammalian cells, it has been consistently reported that these virus variants replicate, propagate in mosquito cells but produce lower virus titers compared to wt (bryant et al., ; mondotte et al., ; lee et al., ) . n -glycan on e protein is important but not essential for denv survival in mosquito cells. successive passages (as low as two passages) of denv in c / cells in vitro resulted in mutation at t which ablates the n -glycosylation motif; however, the non-glycosylated variant was able to propagate in mosquito cells (lee et al., ) . n − denv grows in both mammalian cells and c / cells and produce lower virus titer than its wt counterpart (bryant et al., ; lee et al., ) , which could be due to defective virus budding. consistently, in a study using transmission electron microscopy, it was shown that virus budding of wt wnv occurs at the plasma membrane while the mature progeny of n − mutant scatters at the smooth membrane vesicle within swollen er lumen without budding (li et al., ) . n a ns mutant virus of denv (tajima et al., ) and denv (ngc) (pryor et al., ) failed to generate viable virus in both mammalian and mosquito cells. mutation at n in denv ns caused reduced viral growth in mammalian cells and c / cells (pletnev et al., ) . however, n q ns mutant of denv ( ) produced infectious virus with a similar titer as the wt virus in mammalian cells but with a reduced titer in c / cells (crabtree et al., ) . removal of glycan from n in denv and denv ( ) ns protein produced similar growth and virus titers compared to wt in mammalian cells despite a delayed cytopathic effect (crabtree et al., ; tajima et al., ) , which is not consistent with the observation on denv (ngc) (pryor et al., ) . double mutation attempts (n q/n q and t n/t n) failed to generate genetically stable mutant viruses (crabtree et al., ) . taken together, the findings suggest that at least one of the two n-glycosylation sites (probably n ) in ns protein is essential to produce viable virus. similar to e protein, the impact of deglycosylation at this site varies depending on the virus strain and amino acid residue used for replacement. in denv, n -glycosylation is important but not essential for ns secretion in mammalian cells (despres et al., ; jacobs et al., ; pryor and wright, ; crabtree et al., ) . single and double ns mutant proteins are secreted from infected cells even though a reduced secretion yield has been observed (crabtree et al., ; somnuke et al., ) . the impact of deglycosylation on secretion is thought to be associated with the stability of the ns oligomer. mutation of n or n does not affect dimerization of the protein but compromises the stability of the dimer (winkler et al., ; pryor and wright, ) . the dimer appeared more heat sensitive when the n-glycan was removed from the protein especially for n a mutant. the use of tunicamycin, an enzyme targeting the host glycosylation enzymes, allowed confirm that absence of n-glycan was solely responsible for the instability of ns oligomers instead of changes in the polypeptide backbone in the genetically deglycosylated mutants (pryor and wright, ; flamand et al., ) . furthermore, the secretion of ns was reduced when complex glycans maturation was blocked by glycosylation inhibitors swainsonine and -deoxymannojirimycin (flamand et al., ) . consistently, low levels of ns with solely high mannose glycan are secreted from infected mosquito cells, which lack the enzymes to generate complex type glycans. these findings support the proposal that n-glycosylation and complex glycan are important for ns secretion (hsieh and robbins, ; mason, ; thiemmeca et al., ) . in addition, the majority of the secreted wt and n q ns proteins are hexamers (somnuke et al., ) , whereas secreted n q and n /n q ns proteins showed reduced hexamer population and increase in higher order oligomer (> kda) population. as compared to mammalian cell-secreted ns , mosquito cell-secreted ns is less stable and undergoes degradation more rapidly at body temperature (thiemmeca et al., ) . hence, the presence of complex glycan at n is critical for both ns secretion and ns hexamer stability (flamand et al., ; somnuke et al., ) . similarly, it has been proposed that high-mannose glycans at n stabilizes ns dimer (pryor et al., ; flamand et al., ; somnuke et al., ) . reduced levels of denv n q and n q/n q ns proteins were observed in culture supernatant (somnuke et al., ) which could be explained by two possible scenari: ( ) stability of the secreted mutant forms is compromised due to a different protein conformation (somnuke et al., ) . the misfolding of the protein may lead to a less effective secretion of functional hexamer. ( ) transport of the protein from the perinuclear region is affected which in turn compromises the maturation and secretion of the protein (crabtree et al., ) . deglycosylated ns mutant viruses (n − ) are less neurovirulent as evidenced by the reduced mortality observed with mice infected intracranially with the denv and denv mutants (pletnev et al., ; crabtree et al., ) . the reduced neurovirulence of these viruses which lack the complex type glycans may be linked to the reduced levels of extracellular hexameric ns (crabtree et al., ) . the virulence phenotypes observed with n − mutant viruses varied depending on the denv strain. denv n − mutant displayed decreased virulence (pryor et al., ; crabtree et al., ) , whereas the denv n − mutant showed enhanced virulence in mice (pletnev et al., ) . the low to undetectable levels of ns specific antibodies in mice infected with the denv n − mutant suggests that the enhanced neurovirulence could be attributed to the reduced immunogenicity of the virus (pletnev et al., ) . the complement cascade is the central defense mechanism of innate immunity which triggers the immune effector function to remove infectious pathogens and modified self cells upon activation. the activation and amplification of the complement pathway involves a series of sequential events and the whole process is tightly regulated. complement can be activated through three major pathways, namely the classical, lectin and alternative pathways [reviewed in (ricklin et al., ) ]. the classical pathway is triggered by antibody-antigen complexes whereas the lectin pathway is activated by carbohydrate moieties on the microbial surface. the alternative pathway is activated through direct binding of c b at the surface of pathogens, which results from the constitutive basal cleavage of c (ricklin et al., ) . mannose binding lectin (mbl) in the lectin pathway triggers antibody-independent activation of complement (thielens et al., ) . the proposed mbl-mediated virus elimination mechanisms include ( ) direct virus neutralization, ( ) c /c deposition on virus surface and ( ) interference of host cell lectin receptor binding . mbl differentiates self-from non-self-antigens based on a sugar density-dependent recognition mechanism (dam and brewer, ) , and the micro pattern of the oligosaccharides structure in addition to the spatial geometry of the macro sugar pattern (takahashi and ezekowitz, ) . it was proposed that the additional n -glycan in denv (which is absent in other flaviviruses) could promote a more efficient recognition and binding by mbl . this hypothesis is supported by improved mbl binding and in vivo virus clearance of a genetically engineered wnv with additional n -glycosylation site . mbl is reactive to high mannose oligosaccharides and thus can efficiently recognize insect cell-derived denv with high mannose glycans present on its e proteins. hence, the change of n-glycan profile of e protein after one round of replication in mammalian host cells may provide an opportunity to the virus to escape from effective mbl recognition (fuchs et al., ) . however, mammalian cell-derived denv was found to be effectively inhibited and neutralized by mouse mbl . a separate study instead reported preferential binding of recombinant human mbl to insect cell-derived denv , whereas virions produced in monocyte-derived dc were not neutralized by human mbl . it therefore remains unclear whether mbl-mediated virus clearance is optimally engaged during denv infection in humans. furthermore, studies have shown that ns interferes with the complement pathway through binding to a number of its components (muller and young, ) . n q ns was found to bind to c s proenzyme, c , c , and c b with reduced affinity compared to wt and n q ns (somnuke et al., ) , indicating that the n-glycan is required for effective interaction with complement components. the role of ns glycosylation in the ability of the protein to interfere with the complement activation, however, has been largely ignored. the n-glycans were proposed to be involved in ns binding to c (avirutnan et al., ) although direct experimental evidence has been missing. a recent study reported that secreted ns binds directly to c bp, a major inhibitor of the c b component (thiemmeca et al., ) . this binding leads to the recruitment of c bp on cell surface via ns and inactivates c b thereby interfering with the formation of the membrane attack complex (mac). furthermore, the work has demonstrated a competitive binding of ns to mbl, which prevents mbl-mediated denv destruction. the presence of secreted ns in the saliva of aedes mosquito suggests that secreted ns protein could help denv to escape the host innate immune surveillance during virus transmission (thiemmeca et al., ) . severe den (dhf/dss) is characterized by increased vascular permeability and plasma leakage, thrombocytopenia, hemorrhagic fever and circulatory failure in dss (who, ) . the current paradigm proposes that viral-induced proinflammatory cytokine storm drives the disease progression to dhf/dss (pang et al., ) . direct interaction between denv and clec a expressed on macrophages indicates that virus glycosylation plays a role in den pathogenesis wu et al., ) . similar to the engagement to dc-sign, denv binding to clec a relies on sugar moieties and can be inhibited by exogenous fucose and mannose . however, clec a binding does not mediate viral entry into the host cell, instead it serves as a cooperative signaling receptor to mr/dc-sign that activates macrophage inflammasome and triggers the production of pro-inflammatory cytokines wu et al., ; lo et al., ) . consistently, anti-clec a monoclonal antibody reduced denv-induced vascular leakage in a mouse model , which further supports that targeting the viral glycoprotein-host lectin receptor interactions represents a potential therapeutic approach to counteract the excessive inflammatory responses involved in severe den. glycosylation is a post-translational modification which significantly affects the conformation of a protein. it is a heterogeneous process that is highly host-cell specific. viruses have evolved to utilize their host's glycosylation machinery so as to optimize their fitness, infectivity, replication and virulence. the role of glycosylation and glycan structures in denv virulence has yet to be reported with evidence of attenuated phenotypes in symptomatic mouse models. given the impact of glycosylation in virus entry and virus fitness in mammalian cells, it is highly likely that deglycosylated denv mutants will display reduced virulence in vivo. the ability of celgosivir treatment, a bicyclic iminosugar that inhibits glycosylation through negatively binding to er [alpha]-glucosidase ii, to protect mice from a lethal denv challenge indirectly demonstrates the importance of glycosylation in denv virulence (perry et al., ; sayce et al., ; warfield et al., ) . to date, out of the eight denv potential glycoproteins (table ) , only e and ns proteins have been characterized from a glycosylation standpoint and not across all the denv serotypes. despite the biological importance of these structures being recognized, efforts to characterize the nature of the glycan structures in denv have remained timid. there is for example little understanding of how glycosylation impacts denv cell tropism where different glycan variants may influence binding of denv to various host cell receptors and subsequent cell infection. characterization of denv glycoforms has been mainly performed in the mammalian cell line bhk- but no study has been conducted in more relevant primary mammalian cell types including langerhans cells, monocytes, hepatocytes, and endothelial cells. furthermore, in-depth characterization of the glycan structures using the latest glycomics technologies has yet to be reported for denv. associating the contribution of these glycans to the structure and ultimately function of the virion glycoproteins indeed requires glycomics and glycoproteomics. such data need to be modeled using computational approaches as methods for crystallizing glycoproteins remains a complicated feat. furthermore, the role of glycosylation is also very important to recognize in biotherapeutic strategies. while substantial efforts have been devoted to developing neutralizing antibodies against denv, the potency of these antibodies is largely dictated by the accessibility of the epitope that they target which can be influenced by the glycosylated status of the protein (smith et al., ) . consistently, e protein glycosylation site has been reported to modulate the binding of neutralizing antibodies against a highly conserved, serotype cross-reactive epitope (dejnirattisai et al., ) . these challenges have prompted substantial investment into elucidating the three-dimensional conformation of the protein-antibody complexes and more importantly how glycosylation contributes to the tertiary and quaternary arrangements of the different glycoproteins on the virion. this approach is critical for the development of new therapeutics with broader activity and increased efficacy. in conclusion, the den field as a whole would benefit greatly from in-depth understanding and characterization of the glycosylation patterns of den virions. with the recent technical advances in the fields of glycomics and glycoproteomics, this has become possible and will depend on productive interactions between glycobiologists and den virologists. sy, tn-k, and sa wrote the manuscript. pr provided suggestions and edited the manuscript. sa holds a research grant from the national medical research council (nmrc/mohiafcat / / ) to develop dengue mouse models. revisiting dengue virushost cell interaction: new insights into molecular and cellular virology flavivirus ns structures reveal surfaces for associations with membranes and the immune system crucial role of the n-glycans on the viral 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adversely affect dengue virus infectivity but are beneficial for virion release changes in the dengue virus major envelope protein on passaging and their localization on the three-dimensional structure of the protein characterization of n-glycan structures on the surface of mature dengue virus derived from insect cells the glycosylation site in the envelope protein of west nile virus (sarafend) plays an important role in replication and maturation processes structure of the immature dengue virus at low ph primes proteolytic maturation the roles of direct recognition by animal lectins in antiviral immunity and viral pathogenesis dengue virus infection is through a cooperative interaction between a mannose receptor and clec a on macrophage as a multivalent hetero-complex folding and dimerization of tick-borne encephalitis virus envelope proteins prm and e in the endoplasmic reticulum immunolocalization of the dengue virus nonstructural glycoprotein ns suggests a role in viral rna replication a systematic approach to protein glycosylation analysis: a path through the maze maturation of japanese encephalitis virus glycoproteins produced by infected mammalian and mosquito cells glycosylations in the globular head of the hemagglutinin protein modulate the virulence and antigenic properties of the h n influenza viruses analysis of the glycosylation sites of hepatitis c virus (hcv) glycoprotein e and the influence of e glycans on the formation of the hcv glycoprotein complex the mannose receptor mediates dengue virus infection of macrophages the non-structural protein a of dengue virus is an integral membrane protein inducing membrane alterations in a k-regulated manner the glycosylation of the influenza a virus hemagglutinin by mammalian cells. a site-specific study dengue virus ns protein activates cells via toll-like receptor and disrupts endothelial cell monolayer integrity a ligand-binding pocket in the dengue virus envelope glycoprotein structure of the dengue virus envelope protein after membrane fusion essential role of dengue virus envelope protein n glycosylation at asparagine- during viral propagation structure of the dengue virus glycoprotein nonstructural protein by electron microscopy and single-particle analysis the flavivirus ns protein: molecular and structural biology, immunology, role in pathogenesis and application as a diagnostic biomarker utilizing ion-pairing hydrophilic interaction chromatography solid phase extraction for efficient glycopeptide enrichment in glycoproteomics dendritic-cell-specific icam -grabbing non-integrin is essential for the productive infection of human dendritic cells by mosquitocell-derived dengue viruses glycomic characterization of basal tears and changes with diabetes and diabetic retinopathy structural analysis of glycoprotein sialylation -part ii: lc-ms based detection of cascades and perfect storms: the immunopathogenesis of dengue haemorrhagic fever-dengue shock syndrome (dhf/dss) the asialoglycoprotein receptor clears glycoconjugates terminating with sialic acid alpha , galnac an iminosugar with potent inhibition of dengue virus infection in vivo retargeting the coxsackievirus and adenovirus receptor to the apical surface of polarized epithelial cells reveals the glycocalyx as a barrier to adenovirus-mediated gene transfer chimeric tick-borne encephalitis and dengue type viruses: effects of mutations on neurovirulence in mice cryo-em reconstruction of dengue virus in complex with the carbohydrate recognition domain of dc-sign growth restriction of dengue virus type by site-specific mutagenesis of virusencoded glycoproteins the effects of site-directed mutagenesis on the dimerization and secretion of the ns protein specified by dengue virus glycosylation mutants of dengue virus ns protein functional and antigenic domains of the dengue- virus nonstructural glycoprotein ns- modulation of neural carbohydrate epitope expression in drosophila melanogaster cells the glycosylation capacity of insect cells the envelope glycoprotein from tick-borne encephalitis virus at a resolution reconstitution in vitro of the gdp-fucose biosynthetic pathways of caenorhabditis elegans and drosophila melanogaster complement in disease: a defence system turning offensive parasite glycobiology: a bittersweet symphony hybrid and complex glycans are linked to the conserved n-glycosylation site of the third eight-cysteine domain of ltbp- in insect cells rapid, sensitive sequencing of oligosaccharides from glycoproteins iminosugars inhibit dengue virus production via inhibition of er alpha-glucosidases-not glycolipid processing enzymes next generation dengue vaccines: a review of candidates in preclinical development simultaneous glycan-peptide characterization using hydrophilic interaction chromatography and parallel fragmentation by cid, higher energy collisional dissociation, and electron transfer dissociation ms applied to the n-linked glycoproteome of campylobacter jejuni protein n-glycosylation in the baculovirus-insect cell system avian flu: influenza virus receptors in the human airway hcv e glycoprotein: mutagenesis of n-linked glycosylation sites and its effects on e expression and processing synthesis of proteins and glycoproteins in dengue type virus-infected vero and aedes albopictus cells the potent and broadly neutralizing human dengue virus-specific monoclonal antibody c reveals a unique cross-reactive epitope on the bc loop of domain ii of the envelope protein n-linked glycosylation of dengue virus ns protein modulates secretion, cell-surface expression, hexamer stability, and interactions with human complement proteolytic activation of tick-borne encephalitis virus by furin alpha - (alpha - )-difucosylation of the asparagine-bound n-acetylglucosamine in honeybee venom phospholipase a site-specific n-and o-glycopeptide analysis using an integrated c -pgc-lc-esi-qtof-ms/ms approach suppression of antiviral responses by antibody-dependent enhancement of macrophage infection n-glycans modulate the function of human corticosteroid-binding globulin characterization of asn -to-ala mutant of dengue type virus ns protein the role of the mannose-binding lectin in innate immunity n-glycan structures of murine hippocampus serine protease, neuropsin, produced in trichoplusia ni cells dc-sign (cd ) mediates dengue virus infection of human dendritic cells maturing glycoproteomics technologies provide unique structural insights into the n-glycoproteome and its regulation in health and disease interaction of c q and mannan-binding lectin with viruses secreted ns protects dengue virus from mannose-binding lectin-mediated neutralization essentials of glycobiology dengue viremia titer, antibody response pattern, and virus serotype correlate with disease severity glycoproteomics and glycomics investigation of membrane n-glycosylproteins from human colon carcinoma cells virus glycosylation: role in virulence and immune interactions efficacy of a tetravalent dengue vaccine in children in latin america glycan shield and epitope masking of a coronavirus spike protein observed by cryo-electron microscopy glycomic analysis of human respiratory tract tissues and correlation with influenza virus infection prm-and cell-binding domains of the dengue virus e protein inhibition of endoplasmic reticulum glucosidases is required for in vitro and in vivo dengue antiviral activity by the iminosugar uv- variation in distribution of the three flavivirus-specified glycoproteins detected by immunofluorescence in infected vero cells newly synthesized dengue- virus nonstructural protein ns is a soluble protein but becomes partially hydrophobic and membrane-associated after dimerization evidence that the mature form of the flavivirus nonstructural protein ns is a dimer clec a is critical for dengue virus-induced inflammasome activation in human macrophages two distinct sets of ns a molecules are responsible for dengue virus rna synthesis and virion assembly hybrid mass spectrometry approaches in glycoprotein analysis and their usage in scoring biosimilarity n-linked glycan in tick-borne encephalitis virus envelope protein affects viral secretion in mammalian cells, but not in tick cells evidence for a genetic and physical interaction between nonstructural proteins ns and ns b that modulates replication of west nile virus association of the pr peptides with dengue virus at acidic ph blocks membrane fusion structure of the immature dengue virus at low ph primes proteolytic maturation the c-type lectin-like domain superfamily challenges of glycosylation analysis and control: an integrated approach to producing optimal and consistent therapeutic drugs structures of immature flavivirus particles conformational changes of the flavivirus e glycoprotein the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © yap, nguyen-khuong, rudd and alonso. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- -qqhgmqrg authors: nan, yuchen; zhang, yan-jin title: molecular biology and infection of hepatitis e virus date: - - journal: front microbiol doi: . /fmicb. . sha: doc_id: cord_uid: qqhgmqrg hepatitis e virus (hev) is a viral pathogen transmitted primarily via fecal-oral route. in humans, hev mainly causes acute hepatitis and is responsible for large outbreaks of hepatitis across the world. the case fatality rate of hev-induced hepatitis ranges from . to % in young adults and up to % in infected pregnant women. hev strains infecting humans are classified into four genotypes. hev strains from genotypes and are zoonotic, whereas those from genotypes and have no known animal reservoirs. recently, notable progress has been accomplished for better understanding of hev biology and infection, such as chronic hev infection, in vitro cell culture system, quasi-enveloped hev virions, functions of the hev proteins, mechanism of hev antagonizing host innate immunity, hev pathogenesis and vaccine development. however, further investigation on the cross-species hev infection, host tropism, vaccine efficacy, and hev-specific antiviral strategy is still needed. this review mainly focuses on molecular biology and infection of hev and offers perspective new insight of this enigmatic virus. hepatitis e virus (hev) is a positive-sense, single-stranded rna virus, and is classified in the genus orthohepevirus, the family hepeviridae (smith et al., ) . the hev-caused hepatitis e is generally a self-limiting disease with a case fatality rate from . to % in young adults but up to % in infected pregnant women in their third trimester of gestation (jameel, ; nan, ) . world health organization (who) estimates that there are million infections with over million symptomatic cases and , deaths annually across the world (who, ) . hev is primarily transmitted via fecal-oral route. hev infection was previously thought to be a public health problem only for the developing countries. indeed, hepatitis e is highly endemic in east and south asia, as well as africa according to the who (who, ) . hev strains infecting humans are classified into four genotypes. hev strains from genotypes and are zoonotic, whereas, those from genotypes and have no known animal origin. discovery of hev from swine and other species suggests that genotypes and hev has a wide host range (christensen et al., ; dalton et al., ; meng, ; pavio et al., ) . currently, hepatitis e is frequently recognized in industrialized countries, where it was not thought to be endemic (kwo et al., ; erker et al., ; schlauder et al., ; worm et al., ; kabrane-lazizi et al., ; mizuo et al., ; sadler et al., ) . moreover, along with isolation of hev from the pig, chicken, mongoose, rabbit, rat, ferret, bat, fish, and deer (meng et al., ; haqshenas et al., ; li et al., c; cossaboom et al., ; smith et al., ) , cross-species infection of hev from animal reservoirs to humans is thought to be the major cause of sporadic cases of hepatitis e in the industrialized countries (pavio et al., ) . although previously thought to only cause acute infections, hev is found in chronic infections reported both in immune compromised and immunocompetent individuals (hoofnagle et al., ; grewal et al., ) . in addition, extrahepatic manifestations, such as neurological disorders and kidney injury in hev infected patients have been documented (kamar et al., (kamar et al., , b van eijk et al., ; dalton et al., ; geng et al., ) . taken together, current knowledge for hev implies a significant underestimation of hev infection as a public health concern. in the following sections, recent progress in hev biology, functions of viral proteins, cell culture system, epidemiology, viral pathogenesis, treatment, and vaccine development are reviewed and perspective new insights are discussed. hepatitis e was initially designated as enterically transmitted non-a, non-b hepatitis (et-nanbh) due to similar clinical presentations to hepatitis a and b in patients, but the prospective causative agent was initially unknown (balayan et al., ) . early research implied that an rna virus was the potential pathogen for the et-nanbh. by analysis of a cdna library from infectious bile sample, a portion of a highly conserved rnadependent rna-polymerase (rdrp) motif, commonly found in rna viruses, was identified (reyes et al., ). this new virus was designated as hev, which was responsible for the outbreak of et-nanbh. the complete sequence of hev genome was published year later (tam et al., ) . sequence analysis indicated that hev contains a . kb single-stranded positive-sense rna genome, which is capped and poly-adenylated (ahmad et al., ) . there are three partially overlapped open reading frames (orfs) in an order of sequences encoding non-structural proteins (nsps) followed by structural protein (tam et al., ; tsarev et al., ; figure ) . hev orf encodes a non-structural polyprotein that consists of replicase proteins needed for hev replication. orf encodes the capsid protein, which is the major structural protein of the hev virions, which are non-enveloped particles of - nm in diameter (mori and matsuura, ) . orf encodes a small multifunctional protein with a molecular mass of kda (vp ). there are also short untranslated regions (utrs) in both the and -end of the genome. recently, an orf was identified from genotype hev solely ; figure ). expression of orf is cap-independent and driven by a putative ires-like element between and nt of the hev genome . the orf of hev can be translated directly from the genomic rna, whereas orf and orf are translated soly from the sub-genomic rna in alternative frames (graff et al., ) . in an earlier report, three rna species were detected in liver tissue of experimentally infected macaques, with sizes of . , . , and kb (tam et al., ) . the . and kb rna species were thought to be sub-genomic rnas for translation of orf and orf , respectively. however, a later study in huh cells only identified one capped . kb sub-genomic rna, which is a bicistronic mrna for translation of both orf and orf (graff et al., ) . transcription of this sub-genomic rna initiates at nucleotide position in the sar strain, which is located downstream of the first two methionine codons of the initially presumed orf . the same conclusion was drawn from another in vitro study of genotype hev infection in plc/prf/ hepatoma cells . the hev genome contains two cis-reactive elements (cres) that are essential for the viral replication (cao et al., ; parvez, b) . the first cre overlaps the end of orf and the utr and is essential for hev replication. the second cre locates in the intergenic region of the hev genome and forms a stem-loop structure that may be the promoter for synthesis of the . -kb subgenomic rna (cao et al., ) . hepatitis e virus was initially classified as a member of the caliciviridae family. however, sequence analysis of hev orf indicated no similarity to caliciviruses, or other picorna-like viruses. on the other hand, there is limited but significant similarity to the alphavirus-like superfamily of rna viruses, specifically, the rubella virus (berke and matson, ) . consequently, hev was classified into the family hepeviridae (berke and matson, ; emerson and purcell, ) . although hev strains are highly diverse and heterogenic, only one serotype of hev exists. classification of hev strains is under transition due to the different criteria used (smith et al., (smith et al., , . recently, a new proposal for the classification of the family hepeviridae was published (smith et al., ) . in this proposal, the family hepeviridae contains two genera: orthohepevirus (all mammalian and avian hev isolates) and piscihepevirus (trout hev; table ). within the genus orthohepevirus, four different species (a, b, c, d) are designated to include isolates from different hosts (smith et al., ) . all four previously recognized hev genotypes ( - ) that infect humans belong to the orthohepevirus a virus (smith et al., ) . the previously recognized hev genotypes - classification system was based on complete genomic sequences (lu et al., ) . hev genotype is the most conserved among the four genotypes. there is only one full-length genotype sequence available (smith et al., ) . both hev genotypes and are restricted to humans with no known animal reservoirs, whereas genotypes and are zoonotic with an expanded host range ( table ; meng, ; ahmad et al., ) . therefore, genotypes and hev strains are highly diverse (lu et al., ; smith et al., ) . since the constant discovery of new hev or hev-related isolates from rabbit, rat, ferret, bat, moose, farmed mink, camel, and wild boar (lorenzo et al., ; zhao et al., ; johne et al., ; geng et al., ; takahashi et al., ; drexler et al., ; raj et al., ; krog et al., ; lin et al., ; woo et al., ) , four genotypes are no longer satisfying classification of expanding hev isolates. in the new classification system, some hev strains from wild boars in japan with unique viral nucleotide sequences are designated as genotypes and , while hev from camel is classified as genotype of orthohepevirus a (smith et al., ) . figure | schematic illustration of hepatitis e virus (hev) genome, subgenomic rna, and orfs. orf (nt - ) is labeled above the genomic rna box. orf (nt - ) and orf (nt - ) are encoded by the same subgenomic rna. the newly identified isre like sequence (nt - ) and orf (nt - ) which are overlapped with orf are listed as well. moreover, the numbers above or below the rna boxes indicate nucleotide numbers of the cdna of hev sar (genbank accession number af ) genomic rna. the genotypes that infect humans include , , , , and ( table ; smith et al., ) . hepatitis e virus-like virus isolated from avian species is called avian hev, which shares less than % nucleotide identity but common antigen epitopes in the capsid protein with mammalian hev (haqshenas et al., (haqshenas et al., , huang et al., ) . currently, avian hev is classified into the species orthohepevirus b (smith et al., ) . hev strains from rat, ferret and bat are classified into the species of orthohepevirus c and d, respectively (smith et al., ) . the cutthroat trout virus (ctv) is identified as an hev-like virus in retrospective studies. ctv shares even lower sequence identity with mammalian and avian hev and is now classified as a member of the genus piscihepevirus (batts et al., ; smith et al., ) . for the four genotypes ( - ) of hev infecting humans, there are differences in their geographic distributions. genotype hev mainly includes strains from asia and africa including the sar isolate, while genotype contains a mexican strain and variants from africa. genotypes , including human and swine hev, is mainly found in the industrialized countries (purcell and emerson, ) . the genotype is previously thought to be found only in china (purcell and emerson, ) , however, recent reports show that genotype hev strains are also isolated in other countries, including india, indonesia, japan, vietnam, spain, france, and italy (okamoto, ; midgley et al., ; lapa et al., ) . for details about molecular epidemiology and viral evolution of hev, please refer to the article by purdy and khudyakov ( ) . the orf is the largest orf in the hev genome and has nt in length according to the sar strain (tsarev et al., ; emerson et al., ) . it starts at the end of the genome after a nt non-coding region and can be translated directly from the hev genome. orf encodes a amino acid (aa) polyprotein, which is needed for hev replication. bioinformatics analysis for the protein sequence encoded by orf found eight putative domains according to their similarity to counterparts in the other viruses . moreover, the orf sequence is highly related to the group of rubi-like viruses including rubivirus, betatetravirus, benyvirus, and omegatetravirus (koonin and dolja, ; liu et al., ). these functional domains include methyltransferase domain (met), y domain (y), papain-like cysteine protease (pcp or plp), hypervariable region (hvr), proline-rich region (pro), x domain, helicase domain (hel) and rdrp (figure ) . in recent publications, the proline-rich region is frequently named together with hvr as the hvr. the current data are conflicting about whether the hev orf product functions as a single polyprotein or needs to be further processed into smaller units by viral or cellular proteases ropp et al., ; sehgal et al., ; suppiah et al., ; perttila et al., ) . one study using a vaccinia-derived expression system demonstrated that the orf polyprotein could be cleaved by the pcp within it (ropp et al., ) . more than years after that publication, the same group showed a lack of processing of the orf polyprotein in hek t cells (suppiah et al., ) . two fragments were found in another study on in vitro translation of full-length orf , but they were not observed in pulse-chase assay in human cells and their production was not dependent on the predicted protease domain in orf product (perttila et al., ) . furthermore, in escherichia coli and a cell-free system based on hepg cells, orf was expressed as a kda protein without further processing detected . on the other hand, other studies demonstrated contrasting results. transfection of hepg cells with in vitro transcribed rna from hev cdna produced cleaved products with sizes of , , and kda for the met, hel, and rdrp domains, respectively (panda et al., ) . another study focusing on the analysis of the orf functional domains also observed proteolytic processing of the hev orf fragment in insect cells (magden et al., ) . in a later study, the orf product expressed in insect cells by baculovirus expression system was shown to exist as smaller fragments and this proteolytic processing could be inhibited by e- d, a cell-permeable cysteine protease inhibitor (sehgal et al., ) . a recent publication reported that the refolded pcp domain expressed in e. coli is able to process orf polyprotein in vitro (paliwal et al., ) . moreover, based on an hev-sar replicon system in s - cells (a subclone of huh cells with improved hev replication; graff et al., ) , the putative catalytic aa residues in the orf protease domain are indispensable for hev replication (parvez, ) . overexpression of orf from hev sar strain in s - cells also resulted in cleaved products (parvez, ) . thus, despite the lack of conclusive data, the majority of studies so far are in favor of the polyprotein proteolysis. the cleaved orf products could be possibly detected in the hevinfected cells if effective and specific antibodies against the domains are available. a recent study employing yeast two hybrid (y h) demonstrated intraviral interactome within the domains from orf , further supporting the proteolysis of orf polyproteins (osterman et al., ) . moreover, orf could be a determining factor for host tropism as a recombinant hev harboring orf of a genotype hev strain and the rest genome from genotype strain replicates in transfected porcine kidney cells (chatterjee et al., ) . therefore, orf products involve in determination of hev host tropism and should be further investigated. it is possible that proline rich region or hvr may be involved in host tropism determination since other domains are more conserved among the four hev genotypes. the met domain is the first one at the n-terminus of the orf -encoded polyprotein. as the hev genome is capped and the capping is crucial for its infectivity, a viral-specific frontiers in microbiology | www.frontiersin.org methyltransferase was expected zhang et al., ) . based on sequence analysis, the region in aa residues - was assumed to be a putative methyltransferase . the hev met domain is similar to that of tricornaviruses, which belong to the alpha-like supergroup of rna viruses (van der poel et al., ) . there are an invariant his residue, an aspxxarg signature and an invariant tyr residue in methyltransferase motifs i, ii, and iv, respectively . expression of hev orf cdna (aa residues - ) in insect cells yields a kda protein (p ), along with a kda protein that is believed to be the proteolytic product of p (magden et al., ) . in vitro assays shows that the p possesses guanine- -methyltransferase and guanylyl transferase activity (magden et al., ) . the second domain after the methyltransferase is the y domain, which is assumed to start from aa residue and ends at aa . it is highly similar to that of the rubella virus . currently, there is no information available for the function of this y domain in either hev or the rubella virus. papain cysteine protease domain is downstream of the y domain. the pcp domain demonstrates moderate similarity to the protease domain in the rubella virus . in the rubella virus, the pcp domain is responsible for the proteolytic processing of its nsp (marr et al., ) . mutation of the catalytic residue within the pcp (cys ) abolishes its protease activity and results in inhibition of the nsp processing. it is also involved in trans and cis cleavage of the rubella virus nsp (liang et al., ) . however, regarding the function of the hev pcp domain, the current data are incomplete and controversial. in the vaccinia-mediated orf expression system, mutation of the putative catalytic core (cys ) of hev pcp had no effect on proteolytic processing of the orf product (ropp et al., ) . another putative catalytic site of his in pcp is not conserved among different hev strains. later studies showed controversial data for the processing of the hev orf product ropp et al., ; sehgal et al., ; suppiah et al., ) . this leads to the speculation that whether hev pcp is a real cysteine protease. recently, parvez demonstrated that the mutation of six cystine residues (c a, c a, c a, c a, c a, c a) and three histidine residues (h l, h l, h l) in the pcp domain completely abolished hev rna replication in a sar -based replicon system in s - cells. notably, of these essential cys and his residues, c and h were previously predicted as putative catalytic residues in the pcp domain (parvez, ) . furthermore, the pcp domain expressed in the e. coli c strain (resistant to toxic protein expression) possesses protease activity (paliwal et al., ) . the purified protein cleaves both hev orf and orf products that are in vitro translated. protease inhibitor assay indicates the hev pcp domain is a chymotrypsin-like protease (paliwal et al., ) . this observation suggests that hev pcp is a real protease for hev orf polyprotein processing. in recent years, the connection between ubiquitination and innate immunity signaling has been demonstrated (zeng et al., ; mao et al., ; liu et al., ) , and the antiviral function of some ubiquitin-like molecules, such as interferon-stimulated gene (isg ) and small ubiquitin-like modifier (sumo), has been described (liu et al., ) . some studies indicate that viral coded cysteine proteases possess deubiquitinase activity to inhibit host innate immunity, such as arterivirus papain-like protease (van kasteren et al., ) and pcp from porcine reproductive and respiratory syndrome virus (prrsv; li et al., ; sun et al., ) . similar research performed on the hev pcp domain suggests that it acts as an antagonist to isg function to inhibit host innate immunity when expressed together with met as the met-pcp protein (karpe and lole, ) . moreover, study from our laboratory demonstrated that the pcp domain from hev genotype sar strain is able to inhibit ubiquitination of rig-i and tbk , therefore resulting in the inhibition of rig-i mediated signaling in innate immune responses (nan et al., b) . between the pcp domain and the x domain, there are hvr and pro domains. these two regions were first named as hvr due to the extreme divergence in sequence between nt and (corresponding to residues aa - ) when the hev sar was compared with two other strains (tsarev et al., ) . in a later study, aa - in this region were designated a prolinerich region, which could be found in rubella virus as well. it was also considered to serve as a hinge between the x domain and its upstream domains because multiple proline residues in a protein or polypeptide may result in an unstable tertiary structure tsai et al., ; dosztanyi et al., ; dunker et al., ) . the length and sequence of hvr and pro is highly variable among different hev strains (pudupakam et al., ; smith et al., ) . currently, there is some confusion regarding the nomenclature of those two regions. some of the recent publications designated the region of aa - as the hypervariable domain, which was originally referred to proline-rich region and left out the immediately upstream domain (aa - ; pudupakam et al., pudupakam et al., , , whereas others still designate the aa - as the proline-rich region (purdy, ) . current research mainly focuses on the pro region and pays less attention to the upstream hvr domain. as a result, the function of hvr is unknown. however, data gained from the rubella virus shows that deleting part of the hvr domain along with part of the pro region renders the mutant non-viable (tzeng et al., ) . the pro domain is considered to be an intrinsically disordered region (idr) with flexibility for insertion and deletion (purdy, ; purdy et al., ) . data from its counterpart in the rubella virus indicates that this region is not required for viral replication (tzeng et al., ) . as expected, deletion and mutation of this region in hev indicates that it is not required for viral replication and infectivity, but it plays a role in replication efficiency in vitro (pudupakam et al., (pudupakam et al., , . it was also demonstrated that the pro domain is interchangeable between genotypes with genotype-specific differences (pudupakam et al., ) . more interestingly, a remarkable hev strain kernow-c , which was originally isolated from an hiv-positive patient with chronic hev infection, contains an insertion of a nt gene fragment of human ribosomal protein s in the pro region (shukla et al., ) . this recombinant virus was adapted in culture cells and is able to propagate in cells from different species. it was speculated that insertion of the s fragment occurred in the host but was selected in cultured cells. this speculation needs further verification as direct detection of the inserted fragment from the host sample was not successful. experimental insertion of the s fragment into the pro domain of the sar strain also generated a viable chimeric virus (shukla et al., ) . the s sequence insertion in hev correlates with novel nuclear/nucleolar trafficking capabilities to the orf protein of hev kernow c- p and the enhanced replication of this strain (kenney and meng, a,b) . although the pro domain is considered highly diverse, some motifs are found in the idr. based on computer analysis and comparison with other idrs, purdy et al. identified several linear motifs (lms), including two protease cleavage sites, three ligand binding sites and two kinase phosphorylation sites across all four genotypes . the putative protein-protein interactions of the pro domain were proposed in the same report as well, but need experimental verification. nevertheless, this report provides some assumptions about the disorder-to-order state of the pro domain. in another study, alignment of the pro domain from different genotypes indicated the sequence is more conserved in genotypes and than genotypes and (purdy, ) . adaptation to a wide host range for genotypes and is a possible reason. the authors also assessed the diversity of the pro domain due to the higher rate of substitutions at the first and second codon positions, leading to a shift in translation to be more proline, alanine, serine, and threonine rather than histidine, phenylalanine, tryptophan, and tyrosine. this pattern matches the aa usage in proline-rich idrs (purdy, ) . furthermore, the c-terminus of this domain can tolerate more mutations than the n-terminus. recently, the heterogeneity of the pro and x domains is implicated in hev persistence, which was revealed in an investigation into the association between the genetic heterogeneity of hev quasispecies in orf and the outcome of infection in solid-organ transplant patients ). the x domain is located immediately downstream of the pro domain. in hev, its function is unknown. the hev x domain homologs in other viruses such as rubella virus, alpha virus and coronavirus, are commonly identified as domain flanking the pcp domain (gorbalenya et al., ; koonin et al., ) . it is also known as macro domain, due to its similarity with non-histone domain of the histone macroh a. macro domain has been identified in a variety of bacterial, archaeal, and eukaryotic organisms (pehrson and fried, ; pehrson and fuji, ) . early studies of the human macro domain indicate that it is enriched in inactive mammalian x chromosomes, suggesting a role in gene silencing and inactivation (costanzi and pehrson, ) . the macro domain inhibits transcription and binds to the transcription activator nf-κb (perche et al., ; angelov et al., ) . crystal structure analysis identifies a dna binding motif in the macro domain, suggesting that it might interact with nucleic acids (allen et al., ) . a biochemical functional analysis indicates that the macro domain is involved in the downstream processing of adp-ribose -phosphate, a side product of cellular pre-trna splicing (martzen et al., ) . furthermore, the macro domain is found in association with proteins involved in poly(adp-ribose) polymerization, adpribosylation and atp-dependent chromatin remodeling (aguiar et al., ) . information about the function of viral macro domains is limited. adp-ribose -phosphatase activity has been demonstrated in the macro domain from coronavirus (martzen et al., ; putics et al., putics et al., , saikatendu et al., ) . crystal structure analysis and in vitro assays on the macro domain of the sars virus indicate that the viral macro domain has relatively poor adp-ribose -phosphohydrolase activity, but can bind free adp-ribose and poly(adp-ribose) efficiently (egloff et al., ) . in another report, the macro domains from semliki forest virus, hev, sars virus and yeast were compared with the human macro domain (neuvonen and ahola, ) . the viral macro proteins bind poly (adp-ribose) and poly (a), but have a low affinity for monomeric adp-ribose. this implies that viral macro domains are functionally different from human homolog and may participate in cellular pathways involving rna rather than adp-ribose derivatives. however, a recent study shows that viral macro domains (hev, coronavirus, and venezuelan equine encephalitis virus) can reverse protein adp-ribosylation by acting on adp-ribosylated substrates through the hydrolytic activity of their macro domains (li et al., ) . furthermore, other studies indicate that the expression of viral macro domain in liver cells inhibits apoptosis since it is functionally related to poly(adp-ribose) polymerase- (parp- ; allen et al., ; chen et al., ) , suggesting a role in apoptosis during viral infection. recently, a highly conserved "glycinetriad" (gly -gly -gly ) was identified downstream of the macro domain of hev, which is homologous to the rubella virus protease-substrate (g -g -g ; parvez, ) . mutagenesis study indicates that g v and g v mutations in the macro domain are lethal for sar replication in s - cells. further analysis identified the n-terminus residues asn , asn , his , gly -gly -and gly formed a potential catalytic-site homolog of coronavirus adp-ribose- -monophosphatase, which has essential role in viral replication (parvez, a) . as mentioned above, a recent report suggests that the quasispecies heterogeneity in the macro domain might facilitate hev persistence in solid-organ transplant patients . study from our laboratory demonstrates that x domain of hev sar strain inhibits the phosphorylation of irf , which is a key transcription factor for type i ifn induction (nan et al., b) . moreover, except interacting with light chain subunit of human ferritin and inhibiting ferritin secretion, the x domain interacts with hev met and vp ojha and lole, ) . the rna helicase domain is downstream of the x domain. it is encoded by many positive-stranded rna viruses and is essential for their replication (kadare and haenni, ) . helicases are motor proteins that are able to unwind nucleic acid strands by using energy from atp hydrolysis (kadare and haenni, ) . helicases can be divided into six superfamilies (sf - ; singleton et al., ) . rna virus coded helicases are mainly classified into sf and sf . helicases sf and sf contain seven signature motifs (i, ia, ii, iii, iv, v, and vi) that form the core of the enzyme (kadare and haenni, ) . the hev helicase belongs to helicase superfamily sf and is proposed to possess both ntpase and rna unwinding activities kadare and haenni, ) . in vitro experiments demonstrate that the hev helicase purified from e. coli expression has both of the activities. it drives the hydrolysis of rntps but also dntps at a lower efficiency, as well as unwinds rna duplexes with overhangs (karpe and lole, a) . rna -triphosphatase activity has also been observed in the hev helicase domain, which is proposed to function along with methyltransferase for catalyzing rna capping (karpe and lole, b) . recently, a mutagenesis study on hev helicase demonstrated that motifs i, iv, and vi are dispensable, while motifs i and iii are crucial and unique for hev helicase function (mhaindarkar et al., ) . a recent study shows that a v a substitution in the helicase domain of a swine hev strain is potentially associated with increased virulence (ward et al., ) . however, no human infection was reported to be associated with this strain. the last domain of hev orf polyprotein is the rdrp. all positive-stranded rna viruses code an rdrp, which is necessary for viral replication (o'reilly and kao, ) . the rdrp from all positive-sense rna viruses are classified into three large supergroups. all rdrp domains contain approximately amino acid residues, with the central and c-terminal parts showing high similarity between each other (koonin, ) . rdrp from hev belongs to supergroup iii and has the highest similarity to the domains in rubella virus and beet necrotic yellow vein virus (bnyvv; koonin et al., ) . all eight conserved motifs can be found in hev rdrp, including an mg + binding sequence (gdd), which is essential for rdrp activity. the purified hev rdrp is able to bind the end of hev rna, and needs two stem-loop structures at the end of the poly(a) stretch for this binding (agrawal et al., ) . expression of the rdrp in mammalian cells as a gfp fusion protein indicates that it localizes in endoplasmic reticulum (er), which could be a potential replication site for hev (rehman et al., ) . recent studies indicate that the emergence of g r mutation in the hev rdrp is possibly due to ribavirin-induced mutagenesis and is associated with treatment failure of ribavirin monotherapy in solid-organ transplant patients (debing et al., b; lhomme et al., ; todt et al., ) . the capsid protein is the major component of hev virions. orf is nt in length beginning from nt downstream of orf and ending at nt upstream of the poly-a tail (reyes et al., ; figure ) . the deduced full-length orf product has aa residues with a predicted molecular mass of kda (robinson et al., ) . recombinant orf protein can bind to the region of hev genome . it was first shown that the orf product exists as an kda protein, which carries n-terminal linked glycans and a potential er-directing signal about aa from its n terminus (jameel et al., ) . this kda protein can be further processed and has the potential to form non-covalent homodimers. a further study from the same group demonstrated asn in orf product to be the major site for glycosylation (zafrullah et al., ; figure ) . a mutagenesis study indicated that the n-terminal signal peptide is required for its cell surface expression via er transition, but glycosylation of the capsid protein is not required (zafrullah et al., ) . since glycosylation of the capsid protein in non-enveloped viruses is not common, it is not known whether these modifications have biological significance for hev infection. mutations in the putative glycosylation sites (aa , aa , aa ) prevent formation of viral particles and infection of rhesus macaques but without an effect on genome replication in cells (graff et al., ) . mutation in the first two glycosylation sites prevents virion assembly, while mutation of the third site allows virion particle formation and rna encapsulation (graff et al., ) . hev particles released from cultured cells have a lipid component (quasi-enveloped), which can be removed by detergent treatment (qi et al., ) . the relationship between glycosylation of orf and lipid envelope of viral particles needs to be clarified. on the other hand, data acquired from studies using insect cells provide a different conclusion regarding orf expression and processing. when expressed in insect cells, orf product can be an insoluble full length protein of about kda, and a soluble form of . kda, a processed product of the intact form (mcatee et al., ) . another group shows that when orf is expressed in sf cells, a kda product is detected while lacking the first aa residues of the putative orf polypeptide (zhang et al., ) . further studies in two different insect cell lines (sf and tn ) show a soluble form of orf product with a molecular mass of kda, which lacks the first aa and the last aa of orf polypeptide but retains the ability to form virus-like particles (vlps; li et al., li et al., , d . vlp assembly is thought to involve dimer formation, and the c-terminus of the recombinant orf protein is believed to be responsible for homo-oligomerization (tyagi et al., a; xiaofang et al., ; li et al., a) . a . -Å resolution crystal structure obtained from hev vlp indicates that the truncated hev capsid protein has three domains designated as s (shell, aa - ), m (middle, aa - ), and p (protruding, aa - ; yamashita et al., ; figure ) . the vlp is composed of subunits of the truncated capsid protein, forming icosahedral , , and -fold axes (yamashita et al., ) . mutational analyses indicate that the protruding domain is involved in binding to the susceptible cells and contains neutralization epitopes (yamashita et al., ) . moreover, the hev vlp can be used as a delivery system to display foreign epitopes on its surface (xing et al., ) . the orf product expressed in insect cells is reactive with anti-hev antibodies (tsarev et al., ) . genetic analysis of orf showed over % similarity among the four major hev genotypes in mammalian hosts (mori and matsuura, ) . amino acid alignment indicates that divergences are mainly in the first aa of the n terminus, which is not a component of the virions (mori and matsuura, ) . a study manipulating a phage display system for overlapping peptides and truncated orf proteins maps the major neutralizing domain to residues - , which matches the location of the p domain (schofield et al., ; meng et al., ; zhou et al., ) . both conformational and linear neutralizing epitopes have been identified from the hev capsid protein tang et al., ) . these data provide valuable information for vaccine development. the orf truncated proteins generated by baculovirus or bacterial expression systems have been tested in clinical trials (shrestha et al., ; zhu et al., ) . however, a recent study that evaluated cross-protection against heterologous hev indicates that vaccination of pigs with truncated capsid proteins derived from swine, rat and chicken hev only elicits partial protection against a genotype mammalian hev (sanford et al., ) . study of avian hev capsid protein indicates that the n-terminal aa residues react with swine and human anti-hev sera (wang et al., b) . moreover, a recent study suggests that antigenic composition and immunoreactivity differed between hev recombinant capsid proteins from different genotypes (behloul et al., ) , which raises the concerns about the efficacy of the current hev subunit vaccine marketed in china. on the other hand, experiments based on the newly identified quasi-enveloped hev particles indicate that lipid membrane protects the virions from neutralizing antibodies against the capsid protein (yin et al., ) . additionally, as a structural protein, the hev capsid protein has been found to interact with some cellular proteins and plays a role in cell signaling. in one study, the capsid protein activates the pro-apoptotic gene chop, and increases the expression of hsp , hsp b and hsp , and interacts with hsp (john et al., ) . in addition, the capsid protein interacts with β-trcp, a component of the ubiquitination complex that inhibits iκbα ubiquitination-mediated nf-κb activation (surjit et al., ) . however, these data are all based on overexpression of orf in mammalian cells, and need to be further verified in whole virus infection. the orf is the smallest among the three orfs of hev and overlaps with orf by approximately nt in a different reading frame. however, it does not overlap with orf (graff et al., ) . the overlapping region with orf (nt - ) was found to be the most conserved region between the sar and bur strains (tsarev et al., ). an early study proposed that orf encodes a protein with aa and comes from a different subgenomic rna other than that encoding orf (tam et al., ) . however, a later study based on an hev replicon shows that orf is translated from the bicistronic subgenomic rna and initiates at the third aug of the presumed orf at nt for sar and the product is a protein with or aa and molecular size of kda (vp ), which is aa shorter than the earlier predicted version (graff et al., ) . this observation has been confirmed by another study using a different hev strain (huang et al., ) . sequence analysis has indicated that vp is unique and has no similarity to any other proteins known. it contains two hydrophobic domains in its n-terminal half and two proline-rich domains in its c-terminal portion (kannan et al., ; holla et al., ) . a phosphorylation site (ser ) was identified in the first proline-rich domain and can be phosphorylated by map kinase (zafrullah et al., ) . furthermore, two psap motifs have been identified in genotype vp , with the first psap motif located at aa - and the second at aa - , whereas genotypes , , and have only one psap motif at aa - (nagashima et al., b) . the second psap motif is needed for hev virion release. interestingly, among the four genotypes that infect humans, only genotype vp has an additional prolinerich region that contains a pxxp motif in aa - , which is linear and surface-oriented (nan et al., a (nan et al., ,b, . the unique motif reacts with a genotype vp -specific monoclonal antibody. this proline-rich region contains residues pmsplr, a typical motif (pxxpx+) (+ is either arginine or lysine, x can be any aa) for class ii src homology (sh ) domains. sh domains are known to bind to proline-rich sequences containing a core pxxp motif flanked by a positively charged residue (baumann et al., ; raeder et al., ) . sh domains comprise of about residues and proteins containing sh domains typically play a role in signaling pathways involved in cell growth, differentiation and other regulatory functions (zarrinpar et al., ) . the next proline-rich region spanning aa - are rpsapplp, containing an additional residue than the typical motif (+xxpxxp) for class i sh domains. but interestingly only the pxxp motif in the second proline-rich region is known to interact with sh domains (korkaya et al., ) . the function of the pxxp motif in the first proline-rich region (aa - ) of vp of genotype hev is unknown. it might play a role in cellular signaling as proline-rich motifs are also involved in interacting with other domains besides sh (zarrinpar et al., ) . although the full function of hev vp has not been defined yet, some studies have suggested that vp plays multiple roles during hev infection. early studies focusing on vp antigenicity and epitope mapping demonstrated that the last aa of vp are an immunodominant region, and a synthesized peptide from that region is reactive with anti-hev serum from a recovered patient (semiletov et al., ; dement'eva et al., ) . however, another study mapping the t cell epitopes in orf and orf products indicated that no t cell proliferation was observed when cells were stimulated with peptides from vp (aggarwal et al., ) . a recent study based on genotype hev shows that continuous amino acid motif, vdlp, at the c-terminus of genotype hev vp , is a core sequence of a vp epitope . although vp is dispensable for viral replication in cultured cells , it is indispensable for hev infection in vivo, implying an important role for vp in host invasion (graff et al., ; huang et al., ) . a yeast two-hybrid system is employed to screen for the interaction partners for vp . the vp can bind to inactive mitogen-activated protein kinase (mapk) phosphatase and lead to activation of the mapk (kar-roy et al., ), suggesting that vp can modulate host gene expression since mapk is related to cell signaling and gene expression. another study shows that vp inhibits the nuclear translocation of stat and down-regulates stat -mediated gene expression, such as acute-phase response proteins (chandra et al., ) . the vp can also increase the expression of glycolytic pathway enzymes by increasing the phosphorylation and transactivation activity of p /cbp (moin et al., ) . furthermore, microarray analysis of huh cells with vp expression suggests that liver-specific genes can be modulated, as vp is able to modulate the phosphorylation of hepatocyte nuclear factor (chandra et al., ) . the vp can also up-regulate mitochondrial voltagedependent anion channel genes, which can protect cells from mitochondrial depolarization and death (moin et al., ) . this result implies that vp is able to inhibit the mitochondrial apoptosis pathway. the pro-survival role of vp is also demonstrated in another study showing vp delays the trafficking and degradation of the activated hepatocyte growth factor receptor to prolong endomembrane growth factor signaling (chandra et al., ) . additional interacting molecules have been identified for vp by yeast two-hybrid screens, including α- -microglobulin, bikunin, and bikunin precursor protein (ambp), fibrinogen β chain and hemopexin (tyagi et al., (tyagi et al., , ratra et al., ratra et al., , . moreover, a recent study screening for intraviral protein interactions identified the met, pcp, x, helicase, and rdrp domains as interacting partners for vp as well (osterman et al., ) . besides yeast two-hybrid screens, overexpression of vp coding plasmid in mammalian cells was also employed to elucidate the function of vp . the vp associates with the cytoskeleton fraction when expressed in cells, and deletion of the n-terminal hydrophobic domain of vp abolishes this association (zafrullah et al., ) . in a more detailed study, gfp-tagged vp is found to interact with microtubules to form a filamentous pattern in cells and modulate the microtubule dynamics (kannan et al., ) . vp leads to an elevation of acetylated α-tubulin, indicating increased microtubule stability (kannan et al., ) . since there are two hydrophobic domains located in the n-terminus of vp , truncation analysis indicated that both the hydrophobic domains are required for its association with the microtubules. moreover, salt extraction studies have suggested that the vp -microtubule interaction is electrostatic and motor protein dynein is needed for the interaction (kannan et al., ). an earlier study showed that vp cannot be co-precipitated with tubulin by anti-tubulin antibody (zafrullah et al., ) . these results suggest that vp may associate with microtubules through interaction with another protein. this microtubule-like distribution of vp suggests that it may play a role in promoting virus egress, as the pul protein of herpesvirus can interact with dystonin, an important cytoskeleton cross-linker involved in microtubulebased transport, in order to promote capsid transport on microtubules during egress (pasdeloup et al., ) . besides cellular proteins, vp has been shown to interact with viral helicase, pcp and methytransferase from hev orf , which suggests a regulatory function for vp in orchestrating the formation of the replicase complex (osterman et al., ) . more interestingly, another study using monoclonal antibody against vp to capture hev particles showed that vp can associate with virions and support virus release (takahashi et al., ) . the requirement of vp for virion release was later confirmed by a cell culture-adapted genotype hev strain with vp deletion . studies in caco- cells and huh cells for the sar , a genotype hev strain, showed that the intact psap motif spanning aa - in vp is required for virion release (emerson et al., ; nagashima et al., b) . for avian hev, the psap motif in vp has also been found to play a role in virus release . the psap motif in vp is required for the formation of membrane-associated hev particles with the vp protein itself associated with lipids. this process is mediated by the cellular tsg protein (nagashima et al., a,b) . replacement of the vp psap motif with heterologous late domain motifs (pppy, ypdl, and psaa) affects the virus release (kenney et al., ) . the specific interaction between vp and tsg as well as involvement of endosomal sorting complex required for transport (escrt), which commonly participates in budding of many enveloped viruses, leads to the biogenesis of membrane-associated, "quasi-enveloped" hev particles (hurley, ; feng et al., ; nagashima et al., ; yin et al., ) . therefore, vp is associated with virion during egress and anti-vp antibodies are able to capture hev virions from the serum and cell culture supernatant, but not fecal samples from patients (takahashi et al., ) . a possible explanation is that viral particles could lose lipid-associated vp after passing through the gut (takahashi et al., ) . the role of vp in virus release may be one of its functions during hev replication in vivo, indispensable for viral spread during infection. on the other hand, as a small phosphorylated protein, vp can be phosphorylated at ser by mapk when expressed in cos and huh cells (zafrullah et al., ) . a later study indicates that the ser phosphorylation site is required for the interaction of the capsid protein and vp as vp can interact with the capsid protein in a yeast two-hybrid screen, especially for the non-glycosylated capsid protein (tyagi et al., ) . this finding also supports a role for vp in hev structural assembly. however, a mutagenesis study shows that hev lacking the phosphorylation site in vp is able to replicate its genome in cultured cells, and to infect rhesus monkeys similarly to wild type hev in viremia and seroconversion (graff et al., ) . these data suggest that phosphorylation of vp is not necessary for genome replication or for the production of infectious virions. moreover, in addition to phosphorylation and interaction with the capsid protein, vp can form a homodimer via the aa domain located in the c-terminus (tyagi et al., b) . on the other hand, vp has been reported to activate mapk-jnk / in hepatoma cells (parvez and al-dosari, ) . besides the functions mentioned above, vp also plays a role in the interferon induction and signaling. data from our laboratory show that vp is able to enhance rig-i activation, which leads to enhanced rig-i signaling (nan et al., a) . the vp extends rig-i half-life and interacts with the n-terminal portion of rig-i to enhance its activation by polyi:c. interestingly, there is a genotype difference in the enhancement of rig-i: genotypes and vp but not genotypes and vp have the role, implicating that vp may relate to hev virulence and pathogenesis. on the other hand, another study demonstrate that vp of a genotype hev strain is able to interact with the stat (signal transducer and activator of transcription) to inhibit interferon-α mediated signaling in a (human lung adenocarcinoma epithelial cell line; dong et al., ) . in summary, as the product of the smallest orf of hev, vp has multiple functions and plays an indispensable role in infectivity in experimentally infected animal models. however, it is not required for hev replication in cultured cells. our current knowledge indicates that vp is a multifunctional protein in interacting with many cellular proteins, modulating host gene expression and involved in virion release. recently, a novel orf (nt - ) was identified from genotype hev . unlike other orfs in hev, translation of orf is driven by an ires-like sequence located in nt - of hev genome . the orf product interacts with multiple viral proteins to form a protein complex consisting of viral rdrp, helicase and x, and the orf product stimulated viral rdrp activity to promote viral replication. expression of the orf was verified in a cellfree system and antibodies against this protein were determined from hev-infected patients . however, analysis of hev sequences from other genotypes suggests orf is not conserved across genotypes . therefore, more investigation is needed to elucidate the exact function of orf . due to the lack of an effective in vitro cell culture system for hev, the replication cycle of hev is largely unknown. the capsid protein is believed to bind to an unidentified cellular receptor to initiate viral entry. hev-vlps generated from recombinant orf protein attach to cells via heparin sulfate proteoglycans (hspgs; kalia et al., ). moreover, one study based on a viral overlay protein binding assay (vopba) suggests that a protein with molecular weight about kda could be the candidate receptor for hev entry; but mass spectrometry revealed that this virus binding band contained different proteins . another study suggests that aa - located in the c-terminal region of the capsid protein (m domain) may be the putative receptor binding site of hev virions (he et al., ) . moreover, structure and sequence analyses suggest that the putative binding motif of the capsid protein is conserved among all four major mammalian hev genotypes (guu et al., ) . heat shock cognate protein (hsc ), hspgs and grp are found to be involved in either cell surface binding with hev capsids or intra-cellular transport in different models and are potential cellular receptors or essential factors for hev proliferation (kalia et al., ; yu et al., ; cao and meng, ) . however, further investigation is needed to confirm if these molecules truly act as receptors for hev. after binding with its receptor, hev particles are internalized via a dynamin- , clathrin, and membrane cholesterol-dependent pathway (kapur et al., ; holla et al., ) . in addition, a recent report suggests the quasienveloped hev particles enter cells via a distinct pathway that involves in degradation of the lipid membrane in the lysosome (yin et al., ) . after entry into permissive cells, the hev capsid is uncoated by unknown mechanisms. in one study utilizing vlp from the truncated capsid protein hev , an hsp -specific inhibitor (geldanamycin) blocks the intracellular transport of the hev vlp without affecting its entry . this suggests that hsp may play a role in the intracellular transport of hev particles. after uncoating, the hev orf translation is followed. hev genomic rna replication relies on the replicase encoded by orf . along with the generation of the sub-genomic rna, translation of orf and orf occurs, followed by virion packing and egress. for the release of hev particles, multivesicular body (mvb) pathway and escrt machinery in the cytoplasm are used (nagashima et al., ) . since the discovery of hev, many efforts have been made to develop a rigorous in vitro cell culture system. however, the cell culture system of hev is still limited and relatively ineffective, especially for genotype hev. an early study tried to use primary hepatocytes from macaques with serum-free medium for hev propagation; however, hev replication was limited and the detection of hev in the medium relied on pcr amplification (tam et al., ) . a group from japan reports that hev isolate a is able to replicate in a cells; however, pcr was also used to detect viral rna in the cell culture supernatant (huang et al., ) , instead of immunofluorescence assay to detect viral proteins. another group also showed that the a cell line could be used effectively for passaging two chinese hev isolates (wei et al., ) . on the other hand, as the commonly employed method for single-stranded, positive-sense rna virus, transfection of capped rna from an hev cdna infectious clone via in vitro transcription to plc/prf/ (hepatocellular carcinoma) and huh cells demonstrates limited replication of hev . although cell lysates from the rna transfected cells is infectious in rhesus monkeys, cell to cell spread of the virus in cultured cells is not observed . s - cell line, a subclone of huh hepatoma cell line, has improved replication efficiency of hev for the sar strain (graff et al., ; shukla et al., ) . but this assay still relies on transfection of the cells with full length hev rna. a recent report suggests that replication efficiency of genotype hev in human hepatoma cell lines (huh , huh . , and hepg /c a) is affected by innate immune response . a japanese group reports that a genotype isolate from acute hepatitis patient propagates in plc/prf/ and a cells (tanaka et al., ; okamoto, ) . after a cells were seeded in a six-well plate and inoculated with hev at . × and . × rna copies per well, hev rna reached the highest titer of copies/ml at days post-inoculation. however, plc/prf/ cells could only support efficient growth as a with a higher moi ( . × viral rna copies per well). moreover, in this hev cell culture system, hev infected cells need to be maintained at . • c and cultured with a mixed cell culture medium ( % dulbecco's modified eagle medium and % medium ) supplemented with % (v/v) fetal bovine serum and mm mgcl . the same group also reports that a genotype hev from a fulminant hepatitis patient can grow in plc/prf/ and a cells and reach a titer of . × copies/ml within - days incubation period ). moreover, a human hepatoma-derived cell line heparg and a porcine embryonic stem cell-derived cell line picm- , which have morphological and functional properties similar to primary hepatocytes, were shown to support hev replication (rogee et al., ) . however, the hev replication level in these two cell lines is very low and requires month incubation (rogee et al., ) . in hev kernow-c p cell culture system, the recombinant virus with human s gene insertion was presumed as a minor species in the host but was selectively adapted to cells after six passages (shukla et al., ) . replication of kernow-c p is . -fold higher in hepg /c a human hepatoma cells than in huh . , plc/prf/ , a , caco- or rhesus kidney cells in a -day incubation period, suggesting that hepg /c a cell line is the most permissive. moreover, this hev isolate is also able to infect a variety of non-primate cells, including cow, mouse, chicken, cat, dog, and rabbit cells, albeit with lower efficiency. although it is still unclear how the insertion of s occurred in hev infected patient, okamoto's group demonstrated that two cell adapted hev strains hev je - f (genotype ) and hev jf / f (genotype ) did not shown any recombination with cellular s gene after and generations of passages in plc/prf/ and a cells, respectively (okamoto, ) , which suggested the recombination and insertion of s may occur in patients rather than in cultured cell. besides human hev isolates, animal hev strains from domestic pigs, wild boars, rabbits, and rats can be propagated in human hepatoma cell lines as well takahashi et al., ) . a recent report demonstrates that pluripotent stem cell derived hepatocytes support hev replication in vitro (helsen et al., ) . in summary, the current cell culture systems for hev have limitations. so far, only one report shows limited replication of a genotype hev strain from serum sample in cell culture without rna transfection (takahashi et al., ) . on the other hand, although several groups have demonstrated that genotypes or hev strains can be adapted to cultured cells and are able to reinfect new cells, a long incubation time is needed in comparison to other rna viruses with good cell culture systems. moreover, the cell culture adapted kernow-c virus may have a different phenotype compared with its parental wild type virus as this cell culture adapted virus contains host gene sequence. the hev is primarily transmitted via fecal-oral route. the most common source of infection is contaminated drinking water in developing countries. for a long time, hepatitis e was thought to be a public health problem only for developing countries. however, hepatitis e is now frequently recognized in industrialized countries where it was not thought to be endemic previously (kwo et al., ; erker et al., ; schlauder et al., ; worm et al., ; kabrane-lazizi et al., ; mizuo et al., ; sadler et al., ) . world health organization estimates that there are million hev infections annually across the world. among these cases, there are over million symptomatic cases and , deaths (who, ) . hepatitis e is highly endemic in east and south asia. data indicates over % of global hepatitis e deaths occur in this region. in east asia, large outbreaks of hepatitis e have only been described in china. hepatitis e accounts for - % of acute hepatitis cases in this region. the seroprevalence of anti-hev antibodies in the region varies from to %, indicating that hepatitis e is hyperendemic in this region. in south asia, outbreaks of hepatitis e have been reported in most countries in this region, but variable in scale (who, ) . hev accounts for - % of sporadic acute hepatitis and fulminant liver failure in this region. in particular, the rates of fulminant liver failure are usually higher in pregnant patients. a recent paper reported that hev infection causes % acute viral hepatitis and % fulminant hepatic failure in pregnant women in one area in india . however, the seroprevalence rates of prior exposure to hev are relatively low, ranging from to % in most studies. in the developed countries, such as north america, western europe and japan, no outbreaks have been reported. these areas are considered as low or non-endemic for hev. however, sporadic cases of hepatitis e have been reported. transmission of hev from animal reservoirs to humans is assumed to be the major cause of those sporadic cases. a series of cases of hev infection in people who ate undercooked deer meat - weeks before the onset of disease have been reported (tei et al., ; yazaki et al., ; li et al., c) . hev rna recovered from the leftover deer meat was found to be identical in sequence to the hev rna recovered from the patients (takahashi et al., ) . consumption of shellfish is considered a risk factor in a documented case (koizumi et al., ) . thus, foodborne infection may occur from the consumption of uncooked/undercooked products from infected animals. moreover, blood transfusion and solid organ transplant mediated hev transmission are reported wedemeyer et al., ; sue et al., ) . igm and igg against hev are detected in recipients of blood transfusions (wedemeyer et al., ) . hepatitis e virus genotype is responsible for most endemic and epidemic cases of hepatitis e in asia, and genotype is prevalent in central america and africa (purcell and emerson, ) . there is no known animal reservoir for hev genotypes and (wedemeyer et al., ) . genotypes and are zoonotic and can cause hev infections in the developed countries. for the detailed geographical distribution of hepatitis e virus genotypes, please refer to these reviews (dalton et al., ; kamar et al., a) . hepatitis e virus infection mainly causes acute hepatitis with a case fatality rate from . to % in young adults (jameel, ) . remarkably, case fatality rate resulting from hev-related fulminant liver failure can reach up to % in infected pregnant women in their third trimester of gestation (jameel, ) . generally, hev has an incubation period of - weeks (purcell and emerson, ) . the initial symptoms of acute hepatitis e are unspecific and flu-like, such as myalgia, arthralgia, and weakness. after this short prodromal phase, a period of symptoms such as vomiting, itching, uncolored stools, darkened urine and jaundice could last for days to several weeks accompanied by increased levels of liver transaminases, bilirubin, alkaline phosphatase, and γ-glutamyltransferase (hoofnagle et al., ; wedemeyer et al., ) . current case reports indicate that most cases are selflimited and do not result in chronic hepatitis (hoofnagle et al., ). an investigation on pregnancy outcomes in hepatitis e shows higher hev loads in pregnant women with acute viral hepatitis and fulminant hepatic failure, and higher levels of tnf-α, il- , ifn-γ, and tgf-β than non-pregnant women, which suggests that high cytokine levels are correlated with severe liver injury in hev infection . a recent study highlights the role of tlr and ifn-γ in hev pathogenesis. patients with high levels of tlr and robust ifn-γ response are observed in self-limiting acute viral hepatitis cases, and are able to limit the disease and recover uneventfully (majumdar et al., ) . however, patients with lower expression of tlr and ifn-γ progress to acute liver failure (majumdar et al., ) . hev can cause chronic infection as well. although chronic hev infection was initially reported only in immunocompromised persons, such as organ transplant recipients, patients receiving cancer chemotherapy and hiv-infected persons (hoofnagle et al., ) , latest reports show that chronic hev infection also occurs in an immunocompetent individual with systemic lupus erythematosus (sle; grewal et al., ) . however, since this kind of cases are rare, data available so far are not sufficient to consider this patient as an immunocompetent individual . in organ transplant recipients, the chronic course leads to persistent increases in levels of alanine aminotransferase, significant histological activity and fibrosis in some cases (wedemeyer et al., ) . hiv-infected individuals have higher positive rate of anti-hev antibody than individuals without hiv infection (wedemeyer et al., ) . besides hepatitis, extrahepatic manifestations have been documented. neurological disorders, such as polyradiculopathy, guillain-barré syndrome, bilateral brachial neuritis, encephalitis and proximal myopathy, and neuralgic amyotrophy are reported in patients with acute and chronic hev infections (kamar et al., van den berg et al., ; van eijk et al., ; dalton et al., ; drave et al., ) . the kidney injury caused by hev infection is reported and also documented in monkeys infected experimentally with hev as well (kamar et al., (kamar et al., , b geng et al., ) . furthermore, a recent report provides evidence that extrahepatic replication of hev in the placenta of infected mothers, which may be associated with fetal mortality (bose et al., ) . it also raises the concern for the vertical transmission of hev to fetus and newborn by an infected mother (krain et al., ) . a report suggests the association between the outcome of hev infection in solid-organ transplant patients and the genetic heterogeneity of hev quasispecies in orf . analysis of the viral genetic heterogeneity indicates that both nucleotide complexity and genetic distance of the orf proline-rich domain in patients whose infection became chronic are higher than the patients who cleared the virus . although diagnostic tests for hev are commercially available, none of them have been formally approved in the united states by the food and drug administration (fda; hoofnagle et al., ) . current tests mainly target anti-hev antibodies, including igg and igm. however, several assays are based on antigens expressed by a single hev genotype, especially genotype , and might be limited for the detection of all hev genotypes. indeed, there are variations in sensitivity, specificity and agreement in the results of these assays, which may account for the discrepancies among positive rates of anti-hev antibodies in various populations (mast et al., ; herremans et al., ; drobeniuc et al., ) . it is also notable that a recent study demonstrates the false positive result in hev igm test due to cross reaction with ebv and cmv, which heavily affects the accuracy of hev serology testing (hyams et al., ) . only . % of the total samples with the positive hev igm were pcr positive for hev rna. the cross reactivity of igm against hev, ebv, and cmv is very high. these data suggest that to confirm hev infection in patients, clinical features, blood alt level and pcr testing should be all included in addition to serological test alone. on the other hand, although hev rna can also be detected in blood and stool for several weeks after acute hev infection, in addition to a narrow detectable window of hev viremia (hyams et al., ) , current hev rna tests are still experimental since they have not been standardized yet (wedemeyer et al., ) . furthermore, diagnostic support for igm and igg anti-hev detection in clinical samples using commercially available kits and pcr assay for detection of hev rna in serum and stool samples are also available from the division of viral hepatitis in the centers for disease control and prevention (cdc, ). hepatitis e virus infection mainly causes a self-limited disease and most infected individuals are able to clear it spontaneously. although the case fatality rate in adults is . - %, the rate can increase to % in pregnant women during their third trimester of gestation in south asia (jameel, ) . therefore, antiviral therapy is needed. although no specific treatment has been approved for hev, off-label application of ribavirin as monotherapy for hev has demonstrated promising results in both acute and chronic hepatitis e patients (kamar et al., ; mallet et al., ; gerolami et al., ) . in vitro assay showed that ribavirin could inhibit replication of genotypes - hev through the depletion of intracellular gtp pools in hev infected cells (debing et al., a) . for immunosuppressed patients, a reduction of immunosuppression has shown efficacy in the treatment of chronic hev infection (wedemeyer et al., ) . moreover, application of pegylated interferon in combination with ribavirin has been reported as a treatment for chronic hev infection but only shown moderately synergistic effect (wedemeyer et al., ; debing et al., a) . however, due to the evidence of embryolethality and teratogenicity revealed by animal study, ribavirin has been assigned to pregnancy category x by the fda and contraindicated in women who are pregnant and in the male partners of women who are pregnant (sayed et al., ) . moreover, ribavirin-induced g r mutation was reported and associated with treatment failure of ribavirin monotherapy in solid-organ transplant patients (debing et al., b; lhomme et al., ; todt et al., ) . therefore, viral specific treatment for hev is needed. our laboratory has successfully tested application of peptideconjugated morpholino oligomers (ppmos) as novel anti-hev compounds (nan et al., ) . ppmos are water soluble, nuclease-resistant single-stranded dna analogs containing a backbone of morpholine rings and phosphorodiamidate linkages along with conjugation of arginine-rich cell penetrating peptide for facilitating cell delivery (summerton, ; abes et al., ) . ppmos bind to mrna by watson-crick base pairing and interfere with translation through steric blockade of the augtranslation initiating region. antisense morpholino oligomers are currently tested in clinical trials for treating duchenne muscular dystrophy in humans and has been documented as effective against numerous types of viral infections in experimental animal models (anthony et al., ; mendell et al., ; moulton, ) . importantly, upon systemic administration, ppmos distribute to liver, remain pharmacologically viable, and are effective at reducing viral titers (amantana et al., ; burrer et al., ; paessler et al., ) . in our study, ppmo hp targeting utr of hev genotype sar strain demonstrates strong inhibition of hev replication (nan et al., ) . since the utr of hev genome is highly conserved among different hev genotypes infecting humans, the ppmo hp may be an hevspecific inhibitor with antiviral activity across multiple hev genotypes (nan et al., ) . these qualities, along with the in vitro efficacy against hev (nan et al., ) , make ppmos be appealing for consideration as a novel inhibitor of hev infections. another nucleic-acid based strategy, sirna, has also been reported to be effective in inhibiting hev replication. an sirna targeting hev rdrp was reported to inhibit hev replication in a cells and in piglets . in another report, sirna targeting a cis-acting element and viral nucleotide sequences coding for helicase and rdrp are effective against hev in hepg cells (kumar et al., ) . however, it is generally acknowledged that sirna needs considerable improvements in their delivery to relevant targets in vivo before they can be considered for clinical applications involving systemic delivery against virus infections. current prevention for hev relies on sanitary measures, such as providing clean water, and appropriately cooked food to avoid transmission from undercooked food (kamar et al., a) . since in vitro culturing of hev is limited and ineffective, hev vaccine development mainly focuses on the expression of the capsid protein as a subunit vaccine. the capsid protein shares over % identity among the four major hev genotypes in mammalian hosts (mori and matsuura, ) . the capsid protein from genotype hev expressed by baculovirus or bacterial vectors has been tested in clinical trials. the first candidate was a kda protein expressed in insect cells. in a phase trial in nepal, the vaccine is well-tolerated and highly immunogenic, with % efficacy for protection against hepatitis e (shrestha et al., ) . the second vaccine, hev , encompasses aa - of orf product, is a kda truncated protein expressed in e. coli (li et al., b) . this vaccine is well-tolerated with an efficacy of % protection after three doses in a population tested in china, which included both men and women aged - years (zhu et al., ) . the hev vaccine was approved and marketed in china in . whether it will be endorsed in other countries or how effective it is against all other genotypes of hev infecting humans remains unknown. moreover, a study shows that hev vaccine could protect rabbits against homologous and heterologous hev challenge (liu et al., ; zhang et al., ) . a recent report demonstrates that a hybrid protein fusing protruding (p) domains from capsid proteins of both norovirus (nov) and hev induces a higher antibody titer than either p domain alone . subunit vaccine candidates containing antigens of hev, rotavirus, and astrovirus are reported as well (xia et al., ) . more than years have passed since the discovery and complete genome sequencing of hev. our understanding of hev is still limited, though ongoing research continues to reveal more and more information about this virus. currently, we know that hev is not only a public health concern in developing countries as previously thought, but also a concern with a more complicated scenario in the developed countries. more and more animal reservoirs are revealed and we now understand that genotypes and hev are zoonotic and foodborne pathogens. however, the cross-species transmission and host tropism of different hev genotypes are still elusive. current data imply certain viral proteins such as orf product plays a role in the host tropism of hev. further investigation is needed to elucidate the basic biology of hev. on the one hand, although approved in china, the hev vaccine is still unavailable to most of the world, despite the fact that serum surveillance indicates a high prevalence rate of hev throughout the world. moreover, recent discoveries about the antigenicity variation between hev genotypes and quasienveloped viral particles hidden from neutralizing antibody suggest new challenges and questions about the efficacy of the approved vaccine. further investigation about the vaccine efficacy against multiple hev genotypes or seeking for an improved vaccine is needed. in addition, virus specific treatment for hev infection is not available yet. although, the off-label using of pegylated ifns and antiviral drugs for general purposes have demonstrated efficacy against hev, safety is still a concern as no validation has yet been conducted for these treatments. therefore, a hev-specific treatment such as ppmos is needed. due to the absence of a suitable animal model and a simple cell culture system, many details about this virus and its infection, such as its biology, pathogenesis, strain variances, genotype differences, molecular mechanisms and vaccine efficacy for cross protection are still incomplete. however, current information also indicates that it is possible to establish a useful cell culture system using certain hev strains such as the cell culture-adapted kernow-c strain. these recent advances will facilitate further studies, which hopefully will reveal more insights about the basic biology of hev, such as proteolytic processing of orf product, functions of the viral proteins, hev pathogenesis, effective therapeutics and a better vaccine. vectorization of morpholino oligomers by the (r-ahx-r) peptide allows efficient splicing correction in the absence of endosomolytic agents t-cell epitope mapping of orf and orf proteins of human hepatitis e virus the end of hepatitis e virus (hev) genome binds specifically to the viral rna-dependent rna polymerase (rdrp) b-aggressive lymphoma family proteins have unique domains that modulate transcription and exhibit poly(adp-ribose) polymerase 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hepatitis e virus interacts with liver-specific alpha -microglobulin and its precursor alpha -microglobulin/bikunin precursor (ambp) and expedites their export from the hepatocyte rubella virus di rnas and replicons: requirement for nonstructural proteins acting in cis for amplification by helper virus hepatitis e virus sequences in swine related to sequences in humans neuralgic amyotrophy and hepatitis e virus infection deubiquitinase function of arterivirus papainlike protease suppresses the innate immune response in infected host cells a prolinerich domain in the genotype hepatitis e virus orf c-terminus is crucial for downstream v dlp immunoactivity a dual vaccine candidate against norovirus and hepatitis e virus identification of an antigenic domain in the n-terminal region of avian hepatitis e virus (hev) capsid protein that is not common to swine and human hevs analysis of complete genome sequences and a v a substitution in the helicase domain of swine hepatitis e virus strains 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hepatitis e virus prevalent among farmed rabbits in china role of heat-shock protein in hepatitis e virus capsid trafficking an elisa for putative neutralizing antibodies to hepatitis e virus detects antibodies to genotypes , , , and efficacy and safety of a recombinant hepatitis e vaccine in healthy adults: a large-scale, randomised, double-blind placebo-controlled, phase trial the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © nan and zhang. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- -vcph gn authors: chan, shiu-wan title: the unfolded protein response in virus infections date: - - journal: front microbiol doi: . /fmicb. . sha: doc_id: cord_uid: vcph gn nan unfolded protein response (upr) is a cellular homeostatic response to endoplasmic reticulum (er) stress. increasing evidence suggests an intimate relationship between virus and upr. this research topic collated a number of review articles and original research article, in an attempt to highlight how viruses interact with the host upr in the establishment of acute, chronic and latent infections. virus infection represents an arm race between virus and the host. on one hand, the host mobilizes the upr in an attempt to restrict virus infection. on the other hand, virus subverts or even manipulates the upr to assist in its own infection. the consequence of this is that the upr is often skewed during virus infections to either favor virus elimination or virus invasion. whoever won, the outcome could be pathogenic. the relationship between virus and upr and its associated autophagy is being addressed in three reviews focusing on rna viruses, as their life cycles are closely associated with the er (blazquez et al., ; fung and liu, ; jheng et al., ) . miguel martin-acebes and his group focuses on flaviviruses whereas to s. fung and ding x. liu focus on coronaviruses. jim-tong horng's group takes a closer look at virus interaction with autophagy and also discusses the potential of targeting upr and autophagy as novel anti-virals. in contrast to acute virus, one can only imagine that virus establishing a life-long chronic infection may interact with the host upr in a completely different way to maintain an environment favorable for virus survival. two reviews presented by shiu-wan chan and norica branza-nichita's group on hepatitis c virus and hepatitis b virus, respectively, shed light on how persistent virus interacts with the host upr to benefit establishment of a chronic infection and how chronic activation of the upr leads to diseases (chan, ; lazar et al., ) . upr is prevalent in viruses establishing latent infections such as herpesviruses. herpesvirus is an ancient virus. during its course of millions of years of co-inhabitation with its host, herpesvirus has borrowed a number of molecules from its host to be used in its life cycle. there is no exception in upr, in which herpesviruses also share molecular mimicry with the upr molecules and utilize upr to set up lytic infection and to break dormancy, suggesting that interaction of virus with host upr may be very ancient. varicella-zoster virus (vzv) possesses the smallest genome of human herpesviruses and lacks some genes used by other herpesviruses to manipulate the upr. the key question is therefore whether vzv upr induction is merely a host response or a result of viral manipulation. by using a upr pcr array, john carpenter and charles grose demonstrated vzv differentially induced the upr to expand the er to cope with viral glycoprotein synthesis (carpenter and grose, ) . this study also uncovered vzv upregulation of an unusual upr molecule, the camp responsive element binding protein h. clearly, this will pave the way to future studies to disclose the relationship between vzv and upr. er-associated degradation (erad) is part of an upr functioning to extract unfolded/misfolded proteins from the er into the cytosol for proteasomal degradation. not surprisingly, this process is also targeted by virus. jaquelin dudley and her group re-captures the erad process in details followed by an illustration of how viruses exploit this process (byun et al., ) . first, viruses can simply mobilize the erad to degrade important immune molecules or viral envelope glycoproteins to evade innate and adaptive immune responses. at a more intimate level, some viruses have actually incorporated erad into their life cycles for viral protein and even virion maturation. it is fascinating how naked polyomaviruses will make a de tour to the er for erad-assisted uncoating before re-entering the cytosol en route to the nucleus. lastly, viruses can interfere with erad tuning and hijack certain erad cargo into forming double membrane vesicles as sites of virus replication. upr has emerged to be more than a homeostatic cellular response to virus infections. upr has been intimately linked to innate immunity; whether by modulating innate immunity or as part of the innate immunity. innate immunity is initiated by the sensing of "danger signals" by host pattern recognition receptors (prrs), culminating in the release of interferon, which in turn activates the professional virus killer, one of which is rnase l. one of the proximal upr sensors, inositol-requiring enzyme (ire ), is evolutionarily related to rnase l. in the review of sankar bhattacharyya, he provides a structural and functional comparison between ire and rnase l and comments on a potential anti-viral function of ire by the creation of "danger signals" via the regulated ire -dependent decay (ridd) pathway (bhattacharyya, ) . an important question remains as to whether upr represents a new tool for sensing viruses or select upr molecules are merely being co-opted in "microbial stress response." this is being addressed in judith smith's review, in which she provides a critique on the intersection of the upr with the inflammatory pathways and innate immunity and offers an insight into upr-prr synergy as an evolutionary adaptation to ensure specificity of anti-viral responses (smith, ) . it is increasingly popular to use viruses in clinical applications such as gene therapy and oncolytic virotherapy. the use of viral vectors/viruses in the clinics will not be valid without a thorough understanding of virus-host interaction. giridhara jayandharan and his group presents a review on the emerging impact of upr on gene therapy and how the understanding of this will allow us to exploit and improve the use of viral vectors in gene therapy (sen et al., ) . to date we are still at the sprouting stage of understanding this virus-host interaction. we hope that this selection of articles will provide a foundation to spark more interest in this research area. this will not only lead to a deeper understanding of virus infection and pathogenesis but will also unravel novel anti-viral mechanisms. eventually it will help to unlock novel anti-viral targets and may also impact on optimizing the use of viruses in the clinics. can't ridd off viruses stress responses in flavivirus-infected cells: activation of unfolded protein response and autophagy erad and how viruses exploit it varicella-zoster virus glycoprotein expression differentially induces the unfolded protein response in infected cells unfolded protein response in hepatitis c virus infection coronavirus infection, er stress, apoptosis and innate immunity er stress, autophagy, and rna viruses modulation of the unfolded protein response by the human hepatitis b virus cellular unfolded protein response against viruses used in gene therapy a new paradigm: innate immune sensing of viruses via the unfolded protein response conflict of interest statement: the author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest key: cord- - zlu g y authors: nan, yuchen; zhang, yan-jin title: antisense phosphorodiamidate morpholino oligomers as novel antiviral compounds date: - - journal: front microbiol doi: . /fmicb. . sha: doc_id: cord_uid: zlu g y phosphorodiamidate morpholino oligomers (pmo) are short single-stranded dna analogs that are built upon a backbone of morpholine rings connected by phosphorodiamidate linkages. as uncharged nucleic acid analogs, pmo bind to complementary sequences of target mrna by watson–crick base pairing to block protein translation through steric blockade. pmo interference of viral protein translation operates independently of rnase h. meanwhile, pmo are resistant to a variety of enzymes present in biologic fluids, a characteristic that makes them highly suitable for in vivo applications. notably, pmo-based therapy for duchenne muscular dystrophy (dmd) has been approved by the united states food and drug administration which is now a hallmark for pmo-based antisense therapy. in this review, the development history of pmo, delivery methods for improving cellular uptake of neutrally charged pmo molecules, past studies of pmo antagonism against rna and dna viruses, pmo target selection, and remaining questions of pmo antiviral strategies are discussed in detail and new insights are provided. researchers realized decades ago that antisense nucleic acids could be used to treat diseases. since then, antisense therapies using a variety of nucleic acids or nucleic acid analogs have been evaluated for use as therapeutic compounds in numerous applications. among these candidate treatments, morpholino oligos, also known as phosphorodiamidate morpholino oligomers (pmo), have demonstrated promising effectiveness in developmental biology research involving gene knockdown as well as clinical trials focusing on treatments of genetic disorders (summerton, ) . specifically, pmos are short single-stranded dna analogs that contain a backbone of morpholine rings connected by phosphorodiamidate linkages (summerton, ) . due to the neutral charged property, they are less likely to interact with proteins while maintain the binding to nucleic acids (moulton and jiang, ). more specifically, pmo bind to complementary sequences of target mrna by watson-crick base pairing and block mrna translation through sequence-specific steric blockade. this process is distinct from the rnase h-dependent mechanism for protein translation inhibition, as induced by other antisense compounds such as phosphorothioate dna (summerton, ) . importantly, pmo are resistant to a variety of enzymes in biologic fluids, which makes them highly suitable for in vivo applications (hudziak et al., ) . to date, pmo-based therapy for duchenne muscular dystrophy (dmd) has shown great success by bypassing effects of a gene mutation underlying a human disease. specifically, this pmo-based therapy restores production of functional dystrophin by altering rna splicing to remove the mutated dystrophin exon that disrupts downstream full-length dystrophin protein translation from the mutated mrna. this drug has been approved by the u.s. food and drug administration and is available in the market under the trade name exondys tm (eteplirsen). approval of exondys tm is a hallmark of pmo-based antisense therapy that demonstrates the potential safety and effectiveness of pmo technology, is driving future development of such therapies for treatment of other diseases. aside from genetic disorders, pmo therapies have also been evaluated as treatments for other broad categories of untreatable diseases, including viral infections, antibiotic-resistant bacterial infections and cancers (enterlein et al., ; warfield et al., ; cansizoglu and toprak, ; chen et al., ) . in this review, pmo background, as well as research focusing on intracellular delivery of pmo, pmo efficacy against both dna and rna viruses, and pmo target selection are outlined. finally, current challenges for development of successful pmo antiviral strategies are discussed in detail and new insights are provided. the concept of an antisense oligonucleotide (on) as a potential therapeutic agent was first demonstrated experimentally through on targeting of a translation initiation site within rous sarcoma virus rna. the on mechanism was initially explained by its participation in formation of an rna-dna duplex with viral rna that sterically blocks viral gene expression and ultimately prevents viral replication (zamecnik and stephenson, ) . subsequent studies revealed that on also participated in a second inhibitory mechanism involving rna:dna duplex recognition by the cellular enzyme rnase h, resulting in rna cleavage and abrogation of virus gene expression as well (tadokoro and kanaya, ). more recently, rnase h-mediated degradation of target rna has been shown to be the main rnainterference mechanism responsible for microrna (mirna), small interference rna (sirna), and dna-directed rna interference (saurabh et al., ) . due to their effectiveness and specificity, such technologies have subsequently attracted the attention of global researchers as tools for therapeutic purposes (davis et al., ; millington-ward et al., ; wittrup and lieberman, ) . the first antisense on tested in clinical trials beginning in was designed to target p for treatment of acute myelogenous leukemia (bayever et al., ) . as a medical milestone for the application of antisense technology, the first antisense drug (fomivirsen) was approved by the fda in for treatment of cytomegalovirus retinitis in immunodeficient patients (marwick, ; wikipedia, ) . since then, numerous antisense drugs have been tested in clinical trials for a variety of other human diseases. to date, five antisense compounds have received marketing authorization from the fda, including eteplirsen. currently, more than ongoing clinical trials of antisense compounds are listed on the website clinicaltrials.gov (godfrey et al., ) . however, compared with traditional drug development, industrial development of therapeutic sirna, dna-based on, or on analogs for control of gene expression have been less successful, as exemplified by the removal of the first commercially available antisense drug, fomivirsen, from the market. a key feature underlying the effectiveness of antisense on is that their nuclease resistance helps them to remain intact for hours in extracellular medium or within cells (summerton, ) . methylphosphonate-linked dna analogs developed in the late s constituted a major advance in the emerging antisense field, resulting in production of the first antisense drug exhibiting acceptable stability in biological systems (hudziak et al., ) . however, in addition to stability, the specificity of rnase-hbased antisense therapy is an important concern, since rnase-h cleaves dna/rna duplexes as short as -or -base pairs in length and is highly active against duplexes that are only - base pairs in length (monia et al., ; summerton, ) . therefore, rnase-h-independent steric blockage of antisense on may be a safer strategy. notably, the effective target region of the steric blocking agent on for inhibiting translation is generally limited to the -utr (untranslated region) and start codon regions of mrna, achieving a good specificity with fewer adverse off-target effects. furthermore, the binding of an rnase h-independent antisense on to a partially matched rna sequence is unlikely to have biological consequences (lebleu et al., ) . oligomers possessing a morpholino phosphorodiamidate backbone, also called pmo (figure ) , constitute a novel type of on analog that is synthesized from ribosides. the ribose ring is opened by oxidation, re-closed using ammonia, which forms a substituted morpholine moiety (summerton and weller, ) . next, the phosphodiester intersubunit bonds are replaced with phosphorodiamidate linkages (summerton and weller, ) . pmo demonstrate excellent resistance to nucleases, figure | comparison of chemical structures of pmo and dna. illustration of phosphorodiamidate morpholino oligomer (pmo) contains backbone of morpholine rings connected by phosphorodiamidate linkages. the ribose ring is opened by oxidation, re-closed using ammonia to form a substituted morpholine moiety. the phosphodiester intersubunit bonds are replaced with phosphorodiamidate linkages. proteases, esterases, and a variety of other enzymes present in biologic fluids (hudziak et al., ) . matrix-assisted laser desorption ionization-time of flight mass spectrometry (maldi-tof ms) analysis has demonstrated that pmo are completely resistant to different hydrolases in serum and plasma (hudziak et al., ) , bolstering pmo suitability for in vivo applications (hudziak et al., ) . furthermore, as uncharged molecules, pmo do not interact strongly with proteins, which minimizes hybridization-independent protein interactions since decreased effectiveness of on is likely due to their charged phosphorothioate backbone (moulton and jiang, ; hagedorn et al., ) . pmos bind to complementary sequence in target mrna by watson-crick base pairing to block translation through rnase h-independent steric blockade (figure ) (lebleu et al., ) . moreover, pmo targeting intron-exon junction sequence is capable to modulate pre-mrna splicing as well (figure ) (havens and hastings, ) . for above reasons, antisense pmos have been a revolutionary tool in developmental biology (eisen and smith, ) . by microinjecting pmos into eggs or single or pauci-cellular zygotes, pmos are apportioned into daughter cells during cell division to ensure their delivery to each cell during subsequent cell proliferation (heasman et al., ) . in addition to their use for studies of gene function during embryonic development, pmo have been also tested as treatments for a broad range of diseases in numerous clinical trials (mainly promoted by avi biopharma, inc., now sarepta biopharma inc.). however, application of unmodified pmo is greatly limited by inefficient in vivo delivery unless a relatively high doses are administrated (moulton and jiang, ). in fact, in the animal dmd model, high doses of unmodified pmo injection in dystrophic muscle were needed to induce functional dystrophin expression in skeletal muscle (moulton and jiang, ). thus, two forms of pmos with distinct chemical modifications were developed to facilitate intracellular delivery of pmos, including peptide-conjugated pmo(ppmo) and vivo-pmo. moreover, pmoplus, another novel form of positively charged pmo with a morpholino oligomer-based backbone, has been recently developed as the latest version of pmo. these novel pmo forms are discussed in detail below. initial techniques to deliver pmo into cultured cells were based on mechanical delivery methods such as microinjection or scraping (moulton et al., ) , with associated limitations. more recently, additional mechanical scraping methodologies, electroporation, or use of endosomal escape reagents have been evaluated to improve delivery of on into cytosolic or nuclear compartments in tissue culture (partridge et al., ; moulton and jiang, ) . currently, the most intensively developed and widely used in vivo pmo delivery strategy is based on the use of arginine-rich cell penetrating peptide (cpp) (lebleu et al., ; morcos et al., ) . cell penetrating peptide, also known as protein transduction domain (ptd) or tat-cpp (dietz and bahr, ) , was originally identified after discovery of an unexpected property of the human immunodeficiency virus (hiv) trans-activator of transcription (tat) protein (dietz and bahr, ) . the novel activity was revealed by observations that tat could transactivate a hiv- ltr promoter after crossing cellular and nuclear membranes (dietz and bahr, ) . subsequent structural analysis pinpointed a short peptide located in aa - of tat (sequence grkkrrqrrrppq) that is responsible for the membrane-crossing activity of the parent tat protein (vives et al., ; abes et al., a) . this novel peptide sequence, later named as cpp, was then evaluated for its ability to confer membrane-crossing abilities to other proteins and compounds, including pmo. after fluorescence microscopy or flow cytometry analysis of cells treated with fluoresceintagged pmo, cpp conjugation remarkably enhances cellular uptake of pmo by -to -fold compared with non-conjugated pmo (alonso et al., ) . moreover, other cationic peptides conjugated pmo were shown to be much less effective than pmo conjugated to tat-cpp, while tat-cpp significantly enhanced delivery of pmo to nearly all cells assayed (moulton et al., ) . furthermore, cpp-mediated delivery is a much simpler procedure to conduct than mechanical delivery methods. however, tat-cpp mediated pmos delivery required high pmos concentrations (above µm) to achieve therapeutic antisense activity with cytotoxicity observed. meanwhile, tat-cpp pmos conjugate studies established that the conjugate associated with cell membranes and that internalized conjugate localized to vesicles, cytosol, and nucleus (moulton et al., ) . therefore, cpp sequence optimization, to reduce cytotoxicity and increase uptake efficiency, should enhance pmo effectiveness. this concept has prompted a comparison of two types of cpp (rxr peptide and r pen peptide) (lebleu et al., ) . the most efficient cpp in this study was (r-ahx-r) r, in which ahx represents a -aminohexanoic acid spacer lebleu et al., ) . importantly, (r-ahx-r) r-pmo conjugates were shown to be effective in several murine viral infection models , as well as in treatment of duchenne muscular dystrophy. unfortunately, in some applications, effective doses have approached cytotoxic levels. this obstacle has limited their use in clinical settings (abes et al., b) . vivo-pmo exploits a non-peptide-based transporter to deliver pmo into cultured cells or tissues (morcos et al., ) . currently, gene tools llc (philomath, or, united states) is the major supplier of vivo-pmo for research and development applications. unlike cpp-conjugated pmos, vivo-pmo are covalently linked to a molecular scaffold that carries a dendritic structure assembled around a triazine core that holds eight guanidinium head groups optimally oriented for cell membrane penetration (morcos et al., ; ferguson et al., ) . vivo-pmo effectively entered in vitro cultured cells as well as a wide variety of mouse tissues in vivo to induce correction of a pre-mrna splicing error, as detected by using an experimental test system designed to detect such an event in cells and tissues (morcos et al., ) . compared with cpp-pmos, vivo-pmo have been less frequently investigated for inhibitory effects against target genes. however, available data suggest that at least a % knockdown of target genes can be achieved using vivo-pmo, with no adverse side effects both in vitro and in vivo (guo et al., ; kang et al., ; nazmi et al., ; reissner et al., ) . meanwhile, mouse model studies have also shown that intravenous (iv) and intraperitoneal (ip) administration of vivo-pmo were equally efficacious (reissner et al., ; sartor and aston-jones, ) . furthermore, it appears that vivo-pmo is less cytotoxic than cpp-pmo, since only one report has demonstrated cytotoxicity of vivo-pmo (ferguson et al., ) . it appears that the dendrimer of vivo-pmo is capable to induce red blood cell sedimentation, prompting ferguson et al. to recommend that oligonucleotide analogs should be analyzed for potential to base pair hybridization that may induce dendrimer clustering. moreover, supplementation of vivo-pmo with physiological saline or anticoagulation therapy holds promise for counteracting vivo-pmo toxicity (ferguson et al., ) . compared to cpp-conjugated pmo and vivo-pmo, pmoplus tm is newer type of charged pmo that contain positively charged piperazine groups within its molecular backbone . pmoplus is the most recently developed form of pmo and studies have demonstrated this type of pmo is well tolerated and exhibits improved efficacy in numerous in in vivo viral infection models relative to other pmo therapies (swenson et al., ; warren et al., warren et al., , warren et al., , meng et al., ) . however, pmoplus is still unavailable to most researchers due to its proprietary status as technology solely owned by avi. however, there are reports available to date suggest that pmoplus may be less cytotoxic than cpp-pmo. in a phase i clinical trial to evaluate pmoplus compounds against ebola virus, healthy male and female subjects between and years of age in six dose-expansion cohorts of subjects per dosage group, each received a single i.v. infusion of the active study drug ( . , . , . , . , , or . mg/kg pmoplus). results demonstrated that pmoplus treatments were safe and well tolerated at these doses studied (heald et al., ) , with safety superior to that of cpp-pmo or vivo-pmo. however, a systematic investigation is still needed to compare vivo-pmo and pmoplus with regard to efficiency, stability, and cytotoxicity. an illustration of chemical structures of cpp-pmo, vivo-pmo and pmoplus tm was listed as pmo have been explored as antiviral compounds against rna viruses, including ebola virus, flavivirus, coronavirus, picornavirus, and others. in this section, major advances in this research field are presented. the filoviruses, including marburg virus and ebola virus (ebov), are negative-sense, single-stranded rna viruses that are highly pathogenic, causing human outbreaks of viral figure | chemical structures of cpp-pmo, vivo-pmo, and pmoplus tm . pmo conjugated with cell penetrated peptide (r-ahx-r) r, in which ahx represents a -aminohexanoic acid spacer. vivo-pmo are covalently linked to a molecular scaffold that carries a dendritic structure assembled around a triazine core that holds eight guanidinium head groups optimally oriented for cell membrane penetration. pmoplus tm is charged pmo that contains positively charged piperazine groups within its molecular backbone. hemorrhagic fever with up to % fatality. the - epidemic of ebola virus disease caused , deaths of , total cases in west africa (fischer et al., ) . because currently no commercial vaccines or effective therapeutics are yet available for filovirus infections (fischer et al., ; reynolds and marzi, ; trad et al., ) , antiviral drugs are urgently needed. thus, both peptide-conjugated pmo (ppmo) and non-conjugated pmos have been tested against ebola virus infection in cultured cells and animal models. an earlier study showed that a mer ppmo targeting the translation start site region of ebov vp positive-sense rna exhibited sequence-specific, time-and dose-dependent inhibition of ebov replication in cultured cells (enterlein et al., ) . moreover, this ppmo provided complete protection of mice when administered before or after challenge with a lethal dose of ebov. interestingly, a corresponding non-conjugated pmo also provided protection of mice when administered prophylactically as well. meanwhile, another report in same year demonstrated that a combination of ebovspecific pmos targeting viral mrnas for vp , vp , and rna polymerase l protected rodents against ebov challenge when administered before and after exposure . in the same study, non-conjugated pmo were also tested in a prophylactic proof-of-principal trial in rhesus macaques whereby the same pmo formulation protected % of macaques from lethal ebov infection. more recently, pmoplus has also been shown to be effective against ebola infection in monkeys. when delivered - min after infection, pmoplus avi- (composed of avi- and avi- that target ebov vp and vp , respectively) (iversen et al., ) protected over % of rhesus monkeys against lethal infection with zaire ebola virus (zebov) (warren et al., ) . similarly, pmoplus avi- (composed of avi- and avi- that target vp and vp of marburg virus, respectively) protected % of cynomolgus monkeys against infection with lake victoria marburg virus (marv) when delivered after infection (iversen et al., ) . therefore, pmoplus holds great promise for treatment of patients infected with these highly pathogenic viruses. in another study, the same research group showed that pmoplus avi- targeting vp alone was sufficient to protect monkeys against lethal ebov infection, whereas pmoplus avi- , targeting vp alone, failed to do so (warren et al., ) . these results thus confirm that vp may be a key ebov virulence factor and may serve as a promising target for further development of effective anti-ebov treatment strategies. picornaviruses are non-enveloped positive-sense, single-stranded rna viruses belong to the family picornaviridae. this family includes more than genera and species (zell et al., ) , with rna genomes length ranging from . to . kb. many picornaviruses are important human and animal pathogens, including poliovirus, coxsackievirus b (cvb ), enterovirus (ev- ), and foot-and-mouth disease virus (fmdv). no vaccine yet exists for picornaviruses other than for poliovirus and fmdv. moreover, no effective antiviral therapy yet exists for treatment of infections caused by any pathogenic picornavirus. however, ppmo targeting conserved internal ribosome entry site (ires) sequences have been shown to be highly effective in protecting cultured cells against infection by human rhinovirus type , coxsackievirus type b , and poliovirus type (pv ) (stone et al., ) , with reduction of pv titers by up to log . this mer ppmo (enterox) targets an ires sequence that is identical for > % of all human enteroviruses and rhinoviruses which has been successfully used to treat poliovirus receptor (pvr) transgenic mice to prevent pv infection after challenge with three times the % lethal dose (ld ). this result also showed that mice receiving ppmo treatment exhibited an approximately % higher survival rate than controls, with significant reduction of viral titers in small intestine, spinal cord, and brain (stone et al., ) . coxsackievirus b (cvb ) is a primary cause of viral myocarditis, without any effective therapy. in study tested eight cvb -specific ppmo in cultured hela cells and hl- cardiomyocytes, as well as in a murine infection model (yuan et al., ). among eight ppmos tested, ppmo- , designed to target the portion of the cvb ires, was especially potent against cvb replication in cultured cells. when cells were treated prior to or shortly after cvb infection, virus proliferation was significantly inhibited, with approximate log decrease of viral titers. in a/j mice, ppmo- intravenous administration once prior to and once after cvb infection significantly reduced cardiac tissue damage, with notable decreases in myocardium virus titers than control (yuan et al., ) . enterovirus (ev- ) generally causes mild hand-foot-andmouth disease, but severe neurological complications with high mortality rates have been reported. in one study, testing of vivo-pmo designed to target the ev- ires and the rnadependent rna polymerase (rdrp) was performed (tan et al., ) . vivo-pmo targeting ev- ires significantly reduced ev- replication in human embryonal rhabdomyosarcoma rd cells. in contrast, vivo-pmo targeting ev- rdrp was less effective. the results suggest that ires-targeting vivo-pmo are potential antiviral candidates that can abrogate early ev- infection (tan et al., ) . fmdv causes a highly contagious viral disease of clovenhoofed animals that can lead to severe economic losses to the livestock industry. six ppmos were designed to target and utrs of the fmdv genome (strain a( ) cruzeiro/brazil/ [a( )cru]) and were evaluated in cultured cells (vagnozzi et al., ) . three of the ppmos, targeting domains including the portion of the ires and the two translational start codoncontaining regions, were highly effective in inhibiting fmdv replication. at low micromolar concentrations, ppmo led to a dose-dependent and sequence-specific virus titer reduction of over log , while three other ppmo that targeted other genome regions were less effective (vagnozzi et al., ) . the order nidovirales includes the families coronaviridae, arteriviridae, roniviridae, and mesoniviridae (cong et al., ) . the members of this group are positive-sense, single-stranded rna viruses. the coronaviridae and arteriviridae include groups of viruses infecting vertebrates (mainly mammalian species), whereas the other two families include viruses infecting invertebrates. pmo have been tested as antivirals against members of both coronaviridae and arteriviridae. members of coronaviridae include severe acute respiratory syndrome coronavirus (sars-cov) and middle east respiratory syndrome coronavirus (mers-cov), which are recently identified as potent human pathogens (cong et al., ) . mouse hepatitis virus (mhv), also belongs to this family, which has long served as a model for understanding viral hepatitis in humans and to assess pmo antiviral compounds (neuman et al., ; burrer et al., ) . ppmo targeting the mhv replicase exhibited low toxicity in dbt astrocytoma cells, a cell line for studying mhv infection (neuman et al., ) . a later study tested ppmos against several mhv strains in cell culture and in vivo using mouse models (burrer et al., ; moulton et al., ) . among ten ppmos against various viral genome target sites, ppmo term, which targeted the terminus of the rna genome, was found to be highly effective in inhibiting six different mhv strains. in mice, term ppmo treatment led to prevention of virus-induced tissue damage. prophylactic treatment with term ppmo also decreased mhv-induced weight loss and prolonged post-challenge survival. this study also showed that no weight loss or detectable histopathologic changes were observed after prolonged ppmo treatment of uninfected mice (burrer et al., ) . besides, ppmo have also been shown to inhibit sars-cov replication as well (neuman et al., ). among all ppmo tested, two ppmo targeting the viral transcription-regulatory sequence (trs) within the utr brought about the most significant inhibition of cpe and reduced cell-to-cell spread when administered prior to peak viral proliferation in cultured cells (neuman et al., ) . members of the arteriviridae include an economically important virus, porcine reproductive and respiratory syndrome virus (prrsv). prrsv causes a contagious swine disease characterized by reproductive failure in sows and respiratory disease in pigs of all ages. the disease has plagued the global swine industry since it was first reported in and current strategies used for prrs control are still inadequate (du et al., ) . research in our laboratory has focused on development of ppmos against prrsv. based on the genome sequence and viral protein function, a series of ppmos with various targets were designed that included three ppmo targeting the end utr of the prrsv genome, namely up , up , and hp, as well as six ppmo targeting the translation initiation regions of orfs - , namely p , p , p , p , p , and p han et al., ) . ppmo targeting the utr were highly effective for inhibiting prrsv replication in cell culture in a dosedependent and sequence-specific manner. specifically, ppmo up or hp caused a . log reduction of prrsv yield. moreover, up and hp also demonstrated broad inhibition of heterogeneous prrsv isolates . in addition, if ppmo up targeting the utr of prrsv genome was paired with ppmo p and p , which targeted translation initiation regions of orfs and , respectively, the combination enhanced inhibition of heterologous strains of the north american prrsv genotype more effectively than individually in vitro (han et al., ) . inhibition was verified at both prrsv rna and protein expression levels. since ppmo up , which targets a highly conserved sequence within the -terminal region of prrsv genome, effectively induces a multi-log inhibition of prrsv replication in vitro, we also conducted an in vivo evaluation of this ppmo (opriessnig et al., ) . prrsv-negative -week-old piglets received ppmo intranasally at h before infection as well as and h after prrsv infection. ppmo treatment was well tolerated in piglets, with no weight change observed across all piglet groups and ppmo administration significantly reduced prrsv viremia and interstitial pneumonia. moreover, in alveolar macrophages isolated at days post-infection, elevated expression of antiviral genes in ppmo-treated piglets was observed as well (opriessnig et al., ) . besides prrsv, antiviral effects of pmo were also evaluated against equine arteritis virus (eav), another member of the family arteriviridae (van den born et al., ) . similar to prrsv studies above, ppmo designed to target the terminal utr of the eav genome remarkably reduced virus replication in a sequence-specific and dose-responsive manner. however, ppmo that targeted -terminal regions of the viral genome or its anti-genome only resulted in moderate reduction of eav replication when relatively high concentrations of the ppmos were applied. moreover, ppmo targeting the eav trs, which is essential for subgenomic rna synthesis, were ineffective to achieve transcription interference (van den born et al., ) . however, ppmo targeting the utr of eav were able to cure viral infection of persistently infected hela cells (zhang et al., ) . flavivirus is a viral genus within the family flaviviridae. this genus includes west nile virus (wnv), dengue virus (denv), yellow fever virus, zika virus, and several other viruses which may cause encephalitis (coffey et al., ; musso and gubler, ) . except for yellow fever virus, no effective vaccine or antiviral drug exists against flaviviruses, prompting evaluation of pmo against several members of flavivirus. among a panel of ppmo against wnv, ppmo targeting the -and -termini of the wnv genome, designated end or csi, exhibited the greatest potency in blocking wnv replication (deas et al., ) . moreover, treatment of wnv-infected cells with either end or csi pmo led to a significant reduction of virus titers by approximately - log without apparent cytotoxicity. ppmo end inhibited wnv translation, whereas ppmo csi suppressed wnv rna replication. meanwhile, ppmo csi also inhibited other mosquito-borne flaviviruses when the targeted csi-like sequences which were relatively conserved to the respective wnv csi target sequence. therefore, ppmo targeting conserved cis-acting elements of flavivirus genomes should be explored as anti-flavivirus therapeutics (deas et al., ) . more recently, both ppmo end or csi were also tested in a mouse model of wnv infection and provided partial protection when administered at or µg/day. moreover, minimal to no ppmo-mediated toxicity observed, while toxicity was observed at a larger dosage of µg/day (deas et al., ) . a panel of ppmo was also tested against denv (kinney et al., ) whereby ppmo targeting -terminal nucleotides of the serotype denv (den- ) virus genome exhibited relatively poor suppression of den- virus titer. however, moderate reduction of titer was observed for ppmo targeting either the aug translation start site region of the single open reading frame or the cyclization sequence region. the most highly effective ppmo were sl and cs (targeting the -terminal nucleotides and the cyclization sequence region, respectively), which reduced viral titer by greater than . log compared to controls at days post-infection with den- virus. notably, treatment with µm cs inhibited replication of all four den virus serotypes by over log , in most cases to below detectable limits (kinney et al., ) . a third ppmo that was designed to target the top of the stem-loop ( slt) inhibited den replication in bhk cells (holden et al., ) . the inhibitory mechanism was studied using a novel den reporter replicon and a den reporter mrna. the results demonstrated that sl inhibited viral translation and cs blocked viral rna synthesis but not viral translation, whereas the slt inhibited both viral translation and rna synthesis (holden et al., ) . more recently, anti-denv ppmo sl and cs were also tested in ag mice before and/or after infection with denv- . intraperitoneal (ip) infection of ag mice with - pfu of denv- (strain new guinea c) shortened survival to - days. when sl or cs were administered before and after denv infection, the average survival time was extended by up to more days. this study also included pharmacokinetic and toxicology analysis of non-infected animals. the results showed that the mice had high concentrations of ppmo in liver following nine consecutive once-daily ip treatments of mg/kg ppmo, with little impact on overall mouse health . pmo and ppmo have also been studied for their ability to prevent or treat japanese encephalitis virus (jev) infection as well. a ppmo (p ) targeting the cyclization sequence ( csi) of jev exhibited significant antiviral activity in vero (epithelial), neuro a (neuronal), and j e (macrophage) cells at non-toxic concentrations (anantpadma et al., ) . addition of p to cells before infection decreased jev replication to undetectable levels in vero cells and resulted in a and % reduction in jev titer in j e and neuro a cells, respectively. in this study, antiviral effects of p were also assessed in vivo. when treated intracerebrally with a mg/kg dose of p every h for days, - % of -week-old mice were protected from a lethal dose of jev (anantpadma et al., ) . meanwhile, testing of vivo-pmo targeting the and utr of the jev genome have also been conducted in mice (nazmi et al., ) . administration of intraperitoneal injections of vivo-pmo ( mg/kg body weight) daily for up to days immediately after jev infection of mice prolonged survival, with reduced viral load and viral protein expression in brain. moreover, proinflammatory cytokine levels in brain, which normally increase after jev infection, were reduced following pmo treatment and align with observations of reduced microglial activation in brain as well (nazmi et al., ) . chikungunya virus (chikv) causes infection in humans that is associated with debilitating and persistent arthralgia and arthritis. two ppmo were designed to target highly conserved sequences present in chikv non-structural and structural polyproteins (lam et al., ) . cpmo , a ppmo that targets the orf aug region, significantly suppressed chikv replication in hela cells when administered before infection. notably, in neonatal mice, administration of µg/g cpmo before infection conferred % survival against chikv disease (lam et al., ) . ppmo have also been tested for antiviral effects toward group v (-)ssrna virus families that include pneumoviridae, paramyxoviridae, orthomyxoviridae, arenaviridae, and others. respiratory syncytial virus (rsv), a member of the family pneumoviridae, is a major cause of lower respiratory tract infections in infants, young children, and high-risk adults. currently, no vaccine exists to prevent rsv infection. however, two antisense ppmos designed to target the -terminal region and the translational start site of rsv l mrna have been tested and exhibited minimal cytotoxicity (lai et al., ) . ppmo aug- inhibited rsv replication by reducing viral titers by over log . when administered before rsv intranasal inoculation, ppmo aug- protected balb/c mice from infection, with reduced viral titers in lung tissue and attenuation of pulmonary inflammation (lai et al., ) . measles virus (mev) is a member of the family paramyxoviridae. mev is a highly contagious human pathogen that can be treated effectively with available antiviral compounds and prevented using a vaccine. five ppmos targeting mev genomic rna or mrna were tested in cultured cells (sleeman et al., ) . ppmo , targeting a conserved sequence in the translation start site of the mrna coding for viral nucleocapsid protein, was highly effective against multiple genotypes of mev (sleeman et al., ). influenza a virus, a member of the family orthomyxoviridae, is a relentless ongoing global public health concern. when delivered by intranasal administration, ppmo inhibited replication of equine influenza a virus fluav a/eq/miami/ / (h n ) in mice and exhibited no toxicity at effective antiviral concentrations in vivo (lupfer et al., ) . meanwhile, a ppmo panel was developed to target rna genome segments encoding polymerase subunits of a highly pathogenic mouse-adapted influenza a virus strain (sc m; h n ) (gabriel et al., ) . in this study, virus replication in mdck cells was significantly inhibited by three ppmo targeting either the translation start site region of pb or np mrna or the -terminal region of np viral rna (vrna). in a mouse model, when ppmo targeting the pb -aug region or np vrna were administered intranasally once h before and once days after intranasal infection with a lethal dose of sc m, treated mice exhibited significantly lower viral titers in lungs and % greater survival versus untreated controls over the -day duration of the experiment (gabriel et al., ) . besides targeting viruses, ppmo have also been tested for efficacy against host mrna encoding proteases crucial for viral infectivity. such ppmo can block host protease cleavage of the influenza virus hemagglutinin (ha) to inhibit viral infectivity. treatment of human calu- airway epithelial cells with a ppmo t-ex , designed to interfere with splicing of hacleaving protease tmprss pre-mrna, resulted in production of tmprss mrna lacking exon that resulted in production of an enzymatically inactive form of tmprss (bottcher-friebertshauser et al., ) . ultimately, t-ex ppmo was shown to prevent cleavage of has of various human seasonal and pandemic influenza a viruses, leading to significant reduction of viral titers by to log (bottcher-friebertshauser et al., ) . junín virus, a threat to human health and a member of the arenaviridae family, can cause meningitis and hemorrhagic fever. ppmo designed to interfere with translation have been shown to be effective in reducing junín virus replication (neuman et al., ) . in cultured cells, ppmo target sequences located at the termini of both genomic segments are highly conserved across the arenaviruses. consequently, these ppmo are effective against junín virus, tacaribe virus, pichinde virus, and also against lymphocytic choriomeningitis virus (lcmv) whereby they suppress viral titers in livers of lcmv-infected mice (neuman et al., ) . the genus alphavirus includes positive-sense rna viruses that threaten human health (paessler et al., ) . sindbis virus (sinv) is a member of the family togaviridae. ppmo targeting both the -terminus and aug translation start site of the sinv genome significantly suppressed sinv replication in tissue culture (paessler et al., ) . venezuelan equine encephalitis virus (veev) is another member of the togaviridae. ppmo targeting veev regions corresponding to sinv regions mentioned above inhibit several strains of veev in vitro. notably, mice pre-treated with pmo were protected from lethal veev infection, while only partial protection was observed for mice receiving only post-infection pmo treatment (paessler et al., ) . noroviruses, which belong to the caliciviridae family, are non-enveloped, positive-sense, single-stranded rna viruses with genomes of approximately . kb in length that encode three orfs. noroviruses cause non-bacterial epidemic gastroenteritis (bok et al., ) . ppmo targeting the first aug region of the orf near the -end of the murine norovirus (mnv) genome effectively inhibited mnv replication in cultured cells (bok et al., ) . moreover, a consensus ppmo targeting the corresponding -end of the genome of several diverse human norovirus genotypes also inhibited norwalk virus protein expression (a species of norovirus) in replicon-bearing cells in a cell-free luciferase reporter assay (bok et al., ) . similar to noroviruses, hepatitis e virus (hev) is a positivesense, single-stranded rna virus containing three orfs and is currently classified within the family hepeviridae (nan and zhang, ) . hev shares a similar genome structure with members of caliciviridae and was previously classified in that family (nan and zhang, ) . our study demonstrated that ppmo targeting the terminal of the hev genome at the start codon of orf are highly effective against both hev genotypes and infection in vitro (nan et al., ) . moreover, ppmo targeting the utr of the hev genome or the terminus of antisense hev rna can also block hev replication, but to a lesser extent than achieved by ppmo targeting the terminus (nan et al., ) . since the utr and terminus of antisense hev rna are generally considered binding sites for hev rnadependent rna polymerase (rdrp), it appears that ppmomediated steric blockade also applies to rdrp as well (nan et al., ) . as compared with pmos' applications against rna viruses, pmos use against dna viruses has been much less studied. to date, only members of herpesviridae have been tested for pmo-mediated inhibition. research from our lab evaluated pmos against kaposi's sarcoma-associated herpesvirus (kshv). kshv, also known as human herpesvirus (hhv- ), is a human oncovirus belonging to the gamma herpesvirus subfamily. kshv is associated with kaposi's sarcoma (ks) and two b-cell lymphoproliferative diseases: primary effusion lymphoma (pel) and multicentric castleman's disease (mcd) (purushothaman et al., ) . these diseases are aids-related malignancies in hiv-infected patients. kshv infection in humans exhibits either a lifelong immunologically silent and latent infection or a transient lytic infection with distinct viral gene-expression profiles (lagunoff et al., ; bechtel et al., ) . during the predominantly latent state of kshv infection, the kshv genome is maintained as circular, extra-chromosomal dna that replicates inside host cells in a cell cycle-dependent manner. expression of a few key viral regulators, such as latency-associated nuclear antigen (lana) encoded by orf , viral cyclin (vcyclin) encoded by orf , and viral flip (vflip) encoded by orf , are observed during latency (uppal et al., ) . upon reactivation to assume a lytic infection state, a full repertoire of lytic viral genes, including orf (transcription activator, rta), orf , orf , orf , orf , orf , orf-k , orf-k (virf- ), orf-k (viral interleukin- , vil- ), orf (viral g protein-coupled receptor vgpcr), and viral chemokines (orf-k /vccl-i and orf-k /vccl-ii) are expressed in a temporally-regulated manner (purushothaman et al., ) . based on the functions of kshv latent and lytic genes, a panel of ppmos was designed against a set of genes including lana, vil- , rta, and virf- (zhang et al., (zhang et al., , (zhang et al., , . treatment of kshv-positive pel cells with an rta-specific ppmo rp not only reduced rta expression but also caused down-regulation of several other early and late kshv gene products, including vil- , virf- , and orf-k . a. moreover, kshv dna copy numbers both in ppmo rp -treated pel cells and culture supernatants were reduced, demonstrating inhibition of kshv lytic replication (zhang et al., ) . furthermore, treatment of bcbl- cells with ppmo against lana reduced lana expression (zhang et al., ) . meanwhile, ppmo against vil- and virf- were also evaluated. the viral homologue of the proinflammatory cytokine il- , vil- , is believed to contribute to vascular permeability and formation of pel effusions (sakakibara and tosato, ) . this cytokine shares low but significant homology to human irf family members and acts as an oncogene to inhibit interferon induction (gao et al., ) . treatment of pel cells with ppmo designed against vil- mrna led to marked reduction in the proportion of vil- -positive pel cells and reduced both the growth of pel cells and kshv dna levels (zhang et al., ) . meanwhile, ppmo targeting virf- have also been shown to inhibit viral dna replication in addition to blocking virf- expression in bcbl- cells (zhang et al., ) . interestingly, reduction of virf- expression in kshv-infected cells resulted in higher expression levels of cellular irf- and of the signal transducer and activator of transcription (stat ) (zhang et al., ) . encouraged by these in vitro data, an in vivo evaluation of ppmo effects on expression of multiple viral genes was conducted (zhang et al., ) . however, in vivo results defied expectations drawn from in vitro studies. specifically, only ppmo against vil- demonstrated promising in vivo inhibition whereby scid mice treated with this ppmo exhibited no engraftment of kshv-infected pel cells and remained healthy throughout the -day study (zhang et al., ) . conversely, in scid mice receiving a combination of pmo against two virf- , there was a trend of less engraftment of kshv-infected pel cells, but the difference in results was not statistically significant between treated and control mice. therefore, ppmo targets selected using in vitro assessments may not achieve expected inhibition in vivo, emphasizing the fact that careful validation using adequate animal models is necessary. acyclovir (acv) is a nucleic acid analog of guanosine that is used to treat hsv. viral resistance to acv has become a recent concern. as a potential treatment for acv-resistant hsv, ppmo against resistant hsv- and hsv were evaluated both in vitro and in a mouse model as well. for hsv- , icp and icp were selected as virus ppmo targets. icp from both hsv- and hsv- acts as an e ubiquitin ligase to antagonize host innate immunity (halford et al., ; lanfranca et al., ) . icp is a multiple function regulator involved in pre-mrna splicing and the host innate immune response (christensen et al., ; tang et al., ) . when ppmo targeting translation start site regions of hsv- icp or icp mrna were applied before or soon after hsv- infection of cultured cells, a - % reduction of hsv- yield was observed, as assessed by reduced plaque formation. moreover, icp -specific ppmo also inhibited acvresistant hsv- plaque formation by - %, while an equivalent dose of acv only led to a - % plaque reduction (moerdyk-schauwecker et al., ) . in vivo data suggest that ppmo are well-tolerated in uninfected mice after days of administration of µg/day (moerdyk-schauwecker et al., ) . topical application of µg icp -specific ppmo into the eyes of hsv- -infected mice reduced the incidence of eye disease by . - % compared to controls. therefore, ppmo holds promise as an antiviral drug for use in treating hsv- ocular infection (moerdyk-schauwecker et al., ) . with regard to hsv- , ppmo targeting icp or icp mrna were also highly effective against either non-acvresistant or acv-resistant hsv- strains. in one in vivo study, ppmo were well-tolerated in balb/c mice and cotton rats. cotton rats receiving icp -specific ppmo h after hsv- inoculation showed a reduction in genital lesions and a . % reduction in mortality at days post-infection. mice receiving a combined regimen of µm of icp -and icp -specific ppmo before hsv- inoculation were completely free from genital viral infection at - days post-inoculation (eide et al., ) . a variety of studies suggest that pmo would be good candidate for antiviral therapeutics against emerging or reemerging viruses in the absence of other effective therapies. however, effective target sequences for pmo design need to be carefully validated. a list of previously published effective virus target regions used for pmo designed against rna viruses are briefly summarized in table . to date, for most pmos evaluated against positivesense rna viruses, targeting sequences of pmo are mainly located in the terminal ends of viral genomes (table ) . notably, it appears mrna-like properties of positive-sense rna virus genomes make them highly susceptible to pmo-mediated translation inhibition if the pmos pairing sequence are located at the terminal end of the viral genomes, with few exceptions. moreover, utr at either end of viral genomes are generally conserved among viral strains. conversely, for some positivesense rna viruses, pmo targeting of the ' terminal ends of the viral genome were tested as well. however, data appears to be mixed for terminal-targeting pmos. on the one hand, for members of flaviviridae, pmos targeting the ' cyclization sequence located within the terminal end are highly effective in inhibiting virus replication (deas et al., (deas et al., , kinney et al., ; holden et al., ; stein et al., ; anantpadma et al., ; nazmi et al., ) . on the other hand, the use of pmo targeting either the -terminal regions of the viral genome or of the negative genomic strand only resulted in moderate reduction of eav (van den born et al., ) . this result suggests that pmo targeting the terminal end are less effective for inhibiting eav replication compared to pmo targeting the terminal utr of the eav genome. therefore, the viral genome end is the preferred target region for antiviral pmo design against positive-sense rna viruses. in addition to pmo targets within the terminal end of positive-sense rna virus genomes, pmo targeting rna secondary structures should also be considered, especially for common conserved elements among rna viruses, such as ires sequences. generally, unstructured regions are more accessible to oligonucleotide binding than are structured regions, since internal structures within target rna can impede pmo binding (childs-disney and disney, ) . however, in some cases, direct targeting of ires sequences could inhibit replication of some viruses, including members of the order picornavirales (yuan et al., ; stone et al., ; tan et al., ) . therefore, a deep analysis of the secondary structures of target rna may aid pmo target selection. for pmos evaluated for antiviral activity against negativesense rna viruses, current reports mainly focus on blocking translation of individual viral genes rather than direct targeting the rna genome. for example, pmo targeting ebov vp alone was sufficient to protect monkeys against lethal ebov infection, whereas a pmoplus formulation designated avi- that targeted vp failed to do so (warren et al., ) . therefore, screening to find effective pmo targets of negative-sense rna viruses may require more careful consideration than needed for positive-sense rna viruses. although not yet extensively studied, pmo target selection appears to be more complicated for large dna viruses such as herpesviruses. due to their large genome size, such dna viruses encode many genes, including indispensable and dispensable genes. on the one hand, some indispensable genes studied so far do not appear to be good pmo targets. for example, hsv- early genes ul and ul encode the viral dna polymerase and the large subunit of ribonucleotide reductase, respectively, which are essential for viral dna replication (eide et al., ) . however, blocking hsv- ul and ul mrna translation by pmo did not significantly reduce viral replication or transmission from infected cells (eide et al., ) . on the other hand, dna viruses encode many dispensable accessory proteins with multiple functions, such as antagonist to host innate immunity. such genes need to be carefully validated both in vitro and in vivo before use as pmo targets. for example, pmo targeting of virf- of kshv demonstrated little protection against kshv-infected pel engrafts in scid mice, in spite of its effectiveness as a pmo target in vitro (zhang et al., ) . aside from challenges regarding target selection, the emergence of resistant virus after sequence-specific therapy is another common challenge faced by antivirals. indeed, a hcmv mutant with sequence-dependent resistance to the phosphorothioate oligonucleotide fomivirsen was discovered almost simultaneously with market approval of the drug (mulamba et al., ) . because pmo-based antiviral therapy is sequence-specific, pmo-resistant target mutations would abolish pmo inhibition, as has been already observed (neuman et al., ) . for west nile virus, the sequencing of ppmo-resistant wnv mutants has demonstrated that viruses resistant to -end ppmo treatments contained two to three mismatches within the ppmo-binding site, whereas csi ppmo-resistant viruses accumulated mutations outside the ppmo-targeted region (deas et al., ) . meanwhile, ppmo-resistant-wnv infection of mice was shown to antagonize pmo-mediated protection against virus (deas et al., ) . similar pmo-resistance has been reported for ebola virus after treatment with pmo targeting ebola virus vp and vp as well (kugelman et al., ) . therefore, mutations within or outside of pmo-targeting sequences might lead to pmo resistance and will be a future challenge. in our research of ppmo inhibition of prrsv, a multiple log reduction of viral rna copies was achieved when pmo sequence was complementary to a conserved region within the utr of the prrsv genome, although low level viral rna replication was still observed. this suggests that pmoresistant mutants might have arisen or that the effective one possible strategy to encounter pmo-resistance mutants would be incorporating of promiscuous bases such as inosine to compensate for predicted base-pair mismatches . however, it is a challenge to accurately predict the potential mutation nucleotides within a pmo-targeting sequence. therefore, the pmo target sequence against any specific rna virus must be carefully selected to minimize generation of mutants. moreover, use of multiple pmos against different targets of the same virus may help to avoid mutant virus generation. an alternative strategy to prevent pmo-resistant virus is indirect inhibition of a host factor essential for virus replication. currently, only one of such study has been conducted on influenza virus and exploits the fact that cleavage of viral hemagglutinin (ha ) by host proteases is crucial for viral infectivity. in this study, treatment of human calu- airway epithelial cells with a ppmo designed to interfere with pre-mrna splicing of tmprss (the host protease responsible for ha cleavage) resulted in tmprss mrna lacking exon , finally led to expression of an enzymatically inactive form of tmprss (bottcher-friebertshauser et al., ) . therefore, blocking of ha cleavage by this ppmo was confirmed in different human seasonal and pandemic influenza a viruses and resulted a significant reduction of viral titers (bottcher-friebertshauser et al., ) . in addition to above investigation, variety host proteins have been identified as essential factors required for virus replication, host defense and viral pathogenesis in recent years (rajsbaum et al., ; tripathi et al., ) . the putative ubiquitin ligase ubr was identified as a novel host factor involved in the budding of influenza virion (tripathi et al., ) , targeting such factor could block the release of influenza virus. although it is unknown if ubr could be employed by other enveloped viruses for budding as well, it is possible that targeting a commonly used host factor which is required for viral replication could broaden the antiviral spectrum of pmo. therefore, application of pmo to target host factor as antiviral strategy may not only avoid generation of pmo-resistant virus, but also expand the antiviral spectrum as long as the targeted host factor is employed by different virus for replication. it has been two decades since the first approval of an antisense-based therapy. pmos have become important on analogs that have driven development of antisense therapies with efficacy and safety demonstrated by the recent fda approval of eteplirsen. indeed, biological stability, neutral charge, and rnase h-independent mechanism of action are all unique pmo features. moreover, besides their development as antiviral compounds, pmo evaluation as anti-cancer or antibacterial agents is ongoing (enterlein et al., ; warfield et al., ; cansizoglu and toprak, ; chen et al., ; summerton, ) . since pmo have a good track record as useful tools in developmental biology and as therapeutic agents, three generations of pmo have been developed (unmodified pmo, conjugated pmo, and pmoplus) to improve intracellular delivery (daly et al., ) . however, several issues remain unresolved before pmo are adopted for widespread use. on the one hand, more studies are needed to establish a convenient route for pmo administration in vivo. in most in vivo studies, pmo are administrated via either intravenous or intramuscular injection. although reports have demonstrated that pmo administered via intranasal delivery can inhibit replication of viruses with a respiratory tract tropism (opriessnig et al., ; rajsbaum, ) , it is notable that in these in vivo studies employed pmos for as antiviral agents against two respiratory viruses, influenza virus and prrsv (opriessnig et al., ; rajsbaum, ) . since there is no investigation conducted to see if intravenous or intramuscular administration of pmo also effectively against influenza virus and prrsv, it is possible intranasal delivery of pmo is preferable for virus causes respiratory infection. moreover, it is also possible that virus-specific delivery routes based on the initial infection sites may offer a better antiviral efficiency rather than using intravenous route as universal way for pmo administration. on the other hand, although pmo is a highly adaptive platform for delivery of nucleic acid sequence-specific drugs, selection of an effective target and avoidance of the emergence of mutant virus also require more investigation. if these issues could be properly addressed, pmo should serve as a promising strategy for treatments of a variety of diseases, including difficult-totreat viral infections. as is generally true for antiviral drug development, high efficacy, low toxicity, good pharmacokinetics, good bioavailability, and low cost are all characteristics sought in a pmo compound. results discussed in this review show great promise and warrant further research to develop safe and effective antisense treatments for a variety of human diseases. yn and y-jz designed this manuscript. yn prepared the main body of the manuscript. y-jz prepared the figure and revised the manuscript. all authors approved it for publication. arginine-rich cell penetrating peptides: design, structure-activity, and applications to alter pre-mrna splicing by steric-block oligonucleotides delivery of steric block morpholino oligomers by (r-x-r) peptides: structure-activity studies inhibition of infectious haematopoietic necrosis virus in cell cultures with peptide-conjugated morpholino oligomers inhibition of japanese encephalitis virus replication 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activator and latency-associated nuclear antigen of kaposi's sarcomaassociated herpesvirus by morpholino oligomers key: cord- - izxqd authors: fouret, julien; brunet, frédéric g.; binet, martin; aurine, noémie; enchéry, francois; croze, séverine; guinier, marie; goumaidi, abdelghafar; preininger, doris; volff, jean-nicolas; bailly-bechet, marc; lachuer, joël; horvat, branka; legras-lachuer, catherine title: sequencing the genome of indian flying fox, natural reservoir of nipah virus, using hybrid assembly and conservative secondary scaffolding date: - - journal: front microbiol doi: . /fmicb. . sha: doc_id: cord_uid: izxqd indian fruit bats, flying fox pteropus medius was identified as an asymptomatic natural host of recently emerged nipah virus, which is known to induce a severe infectious disease in humans. the absence of p. medius genome sequence presents an important obstacle for further studies of virus–host interactions and better understanding of mechanisms of zoonotic viral emergence. generation of the high-quality genome sequence is often linked to a considerable effort associated to elevated costs. although secondary scaffolding methods have reduced sequencing expenses, they imply the development of new tools for the integration of different data sources to achieve more reliable sequencing results. we initially sequenced the p. medius genome using the combination of illumina paired-end and nanopore sequencing, with a depth of . x and . x, respectively. then, we introduced the novel scaff link software to integrate multiple sources of information for secondary scaffolding, allowing to remove the association with discordant information among two sources. different quality metrics were next produced to validate the benefits from secondary scaffolding. the p. medius genome, assembled by this method, has a length of , mb and consists of , contigs and , scaffolds with an ng of mb. at least . % of the assembled sequences is covered by interspersed repeats already described in other species and , coding genes are annotated. phylogenetic analysis demonstrated the clustering of p. medius genome with two other pteropus bat species, p. alecto and p. vampyrus, for which genome sequences are currently available. sars-cov entry receptor ace sequence of p. medius was . % identical with ace of rhinolophus sinicus bats, thought to be the natural host of sars-cov. altogether, our results confirm that a lower depth of sequencing is enough to obtain a valuable genome sequence, using secondary scaffolding approaches and demonstrate the benefits of the scaff link application. the genome sequence is now available to the scientific community to (i) proceed with further genomic analysis of p. medius, (ii) to characterize the underlying mechanism allowing nipah virus maintenance and perpetuation in its bat host, and (iii) to monitor their evolutionary pathways toward a better understanding of bats’ ability to control viral infections. indian fruit bats, flying fox pteropus medius was identified as an asymptomatic natural host of recently emerged nipah virus, which is known to induce a severe infectious disease in humans. the absence of p. medius genome sequence presents an important obstacle for further studies of virus-host interactions and better understanding of mechanisms of zoonotic viral emergence. generation of the high-quality genome sequence is often linked to a considerable effort associated to elevated costs. although secondary scaffolding methods have reduced sequencing expenses, they imply the development of new tools for the integration of different data sources to achieve more reliable sequencing results. we initially sequenced the p. medius genome using the combination of illumina paired-end and nanopore sequencing, with a depth of . x and . x, respectively. then, we introduced the novel scaff link software to integrate multiple sources of information for secondary scaffolding, allowing to remove the association with discordant information among two sources. different quality metrics were next produced to validate the benefits from secondary scaffolding. the p. medius genome, assembled by this method, has a length of , mb and consists of , contigs and , scaffolds with an ng of mb. at least . % of the assembled sequences is covered by interspersed repeats already described in other species and , coding genes are annotated. phylogenetic analysis demonstrated the clustering of p. medius genome with two other pteropus bat species, p. alecto and p. vampyrus, for which genome sequences are currently available. sars-cov entry receptor ace sequence of p. medius was . % identical with ace of rhinolophus sinicus bats, thought to be the natural host of sars-cov. altogether, our results confirm that a lower bats have been reported to be the natural reservoir of several zoonotic viruses that cause severe human diseases, including marburg, ebola, nipah, hendra, sars, and mers viruses (wang and anderson, ) . pteropus medius (also known as p. giganteus and commonly called indian flying fox (mlikovsky, ) is a frugivorous giant bat, widely distributed in southeast asia, and shown to host numerous viral species (anthony et al., ) , including nipah virus (yadav et al., ) . nipah virus is a recently emerged zoonotic paramyxovirus, capturing the attention of both scientific and public health communities due to its high lethality rate, up to % in bangladesh and india epidemics, associated with human-to-human transmission (mathieu and horvat, ; arunkumar et al., ) . although this virus is highly pathogenic in humans and numerous other mammalian species, it is asymptomatic in its natural host, fruit bats (enchéry and horvat, ) . a better understanding of virus-host interactions requires further studies necessitating the availability of the sequenced genome (shi, ) , which has been lacking for p. medius. therefore, our work aims to provide the genome sequence of p. medius as a new resource for genomic studies, analysis of molecular basis of bats' unique adaptation and bats' immunovirology and should help in understanding the underlying evolutionary mechanisms used by emerging viruses, like nipah virus, to cross species barriers and to widespread in the newly introduced host. moreover, recent outbreaks of numerous pathogenic viruses from bats, including sars coronavirus (hu et al., ) , urge improved comprehension and characterizations of bat species. although bats make up more than % of existing mammals with , species (lazzeroni et al., ) , only bat genome assemblies are currently available in the ncbi genbank database. several different sequencing strategies have been used in the past years to sequence bat genomes, including (i) high coverage long-read sequencing strategy, e.g., x of pacbio eonycteris spelaea (wen et al., ) , (ii) illumina sequencing (e.g., x for paired-end libraries and x of matepair libraries) for p. alecto (zhang et al., ) and also (iii) hybrid sequencing, e.g., x of illumina short reads and x of pacbio long-reads for rousettus aegyptecus (pavlovich et al., ) . combined technological developments applied to genome assembly, including hi-c (ghurye et al., ) , optical mapping (jiao et al., ) and synthetic long reads (coombe et al., ) provide a possibility for the high quality chromosomelevel assembly (teeling et al., ) . in contrast to direct sources of evidence used for scaffolding the genome sequence, secondary scaffolding leverages less direct type of information to improve assembly scaffolding. in this report, we focus on a reference-assisted assembly method using ragout (kolmogorov et al., ) and a gene-based method using agouti (zhang et al., ) that require a reference genome and rna-seq data, respectively (zhang et al., ; pavlovich et al., ; wen et al., ) . we used a combination of illumina paired-end reads and nanopore long-reads sequencing with coverage of . x and . x, respectively, based on a genome size estimation of gb. in addition, the sequencing results in this study have benefited from the produced rna-seq data. although the sequencing coverage obtained in this study was lower than in some other recent reports, we show here that it has been enough to provide a good quality genome using current computational method. in the first step, we provide an assembly based directly on sequencing evidence (paired-end and long-reads), where assembled fragments are called solid scaffolds. then, we improve the assembly using secondary scaffolding methods, agouti (gene-based) and ragout (reference-assisted), using rna seq data obtained with an illumina bp paired-end sequencing. finally, we provide an original tool, called scaff link, which integrates both sources of secondary scaffolding to keep only non-discordant linkages, hence avoiding potential errors. the development of these methods is essential for a better use of available resources, particularly when the number of the high-quality genome sequences is expected to increase rapidly. to follow the impact of the different methods regarding the quality of the assembly, we next describe metrics for each mentioned step: solid scaffolds, secondary scaffolding and the use of scaff link. therefore, in the same order, the metric described in this study contains: (i) fragmentation state of the genome sequence; (ii) benchmark using single-copy orthologs (sco), and (iii) dna-seq realignment consistency. to characterize the nature of the sequence provided as a resource, we performed a k-mer analysis on solid scaffolds, describing the state of ploidy. based on quality metrics, we show that the best assembly was obtained after integration of both secondary scaffolding methods with scaff link. finally, we demonstrate the potential use of the obtained genome assembly by providing annotation of both coding genes and interspersed repeats and performing the phylogenetic analysis. this new approach allowed us to assemble the genome of p. medius with a length of , mb, consisting of , contigs and , scaffolds with a ng of mb. identified repeat elements covered . % of the assembled sequences and , coding genes were annotated, thus providing the first genome sequence of this bat species and making it available for further studies and better understanding of the mechanisms of nipah virus emergence. cell culture from pteropus bat flying fox was generated from a wing-membrane skin biopsy of a female specimen of p. medius (known also as indian flying fox and p. giganteus, belonging to yinpterochiroptera suborder) (figure ) , collected in the tiergarten schönbrunn (vienna, austria), during the regularly veterinary check, as previously described (gaudino et al., ) . briefly, sample was washed with sterile pbs and transferred into freezing medium cryo-sfm (promocell bioscience) in dry ice, for the shipment from the zoo. to obtain a primary cell culture (ptgv), samples were thawed and fractioned in a petri dish, then homogenates were harvested in different media and cultured in dmem/f- medium (gibco) supplemented with % fetal bovine serum, % l-glutamine ( mm), , u/ml of penicillin, , u/ml of streptomycin, at • c, at % co . we confirmed that the biological sample belongs to the p. medius species by sequencing a mitochondrial region d-loop (saccone et al., ) and nuclear introns acox , cops a, bgn, rogd , and stat a, suggested to be pertinent to distinguish among closely related species (dool et al., ) . we compared these sequences with those obtained from several individuals phenotypically identified as p. medius, sampled at national institute of high security animal diseases, bhopal, india. genomic dna was purified from a pelleted primary ptgv cells using nucleospin r tissue kit (macherey-nagel, ). a paired-end library was constructed with an insert size of bp in average using nextflex r pcr-free dna-seq kit (bioo scientific) with bioruptor (diagenode) for dna fragmentation step. paired-end short-reads of bp were generated on illumina nextseq platform using a high output flowcell. primary cell cultures of ptgv cells, containing principally fibroblast-like cells, were used as a source of rna. three replicates were obtained for each condition h post-infection, either from nipah virus-infected ptgv cells at three viral particles/cell or from non-infected cells (incubated with mock preparation). the level of nipah virus infection was bellow (aurine et al., ) . rna was extracted using nucleospin r rna (macherey-nagel, ). libraries were generated using nextflex r rapid directional rna-seq library prep kit according to manufacturer's protocol (bioo scientific) using ng of extracted rna as input material. libraries were sequenced via illumina nextseq in paired-end bp using a mid-output flow cell. high-molecular-weight dna was extracted from cell culture using the magattract r hmw dna kit (qiagen). then, genomic dna was sheared using covaris g-tube as per manufacturer's guidelines to obtain approximately kb fragments without any additional enrichment or purification step. dna concentration was measured using the quantus tm fluorimeter with the quantifluor r dsdna kit (promega), and purity was evaluated by measuring a /a and a /a ratios using a nanodrop tm spectrophotometer. the quality of dna and its size were assessed by electrophoretic migration using fragment analyzer with the high sensitivity large fragment kb analysis kit (aati). figure | schematic presentation of the main analytical steps of the assembly workflow of pteropus medius genome. analytic steps, including the applied software (masurca, hisat, progressive cactus agouti and scaff link) are marked in black, input data in dark blue, data obtained from databases in light blue, nomenclature used for produced assemblies in green and produced data in orange. sequencing libraries were prepared using the ligation sequencing kit d (sqk-lsk ) or the d sequencing kit (sqk-lsk ) (oxford nanopore technologies). the libraries were then loaded to minion using a flow cell r . version (flo-min ) (ont) and the sequencing runs were performed under minknow version . . for up to h. raw reads were trimmed using cutadapt (version . . with options: -q -m for dna-seq and -q -m for rna-seq). for dna-seq, resulting reads were corrected using lighter (version . . ). expected genome size has been predicted using kmergenie software (v . ). base calling was done through albacore (v . . ). low quality ends and chimera reads were trimmed using porechop (v . . ). resulting long-reads were filtered using filtlong with short dna-seq illumina reads to estimate quality (v . . with options: -trim -split -min_length -min_mean_q , -length_weight and -window_q_weight ). hybrid assembly using both illumina short-reads and nanopore long-reads was achieved using masurca whole genome assembly software (v . . ) (zimin et al., ) , with a k-mer size of . this value was chosen after preliminary tests (described in the supplementary material). gap filling was performed on this assembly using gapfiller (v . ) with iterations. for gene-based scaffolding, rna-seq data has been aligned using hisat (v . . ) on the assembly. gene annotation have been predicted using augustus (v . . ) with human training set. finally, these data were integrated with agouti (v . . ). for reference-based scaffolding, genome sequences of p. vampyrus (ptevam ), p. alecto (pteale ), and r. aegyptecus figure | fragmentation graph for produced assemblies. the graph presents a scatter plot comparing cumulative length (y-axis) and the fragment length (x-axis) for four different p. medius scaffold assemblies, compared with already published p. vampyrus (ptevam ) and p. alecto (ptealei). for each assembly, cumulative lengths were computed along sorted fragments, from the smallest to the highest. a label box gives information about the total size of the assembly (g) and the ng values, where the genome size has been set to gb for normalization. (raegyp ) were extracted from genbank database and aligned with the solid-scaffold assembly using progressive cactus (https://github.com/glennhickey/progressivecactus). the software ragout v . performed the scaffolding based on the multi genome alignment using both ptevam and pteale assembly sequences as reference, raegyp as outgroup and the solid scaffold assembly as target (kolmogorov et al., ) . we developed an algorithm to integrate, in a conservative way, scaffolding results given by ragout and agouti (available at: https://github.com/jfouret/scaff links). briefly, this algorithm builds a graph with phylogenetic and gene-based linkage information as input for edges, and fasta formatted sequences as input for nodes (supplementary figure s ) . first, linear paths are simplified and then higher level of simplification is added using pattern of directed acyclic graph (dag) (supplementary figure s ). the pseudocode of the algorithm is given in supplementary material. ng have been computed for all assemblies using a genome size of gb. k-mer analysis toolkit (v . . ) have been used to compare -mers presence and multiplicity in illumina reads and in assembly. busco (v ) has been used to control the presence in single copy of sco shared in laurasiatheria super order. illumina dna-seq reads were mapped with bowtie (v . . ) with options to consider an alignment properly paired if fragment size is in range (-i) and , (-x) and "-very-sensitive" mode. more specifically, metrics were computed using "flagstat" tools of the samtools suite (v . ). genomes from p. vampyrus (ptevam ), p. alecto (pteale ), and r. aegyptecus (raegyp ) and the ma_sr-lr_union assembly were aligned using progressive cactus (https://github.com/ glennhickey/progressivecactus) (paten et al., ) . r. aegyptecus was used as outgroup to improve the multiple alignment process and has been chosen because it was a species close to the pteropus genus with a good quality assembly. gene annotation was performed using the comparative annotation toolkit (cat) v . (zhang et al., ) automated pipeline with p. alecto as reference and p. medius as target with a step of ab initio gene prediction using augustus . . (stanke and waack, ) . of note, r. aegyptecus genome assembly has been used solely to improve the multiple genome alignment of the three pteropus species genomes, however, r. aegyptecus was not used directly to infer p. medius genome annotation. figure | quality assessments of produced assemblies. (a) benchmark of universal single-copy orthologs (busco) genes for the laurasitharia super order with comparison to already published assemblies of two pteropus species, p. vampyrus (ptevam ), and p. alecto (ptealei). (b) short-reads mapping against produced assemblies were analyzed. y -axis is the number of reads that are either (i) mapped (ii) properly paired or (iii) mapped with quality superior to five and with a mate mapped to a different fragment. the label box on top of each bar chart presents the evolution of read counts compared to the solid scaffold assembly (ma_sr-lr). figure | genome assembly is representative of only one allele. the x-axis presents the multiplicity of a k-mers. the y-axis is the amount of distinct k-mers present at this multiplicity in reads. fill colors represent the number of time ( x, x, x or x, and more) a distinct k-mer is present in assembly. the right part of the dotted gaussian curve has been hand-traced and a symmetry has been applied to get the left part. cat is using other dependencies: hal v , samtools . - and bedtools v . . . repeated sequences were annotated with repeatmasker (v open- . . ) using rmblast (nucleotide-nucleotide blast with repeatmasker extensions . . +) and repeatmasker combined database (dfam_consensus- , repbase- ) filtered for mammal species. additional option for repeatmasker were "-s -nolow -no_is." proteomes were retrieved from ncbi for miniopterus natalensis (gcf_ . ), eptesicus fuscus (gcf_ . ), myotis brandtii (gcf_ . ), m. lucifugus (gcf_ . ), m. davidii (gcf_ . ), rousettus. aegyptiacus (gcf_ . ), p. alecto (gcf_ . ), p. vampyrus (gcf_ . ), rhinolophus ferrumequinum (gcf_ . ), desmodus rotundus (gcf_ . ), hipposideros armiger gcf_ . ), and phyllostomus discolor (gcf_ . ). more specifically "gcf_ * _ translated_cds.faa.gz" file was fetched from ncbi refseq ftp repository for each species. for p. medius, the proteome was translated from the annotation. in case of isoforms, only the longest isoform per gene was kept. sco were identified thanks to orthofinder (emms and kelly, ) using default parameters. multiple sequences alignment was conducted separately on each locus using mafft v . b (katoh, ) with, generalized affine gap score, blossum (-bl) matrix for scores, an open gap penalty of (-op ) and all other parameters set to default. all positions with at least one gap were removed from the msa along with surrounding five residues, as gaps may be linked with a missing or incorrect sequence. of note, the models implemented in the phylogenetic tools used hereafter do not consider gaps in a multiple sequence alignment. then conserved blocks were extracted from each msa using gblocks . b (−b = , −b = , −b = , −b = ) in order to improve the phylogeny (talavera and castresana, ) . msa were then concatenated. maximum of likelihood searches have been conducted with respectively distinct starting trees to identify the best tree using raxml . . (stamatakis, ) . separately, bootstrapping was done with iterations. the random seed " " was used at all steps with raxml. the tree was rooted manually. a msa subset with , sites was used for estimation of divergence times using soft fossil constraints with paml v . j mcmctree program (yang, ) . configuration files used in paml are available in the supplementary material. the procedure has been repeated with another subset with no significant changes in age time and confidence intervals. the minimum age of fossil data, obtained from paleobiodb database , were used as lower bound; all values used for the calibration were specified (supplementary figure s ). an upper constraint was set for the root using the upper value of the % confidence interval available for the chiroptera order on time tree database (kumar et al., ) . pteropus medius dna was extracted from cultured primary bat cells and sequenced using both paired-end illumina and nanopore sequencing, generating . gb of illumina dna-seq data (after primary analysis; see section "materials and methods"). based on the analysis on distinct k-mer counts in dna-seq short-reads, the software kmergenie (chikhi and medvedev, ) predicted a genome size from . to . gb, corresponding to the range of size of previously published bat genomes (teeling et al., ) . using an alternative method based on the c-value (smith and gregory, ), the genome size was estimated at , gb, with a gc content of about % deduced from dna-seq data. we then used a genome size of gb to calculate the depth of sequencing and subsequently normalized assembly metrics. the sequencing depth obtained for illumina paired-end data was . x. for long-read data, . gb of minion were produced after primary analysis with a mean long-read size of kb, corresponding to a depth of . x. these data altogether were then used for the final assembly. we also isolated the cellular rna and performed the rna-seq analysis using the same biological material, then used the produced data in the secondary scaffolding step. both illumina paired-end and minion long-reads were assembled with the masurca (zimin et al., ) hybrid software and gaps were filled with gapfiller (see section "materials and methods"). the workflow of this step is part of the general workflow presented in figure , where the figure | integrating phylogenetic and gene-based secondary scaffolding with scaff links. scaff link allows the import of linkages established by both agouti for gene-based scaffolding and ragout for reference-assisted scaffolding (see section "materials and methods"). the results of the process of conservative union from ( ) the initial state with links parsed from ragout and agouti to ( ) the linear chain simplification and ( ) the directed acyclic graph simplification is respectively shown on (a), (b), and (c) were connected components resolved up to a single node are no longer displayed. one connected component at the initial step , , and are respectively shown on (d), (e), and (c) and highlighted in blue. graph structure and different steps ( - ) are detailed in supplementary material. assembly produced is named "ma_sr-lr" for (ma: masurca, sr: short-reads, lr: long-reads). metrics describing this assembly are the fragmentation state of the genome sequence (figure ) , benchmark using sco ( figure a ) and dna-seq realignment consistency ( figure b) . previously published pteropus bat genomes from p. vampyrus (lindblad-toh et al., ) and p. alecto (zhang et al., ) were used for comparative analysis. for the fragmentation presented on figure , the ma_sr-lr reached an ng of kb, with a total length of . gb. ma_sr-lr ng metrics was ∼ and ∼ times lower compared to p. vampyrus (ng : mb) and p. alecto (ng : mb) assemblies, respectively (figure ) . focusing on the potential use of the genomes for gene annotation, we have used busco (benchmark of universal single copy orthologs). this approach attempts a homology-based annotation of a group of genes selected because their orthologs are present in at least % of species from laurasiatheria in single copy. for "ma_srlr, " only . % of buscos ( figure a ) have been successfully annotated and confirmed to be present as a single copy, even if it has been lower than in p. vampyrus ( . %) or p. alecto ( . %). the relative difference with other pteropus is lower than the fragmentation of the genomes, indicating that we might have reached a limit of genome contiguity which facilitates gene annotation. indeed, kb (ng of ma_sr-lr) is in order of magnitude ∼ times higher than the median gene size among species of the pteropus genus; . kb for p. vampyrus (refseq ) and . kb for p. alecto (refseq ) (ncbi resource coordinators, ). we then focused on comparing the distribution of k-mer multiplicity between dna-seq reads and the assembly. as shown in figure , large number of k-mers with low multiplicity in illumina dna-seq reads were not present in the assembly (corresponding to the first left peak, close to x); this is most probably an artifact from sequencing errors. this peak is followed by a bimodal gaussian-like distribution, suggesting that those two mixed distributions correspond to the heterozygous k-mers (left curb; mean: ∼ x) and for homozygous k-mers (right curb; mean: ∼ x); we indeed expected homozygous k-mers to be twice more present in sequencing reads. half of heterozygous k-mers were apparently not integrated in the assembly, while the remaining part was integrated only once ( figure ). this suggests that most heterozygous positions were inserted only once in the genome assembly. on the other side, if two alleles were integrated in the assembly for a consequent number of polymorphic positions, we would expect to see a significant proportion of homozygous k-mers present at a multiplicity of two in the assembly; however, only . % of the total k-mers is found twice in the assembly (not visible in the graph). therefore, the assembly presented in this report is very likely to be representative of a haplotype. the workflow used to perform secondary scaffolding is presented in figure . both methods start with the ma_sr-lr assembly. the assembly produced after reference-assisted scaffolding is called "ma_sr-lr_phylo." the number of scaffolds (supplementary table s ), , for ma_sr-lr, was diminished by , and , by gene-based scaffolding (ma_sr-lr_rna) and referenceassisted (ma_sr-lr_phylo) scaffolding, respectively. quality was assessed based on different metrics as explained above. the state of fragmentation of obtained assemblies is presented on figure . while reference-assisted scaffolding showed a very high ng value of mb, the value for gene-based scaffolding was limited to kb (figure ) . interestingly, the value of ng for ma_sr-lr_phylo assembly outperforms the ng of p. alecto assembly ( mb) (zhang et al., ) , while having a smaller number of ns (any nucleotide according to the iupac code) in its sequence (supplementary table s ). during the scaffolding processes, ns are produced when resolving a gapped linkage of two fragments. annotation metrics of benchmark of universal single-copy orthologs (buscos), is presented on the figure a . the percentage of complete and single copy buscos annotated raised from . to . % (+ . %) and . % (+ . %) for ma_sr-lr_rna and ma_sr-lr_phylo, respectively. it appears that in most cases this difference corresponds to fragmented buscos in the solid-scaffold assembly, which can be annotated on a single fragment in ma_sr-lr_rna and ma_sr-lr_phylo assemblies ( figure a) . finally, figure b presents metrics of dna-seq illumina read realignment. there is a slight increase in mapped reads for both ma_sr-lr_rna and ma_sr-lr_phylo assemblies, + . · and + . · mapped reads (out of . · ), respectively. an increase within the same order of magnitude co-occurs for the number of properly paired reads. given options to the aligners define proper alignment with the constraint of the fragment size to be between and , , as expected from sequencing. finally, the number of reads with a mate mapped to different fragments had decreased. all these data together indicate that the most added linkages are consistent with dna-seq realignment. the scaff link steps presented on the general workflow (figure ) combine different pieces of evidence allowing to avoid errors at secondary scaffolding steps (see section "materials and methods"). scaff link allows conservative integration of both linkage information built by agouti and ragout. when importing links from agouti based on rna-seq data, the minimum number of reads to consider a linkage is an option; this option has been tested with and minimum rna-seq reads leading to ma_sr-lr_union and ma_sr-lr_union assemblies, respectively. a general picture of the assembly states during scaff link processing is shown in figure , with intermediary steps presented on figures a,b and the final step depicted in figure c , corresponding to ma_sr-lr_union . in this final step, there were still unresolved links due to contradictory information (potentially linked with scaffolding errors) or forklike graph structures that are impossible to resolve without introducing potential misassembles. then, the quality has been assessed with the same metrics as those used before. first, ng values of reference-assisted scaffolding are high with and mb (figure ) , corresponding to ma_sr-lr_union and ma_sr-lr_union , respectively. these values are close to ma_sr-lr_phylo ( mb) and still higher than p. alecto ( mb). second, in terms of quality for gene annotation (figure a) , results for scaff link assemblies are very similar to the referenceassisted assembly ma_sr-lr_phylo. interestingly, for ma_sr-lr_union compared to ma_sr-lr_phylo, one more busco annotation is complete (from fragmented) and presents as a single copy, indicating a slightly better scaffolding quality. third, regarding dna-seq read mapping (figure b ), results are again very similar to ma_sr-lr_phylo. more detailed changes in read counts are given in table . there are more reads mapped on ma_sr-lr_union assembly than on *more detailed view of dna-seq consistency within scaffold assemblies after secondary scaffolding. the table focus on differences between union and union assemblies which differ in the minimum number of reads to support a linkage through mrna-seq. dna-seq consistency is expressed in terms of shortread alignment statistics. # "mapped" stands for the number of reads mapped. "properly paired" stands for the number of reads that are properly paired (right strand and insert size range). a singleton is a read with an unmapped mate. mapq corresponds to a pared quality score of a read alignment. ma_sr-lr_phylo assembly (+ reads), there are even more reads mapped properly in pairs (+ ). being slightly better than other assemblies, the ma_sr-lr_union was chosen to perform annotation and the identification of repeats, and for the final upload in ena. a total of , coding genes were annotated on ma_sr-lr_union scaffold assembly, as shown in table and compared to , for p. vampyrus annotation (ptevam /refseq ). therefore, in terms of annotated gene count, both are similar ( . k vs . k). more mrnas were annotated for p. medius ( k vs k), this type of difference being expected when different annotation methods are used. finally, as shown in table , genes including introns are covering . % of the genome and cds only (coding sequence) . %. although the majority of cdss are complete, some ( k out of k) miss either a start codon and/or a stop codon. this would require manual inspection for each cds to elucidate the cause of incompleteness; each non-complete cds might be the result of the gap presence (bench of ns), more problematically a mis-assembly or it may be an event of pseudogenization. using repeatmasker, . % of the genome was identified as already known interspersed repeats, discovered and annotated in another species and present in the repeat database (repbase), as shown table . to investigate genetic relationships between p. medius and the other bat species for which the complete genome sequences are available, we performed the phylogenetic analysis with published bat genomes with refseq proteomes available (figure ) . a total of , sco were found thanks to orthofinder. following multiple sequence alignment and postalignment analysis (described in the section "materials and methods"), the concatenated msa contained , , positions of which , were variable in at least one species accounting for , distinct alignment patterns. all bootstrap analyses ( %) were consistent with the topology of this tree. this analysis strongly supports that p. medius is phylogenetically closer to p. vampyrus than to p. alecto. our timescale, based on protein alignments, indicates a divergence time of . my ( . - . ) between both p. medius and p. vampyrus, and . ) between them and p. alecto. finally, as angiotensin converting enzyme (ace ) is considered to be the entry receptor for sars-cov- , the virus responsible for the current pandemic of covid- (zhou et al., ) , we compared the sequences of ace ( amino acids) between p. medius and two rhinolophus bat species, r. ferrumequinum and r. sinicus, thought to be a natural reservoir of coronaviruses (ge et al., ) . we observed . % of identity of p. medius with both *results from repeatmasker are summarized by repeat class and the repeat class below . % is masked but was taken into account for the total counts. rhinolophus bat species. the percentage of similitude (based on blossum matrix) of p. medius sequence is respectively of . and . % for r. ferrumequinum and r. sinicus (supplementary figure s ) . of note, the percentage of similitude between r. ferrumequinum and r. sinicus is . %. in this study, we sequenced and assembled a solid scaffold for the p. medius genome. we obtained ng of kb, on which we were able to annotate most of the genes as shown by busco analysis. in addition, we report here that the process of secondary scaffolding is beneficial in terms of assembly quality ( . x of nanopore long-reads and . x illumina paired-end) with sequencing cost that remains relatively moderate, making thus this approach rather attractive. although it would probably be insufficient to provide a resourceful assembly if closely related species have not been fully sequenced, we show in this study that it is possible to take advantages of published genomes from close species to provide a good quality assembly. in a context where international consortiums provide the scientific community with high quality genome sequences at chromosome level (genome k community of scientists, ; teeling et al., ) , for a genus without available genome sequences, there will be more resources available for reference-assisted scaffolding methods and for sequencing species of interest at lower cost. our results suggest that the combination of different sources of information for secondary scaffolding is necessary to limit the introduction of misassembles and improve quality; we thus developed the scaff link software, which is compatible and reusable with ragout and agouti. we have shown that the ma_sr-lr_union , produced by scaff link, is slightly better than other assemblies. indeed, scaff links prevents the linkage of fragments within a new scaffold if contradictory information exists between gene-based and synteny-based secondary scaffolding. this is expected to limit the number of scaffolds produced with mis-assemblies. finally, our results suggest the advantage of the utilization of the ma_sr-lr_union assembly for gene annotation and subsequent analysis of regulatory features including ngs applications, such as rna-seq. ma_sr-lr_union assembly could also be used to perform genome-wide comparative studies to identify or compare loci, including coding sequences or regulatory motifs. however, the fact that some contiguities in the genome are based on prediction (from secondary scaffolding), rather than on actual data might be problematic for some analysis. as example, for use as a reference genome for ragout, it would be preferable to use ma_sr-lr assembly. similarly to the previously sequenced e. spelaea bat genome (wen et al., ) , the main class of repeats present in p. medius genome are line (long-interspersed nuclear elements), covering ∼ % of the genome. however, the percentage of total genome coverage for interspersed repeats found in this study was lower than previous reports about closely related bat species (wen et al., ) . this difference is linked with the lower sensitivity of the tool used to search similarities between the genome and the repeat database (rmblast, used in this study). to our current knowledge there is no published work with significant statistical support allowing the resolution of phylogenetic relationship of p. medius with p. alecto and p. vampyrus. phylogenetic analysis shown the clustering of p. medius genome with two other pteropus bat species, p. alecto and p. vampyrus. the overall tree topology for bats is concordant with other studies (teeling et al., ; hawkins et al., ) . finally, the tree topology is coherent with the geographical distribution of those bats: p. medius (molur et al., ) and p. vampyrus , which clustered together, are both present on the asian continent. on the other hand, phylogenetically more distant species p. alecto is mainly located in australia (roberts et al., ) . we estimated in this study the divergence time between pteropus species lower than the minimum ranges reported on time tree database. however, some other studies reported divergence times focused on pteropus genus even lower than ours (almeida et al., ) ; in that study the % confidence interval for the divergence time between p. vampyrus and p. medius ( . - . ) overlaps with ours ( . - . ). we conducted our timescale estimation using figure | phylogenetic classification of p. medius among other bats and within pteropus species. the distance scale unit is mya (million years ago). all bootstrap values were %. horizontal blue bars around each node represents the % confidence interval relative to this node age. sco sequences, where the proportion of site under non-neutral selection may vary among branches. we acknowledge that time divergence estimations on branch with a greater proportion of sites under positive selection might be estimated higher than the real divergence time; inversely time divergence estimations on branches with a higher proportion of sites under negative selection might be estimated lower. indian p. medius bats have been suggested to host coronaviruses (anthony et al., ; yadav et al., ) . to underline the importance of having available an annotated p. medius genome, we analyzed the ace gene which is considered to code for the entry receptor for coronavirus sars-cov- , responsible for the current pandemic of covid- (zhou et al., ) . comparison of the sequences of ace between p. medius and r. sinicus, thought to be a natural reservoir of coronaviruses (ge et al., ) revealed . % of identity and . - . % of similarity (supplementary figure s ) . although this is a preliminary result, it emphasizes the interest of having ready-to-use genomic resources for a maximum of species, for the further in-depth analysis. altogether, these results confirm that a lower depth of sequencing is enough to obtain reliable genome sequence using secondary scaffolding approaches and demonstrate the benefits of the scaff link application, described in this article. the genome sequence is now available to the scientific. in addition, as bats display many unique biological features among mammals (racey, ) , growing number of new bat species are expected to be sequenced, some of them within the bat k project for sequencing all bats' species (teeling et al., ) . a meta-analysis of bat phylogenetic and positive selection recently suggested a number of genes known to be primarily related to immune responses (hawkins et al., ) and their further functional analysis will allow the understanding of their role in hosting different viruses by bats. increasing the availability of different bat genomes is indeed essential for a better understanding of the genetic and evolutionary mechanisms that underlie the adaptations specifically to bats, such as their ability to fly which is unique among mammals, their metabolic adaptation, as well as the immuno-virological peculiarities associated to their capacity to both host and transmit nipah virus as well as the other viruses highly pathogenic to humans. the datasets generated for this study can be found in the european nucleotide archive (ena), n • prjeb , erp . some files important for the reproducibility of this work are available at: https://github.com/jfouret/pmed_ genomedata. ethical approval was not required for this study according to local regulations as tests were performed in the course of regular health checks by veterinarians of the vienna zoo. jf, bh, cl-l, and mb-b conceived, designed, and supervised the study. dp performed the sampling of biological material. jf, na, and fe performed the experimental processing of the samples. sc and mg performed, respectively, minion and illumina sequencing with inputs from jf for minion sequencing. jf led the software development and performed the sequence analysis, genome assembly, and gene annotation. mb contributed significantly for genome assembly, evaluation, and genome annotation. ag, jf, and jl brought methodological inputs. fb, jf, and j-nv performed the phylogenic analysis. jf wrote the initial manuscript. fb, ag, bh, cl-l, and jl did the proofreading. all authors read and approved the final manuscript. each flying fox on its own branch: a phylogenetic tree for pteropus and related genera (chiroptera: pteropodidae) a strategy to estimate unknown viral diversity in mammals outbreak investigation of nipah virus disease in kerala, india reprogrammed pteropus bat stem cells present distinct immune signature and are highly permissive for henipaviruses pteropus vampyrus. iucn red list threat. spec. :e.t a informed and automated k-mer size selection for genome assembly assembly of the complete sitka spruce chloroplast genome using x genomics' gemcode sequencing data nuclear introns outperform mitochondrial dna in inter-specific phylogenetic reconstruction: lessons from horseshoe bats (rhinolophidae: chiroptera) orthofinder: phylogenetic orthology inference for comparative genomics understanding the interaction between henipaviruses and their natural host, fruit bats: paving the way toward control of highly lethal infection in humans high pathogenicity of nipah virus from pteropus lylei fruit bats isolation and characterization of a bat sars-like coronavirus that uses the ace receptor genome k: a proposal to obtain whole-genome sequence for vertebrate species integrating hi-c links with assembly graphs for chromosome-scale assembly a metaanalysis of bat phylogenetics and positive selection based on genomes and transcriptomes from species bat origin of human coronaviruses improving and correcting the contiguity of long-read genome assemblies of three plant species using optical mapping and chromosome conformation capture data mafft: a novel method for rapid multiple sequence alignment based on fast fourier transform chromosome assembly of large and complex genomes using multiple references ragout-a referenceassisted assembly tool for bacterial genomes timetree: a resource for timelines, timetrees, and divergence times hibernation in bats (mammalia: chiroptera) did not evolve through positive selection of leptin a high-resolution map of human evolutionary constraint using mammals henipavirus pathogenesis and antiviral approaches correct name for the indian flying fox (pteropodidae) database resources of the national center for biotechnology information cactus: algorithms for genome multiple sequence alignment the egyptian rousette genome reveals unexpected features of bat antiviral immunity the uniqueness of bats pteropus alecto. the iucn red list of threatened species structural elements highly preserved during the evolution of the d-loop-containing region in vertebrate mitochondrial dna bat and virus the genome sizes of megabats (chiroptera: pteropodidae) are remarkably constrained raxml version : a tool for phylogenetic analysis and post-analysis of large phylogenies gene prediction with a hidden markov model and a new intron submodel improvement of phylogenies after removing divergent and ambiguously aligned blocks from protein sequence alignments a molecular phylogeny for bats illuminates biogeography and the fossil record bat biology, genomes, and the bat k project: to generate chromosome-level genomes for all living bat species viruses in bats and potential spillover to animals and humans exploring the genome and transcriptome of the cave nectar bat eonycteris spelaea with pacbio long-read sequencing nipah virus sequences from humans and bats during nipah outbreak detection of coronaviruses in pteropus & rousettus species of bats from different states of india paml : phylogenetic analysis by maximum likelihood comparative analysis of bat genomes provides insight into the evolution of flight and immunity agouti: improving genome assembly and annotation using transcriptome data a pneumonia outbreak associated with a new coronavirus of probable bat origin the masurca genome assembler the work was supported by labex ecofect (anr- -labx- ) at "université de lyon, " within the program "investissements d'avenir" (anr- -idex- ) operated by the french national research agency (anr), by aviesan sino-french agreement on nipah virus study and viroscan d. jf was supported by the doctoral fellowship cifre-défense operated by the direction générale de l'armement (dga). we wish to thank a. weissenbacher and tiergarten schönbrunn (vienna, austria) for the pteropus bat samples. we are also grateful to drs. k. dhondt and m. roche (lyon) for the help to initialize and realize this work; to e. guillot-combe for the tutorship during the jf ph.d. thesis work; to a. raout (bhopal, india) for sharing sequences from p. medius nuclear introns; and to n. lartillot (lbbe, lyon) and a. sadier (ucla) for advice with our phylogenomic analysis. the supplementary material for this article can be found online at: https://www.frontiersin.org/articles/ . /fmicb. . /full#supplementary-material key: cord- -fmobf f authors: sekizuka, tsuyoshi; kuramoto, sanae; nariai, eri; taira, masakatsu; hachisu, yushi; tokaji, akihiko; shinohara, michiyo; kishimoto, tsuyoshi; itokawa, kentaro; kobayashi, yusuke; kadokura, keisuke; kamiya, hajime; matsui, tamano; suzuki, motoi; kuroda, makoto title: sars-cov- genome analysis of japanese travelers in nile river cruise date: - - journal: front microbiol doi: . /fmicb. . sha: doc_id: cord_uid: fmobf f japan has reported cases of coronavirus disease (covid- ) linked to cruise tours on the river nile in egypt between march and , . here, we characterized the severe acute respiratory syndrome coronavirus (sars-cov- ) genome of isolates from travelers who returned from egypt and from patients possibly associated with these travelers. we performed haplotype network analysis of sars-cov- isolates using genome-wide single-nucleotide variations. our analysis identified two potential egypt-related clusters from these imported cases, and these clusters were related to globally detected viruses in different countries. the current pandemic of coronavirus disease (covid- ) is caused by a positive-sense rna virus, named the severe acute respiratory syndrome coronavirus (sars-cov- ) (coronaviridae study group of the international committee on taxonomy of v., ). as of april , , japan has confirmed , cases in total, excluding the cases in diamond princess cruise ship. this makes japan one of the developed countries least affected by sars-cov- . the japanese government has focused on the identification and mitigation of emerging covid- clusters before further expansion, a strategy considered optimal in a low infection rate situation. japan has sustained moderate spread by focusing on covid- outbreak clusters; however, an ever-increasing number of covid- cases has made it difficult to identify all infection routes. from the beginning of march , travelers who returned to japan from abroad were suspected to have imported covid- ; these cases account for roughly % of all new cases recorded in japan. among these imported cases, as many as have been linked to cruise tours on the river nile in egypt between march and , . most of the travelers visited egypt from late february to early march and embarked on nile river cruise ship tours between cairo and luxor for - days. soon after they returned to japan, they experienced the onset of fever and sore throat. they visited their respective local consultation centers for recent arrivals from abroad and underwent pcr testing, which confirmed them as being positive for sars-cov- . a field figure | summary of travel history, clinical course, and pcr testing for sars-cov- -positive travelers who returned to japan from egypt, as well as the associated patients who were their close contacts. epidemiological study was conducted on the people closely associated with them, such as family members, who might have been exposed to the virus. in this study, we have evaluated viral genome sequences from sars-cov- -positive travelers who returned from egypt, and characterized the haplotype networks to demonstrate possible routes of the spread. we evaluated viral genome sequences from sars-cov- positive travelers who returned from egypt, as well as their close contacts, to identify possible routes of spread. the travel histories, clinical courses, and pcr testing results are summarized in figure . to characterize the potential origins and routes of the suspected imported cases, we determined the whole-genome sequence of sars-cov- using a multiplex pcr-based rna-seq by artic network protocol based on primalseq (quick et al., ; grubaugh et al., ) with modified primers and protocol (itokawa et al., ) . for the obtained genome sequences, haplotype network analysis using genome-wide single-nucleotide variations (snvs) on the core regions from positions to , nt in the wuhan-hu- reference genome sequence (gisaid id, epi_isl_ ; genbank id, mn . ) was performed (figure ) . sars-cov- genome sequences with nearly fulllength information (≥ kb) were retrieved from the gisaid epicov database on march , , and we generated haplotype networks by median-joining network analysis using popart software to highlight and trace a potential infectious route among covid- patient populations. patient p (p ; hereafter, patients are designated in this manner) arrived at cairo airport from tokyo on february and embarked on a nile river cruise ship for days (figure ) . the sars-cov- genome sequence of the p isolate shows a close lineage with european isolates, with several snvs (figure ). p - and p - had visited egypt together and traveled aboard the same nile river cruise ship, and sars-cov- genome sequences isolated from them are identical with that of p (figure ) . the couple p - and p - visited egypt together, on the same tokyo to cairo flight as the above p patient but boarded a different nile river cruise ship. the husband, p - , showed flu-like symptoms on march and was confirmed with covid- on march , while the wife, p - , was asymptomatic and pcr negative on march despite their close contact during the trip. days later, however, on march , p - exhibited symptoms and was confirmed with covid- . these two sars-cov- isolates show identical genome sequence (figure ) and are distinct from the genome sequences (p , p - , and p - ) by only one snv (figure ) . meanwhile, compared to the genome sequences of the above five patients, the sars-cov- genome sequence obtained from following p and p patients showed clearly different haplotype lineage, with at least five differential snvs (figure ) . four patients (p - to p - ) had visited egypt at the end of february, a few days after the above-mentioned patients, and exhibited symptoms after returning back to japan. intriguingly, the genome sequences of four additional patients (p - -p - ), having no history of recent overseas travel or contact with the above patients, were markedly close to the p -related isolates. p - and p - are coworkers with p - , and p - is mother of p - , indicating that three patients (p - -p - ) were close contacts to p - as original source. this finding demonstrated the identification of a potential hidden link to the import of infection from egypt. table s ) by median-joining snv network analysis. the numbers on the edges indicate differential snvs between pair-wise nodes (isolates). sars-cov- disseminated from the end of december, , from wuhan city in china, one of the potential origins of wuhan-hu- , isolated on december , (genbank id: mn ). wuhan-hu- is plotted at the center of the haplotype network. currently, at least three clades have disseminated globally in a region-specific manner. p - had visited egypt and embarked on a nile river cruise ship different from the other travelers mentioned above. he showed symptoms and tested positive by pcr after returning home. his relatives, p - (daughter) and p - (sister), who had close contact with him, subsequently showed symptoms and tested positive for sars-cov- by pcr (figure ) . the sars-cov- genome sequence of p - was identical to that of p - , distinct only by one snv from that of p - , indicating direct infections from p - to p - and p - , (figure ) . thus far, two genome sequences of sars-cov- isolates in egypt (isolation date: / / ; gisaid id: epi_isl_ and epi_isl ) are available in gisaid, and the haplotype figure | an excerpt of hn-gsnvs from japanese travelers returning from egypt and patients associated with them. the haplotype of sars-cov- genome sequences for patients (figure ) was found in the two marked clusters, which comprised the most european isolates (figure ) . see the legends in figure for details. network exhibits that p and p patients are closely related to those egypt isolates with or snvs (figure ). in this study, we found two sars-cov- genome lineages from egypt-related imported cases. these virus lineages belonged to a single clade rooted to the major indexed isolates, which diverged from wuhan-hu- by several clades. the members in this clade are considered to be circulating in multiple countries, mainly in the europe and south america. the egypt-related isolates described in this study are divided into two distinct sars-cov- haplotype lineages, with two or three additional snvs from the major indexed isolates; one lineage, including p -p , included most haplotypes isolated from france and egypt (figure ) , whereas the other lineage, including p and p , included the netherlands/belgium/ switzerland isolates. on march, the egyptian health ministry confirmed covid- cases among the egyptian crew staff aboard a nile river cruise ship. on march, the egyptian health authorities announced that people on board that ship had tested positive, and that the ship had been subjected to quarantine at a dock in luxor. it is also speculated that egypt probably has a large burden of covid- cases that are unreported, and egypt might be a source of covid- export that is not yet accounted for by many public health initiatives (tuite et al., ) . since early march this year, the number of reported covid- cases has been rapidly increasing in europe countries. this study suggested that patients with a history of travel to egypt and embarking on nile river cruises between mid-february and early march could be one of the potential sources of covid- cases imported into japan. pharyngeal specimens were collected from patients, and a quantitative reverse transcription pcr (rt-qpcr) testing for sars-cov- (jung et al., ; shirato et al., ) was performed. basically, whole genome sequences of sars-cov- was obtained by primalseq protocol to enrich cdna of sars-cov- genome by multiplex rt-pcr amplicons using a multiplexed pcr primer set which was proposed by wellcome trust artic network. we found particular two amplicons regularly showed low to zero coverage due to primer dimerization as described in itokawa et al. ( ) , we used the modified primer for the multiplex pcr amplifications (itokawa et al., ) . the pcr products from same clinical sample was pooled, purified and subjected for illumina library construction using qiaseq fx dna library kit (qiagen, hilden germany). nextseq platform (illumina, san diego, usa) was used for sequencing the indexed libraries. the ngs reads were mapped to the sars-cov- wuhan-hu- reference genome sequence ( . kb ss-rna; genbank id: mn ), resulting to the specimen-specific sars-cov- genome sequence by fully mapping on the reference. these mapped reads of sars-cov- sequences were assembled using a -miseq v. (coil et al., ) to determine the full genome sequence (see the details in table s ). the snv sites and marked heterogeneity were extracted by the read-mapping at ≥ × depth and from to , nt region of wuhan-hu- genome sequence (see the details in table s ). the nearly full-length genome sequence (≥ kb) of sars-cov- were retrieved from gisaid epicov database in march , , followed by multiple alignment using mafft v . (katoh and standley, ) . the poorly aligned regions in ′ and ′ end were trimmed; we determined that the core regions were from to , nt position against wuhan-hu- genome sequences (gisaid id, epi_isl_ ; genbank id, mn . ). gap-containing sequences in the core region were excluded; sequences of , isolates in gisaid database were eventually used in subsequent analyses (updated on march , . see table s ). the genome sequences were aligned using mafft program together with sequences retrieved from database, followed by extraction of snv and deletion sites. the snv median-joining network analysis was performed by popart software . the new sequences have been deposited in gisaid with accession ids epi_isl_ -epi_isl_ , in addition, in ddbj/ncbi/ebi with accession ids lc -lc . the studies involving human participants were reviewed and approved by the national institute of infectious diseases in japan (approval no. ). it was conducted according to the principles of the declaration of helsinki, in compliance with the law concerning the prevention of infections and medical care for patients of infections of japan. the ethical committee waived the need for written consent regarding the research into the viral genome sequence. the personal data related to the clinical information were anonymized, and our procedure is not to request written consent for all patients suffering from covid- . written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements. ts, ki, and mk designed and organized the genome study. sk, en, mt, yh, at, msh, and tk performed the laboratory detection. ts and ki performed the genome analysis. yk, kk, hk, tm, and msu contributed to the field epidemiological study. mk wrote the manuscript. this study was supported by a grant-in aid from the japan agency for medical research and development (amed) under grant number jp fk and jp fk . the funding agencies had no role in the study design, data collection or analysis, decision to publish, or manuscript preparation. a -miseq: an updated pipeline to assemble microbial genomes from illumina miseq data the species severe acute respiratory syndrome-related coronavirus: classifying -ncov and naming it sars-cov- an amplicon-based sequencing framework for accurately measuring intrahost virus diversity using primalseq and ivar a proposal of an alternative primer for the artic network's multiplex pcr to improve coverage of sars-cov- genome sequencing comparative analysis of primer-probe sets for the laboratory confirmation of sars-cov- . biorxiv mafft multiple sequence alignment software version : improvements in performance and usability multiplex pcr method for minion and illumina sequencing of zika and other virus genomes directly from clinical samples development of genetic diagnostic methods for novel coronavirus (ncov- ) in japan estimation of the covid- burden in egypt through exported case detection we are deeply thankful to the staff of the local public health centers in the prefectures of kochi, ishikawa, chiba, and saitama for the field epidemiological study and for preserving and providing the clinical specimens. we thank the field epidemiology training program (fetp) team; the ministry of health, labor and welfare; and local governments for their assistance with administrative matters, field investigation, data collection, and laboratory testing. we would like to thank all the authors who have kindly deposited and shared genome data on gisaid. a table with genome sequence acknowledgments can be found in table s . the supplementary material for this article can be found online at: https://www.frontiersin.org/articles/ . /fmicb. . /full#supplementary-material key: cord- -purplsjn authors: fernández-ponce, cecilia; durán-ruiz, maria c.; narbona-sánchez, isaac; muñoz-miranda, juan p.; arbulo-echevarria, mikel m.; serna-sanz, antonio; baumann, christian; litrán, rocío; aguado, enrique; bloch, wilhelm; garcía-cozar, francisco title: ultrastructural localization and molecular associations of hcv capsid protein in jurkat t cells date: - - journal: front microbiol doi: . /fmicb. . sha: doc_id: cord_uid: purplsjn hepatitis c virus core protein is a highly basic viral protein that multimerizes with itself to form the viral capsid. when expressed in cd (+) t lymphocytes, it can induce modifications in several essential cellular and biological networks. to shed light on the mechanisms underlying the alterations caused by the viral protein, we have analyzed hcv-core subcellular localization and its associations with host proteins in jurkat t cells. in order to investigate the intracellular localization of hepatitis c virus core protein, we have used a lentiviral system to transduce jurkat t cells and subsequently localize the protein using immunoelectron microscopy techniques. we found that in jurkat t cells, hepatitis c virus core protein mostly localizes in the nucleus and specifically in the nucleolus. in addition, we performed pull-down assays combined with mass spectrometry analysis, to identify proteins that associate with hepatitis c virus core in jurkat t cells. we found proteins such as nolc , pp γ, ilf , and c qbp implicated in localization and/or traffic to the nucleolus. hcv-core associated proteins are implicated in rna processing and rna virus infection as well as in functions previously shown to be altered in hepatitis c virus core expressing cd (+) t cells, such as cell cycle delay, decreased proliferation, and induction of a regulatory phenotype. thus, in the current work, we show the ultrastructural localization of hepatitis c virus core and the first profile of hcv core associated proteins in t cells, and we discuss the functions and interconnections of these proteins in molecular networks where relevant biological modifications have been described upon the expression of hepatitis c virus core protein. thereby, the current work constitutes a necessary step toward understanding the mechanisms underlying hcv core mediated alterations that had been described in relevant biological processes in cd (+) t cells. hepatitis c virus core protein is a highly basic viral protein that multimerizes with itself to form the viral capsid. when expressed in cd + t lymphocytes, it can induce modifications in several essential cellular and biological networks. to shed light on the mechanisms underlying the alterations caused by the viral protein, we have analyzed hcv-core subcellular localization and its associations with host proteins in jurkat t cells. in order to investigate the intracellular localization of hepatitis c virus core protein, we have used a lentiviral system to transduce jurkat t cells and subsequently localize the protein using immunoelectron microscopy techniques. we found that in jurkat t cells, hepatitis c virus core protein mostly localizes in the nucleus and specifically in the nucleolus. in addition, we performed pull-down assays combined with mass spectrometry analysis, to identify proteins that associate with hepatitis c virus core in jurkat t cells. we found proteins such as nolc , pp γ, ilf , and c qbp implicated in localization and/or traffic to the nucleolus. hcv-core associated proteins are implicated in rna processing and rna virus infection as well as in functions previously shown to be altered in hepatitis c virus core expressing cd + t cells, such as cell cycle delay, decreased proliferation, and induction of a regulatory phenotype. thus, in the current work, we show the ultrastructural localization of hepatitis c virus core and the first profile of hcv core associated proteins in t cells, and we discuss the functions and interconnections of these proteins in molecular networks where relevant biological modifications have been described upon the expression of hepatitis c virus core protein. thereby, the current work constitutes a necessary step toward understanding the mechanisms underlying hcv core mediated alterations that had been described in relevant biological processes in cd + t cells. keywords: hepatitis c virus, immune evasion, proteomics, interactome, ultrastructure, regulatory t cells, immune tolerance introduction hepatitis c virus (hcv) infection is an important cause for chronic viral liver disease and one of the main indications for liver transplantation (anzola, ; dustin and rice, ) . hcv affects million people worldwide and in more than % of the patients leads to chronicity (gower et al., ) . the high level of chronicity and the absence of a protective vaccine, makes hcv infection a significant public health problem (anzola, ; dustin and rice, ; gower et al., ) . the molecular mechanisms harnessed by hcv to establish a chronic infection and their implications in the innate and adaptive immune systems have not been fully elucidated. in this regard, several hcv viral proteins have been described as modulators of immunological phenomena (yao et al., ; krishnadas et al., ; tu et al., ; chen et al., ) . among them, hcv core protein has been widely associated with pathogenicity, virulence, immune evasion and immune regulation (dominguez-villar et al., , a waggoner et al., ; tu et al., ; doumba et al., ; fernandez-ponce et al., , . however, the underlying molecular processes, as well as the behavior of hcv core protein or its interactions with host cell components, remain unclear. hcv core is a highly basic protein, which binds and protects the viral rna, multimerizing with itself to form the viral capsid (santolini et al., ) . in mammalian infected cells, hcv core interacts with endoplasmic reticulum membranes, lipid droplets and other cellular and viral proteins to promote assembly of new virions. however, several inhibitory molecules and the lack of some host factors can hamper viral production by disrupting the core multimerization process and the coordinated interactions with other viral proteins (mousseau et al., ; gawlik and gallay, ) . in this way, non-enveloped hcv core proteins can be directed to alternative subcellular locations and also be released into the extracellular space (maillard et al., ; polyak et al., ; tan et al., ) . according to these findings, in hcv chronically infected patients, hcv core protein, has been detected as non-enveloped isolated nucleocapsids, not only in hepatocytes (falcon et al., ) , but also in serum (maillard et al., ) , peripheral blood cd + t cells (fernandez-ponce et al., ) and other nonparenchymal liver cells, such as, lymphocyte, pit, endothelial, stellate, kupffer and polymorphonuclear cells (falcon et al., ) . in t cell lines, non-enveloped isolated nucleocapsids binding and internalization has also been described in vitro (doumba et al., ) . these data further support the presence of hcv core protein inside immune cells, including lymphocytes, during hcv chronic infection. interestingly, hcv core protein intracellular expression in cd + t lymphocytes has been shown to induce modifications in cell proliferation, cell cycle progression, expression of anergy genes, transcription of genes involved in cytoskeleton reorganization, vesicle trafficking, endocytosis, transcription and translation, cytokine production, cell death and generation of a t cell regulatory phenotype with exhausted features (bergqvist and rice, ; bergqvist et al., ; dominguez-villar et al., , a doumba et al., ; fernandez-ponce et al., ) , characterized by an increased expression of foxp (forkhead box p ) and ctla- (cytotoxic t-lymphocyte antigen- ) (dominguez-villar et al., a) , high levels of il- secretion, and decreased il- and ifn-γ production (doumba et al., ; fernandez-ponce et al., ) . it has been described for several viruses that the specific subcellular localization of viral proteins and their interactions with host molecules can alter the spatial distribution and organization of cellular proteins, and in this way, induce diverse molecular and cellular effects (chen et al., ; yoo et al., ; ning and shih, ; bertrand and pearson, ; ponti et al., ; hiscox et al., ; zhu et al., ; raval et al., ) . as studies using the whole virus do not allow for the elucidation of the specific molecular mechanisms in which each protein is implicated, in this work, we focused on a single viral protein, showing that in cd + t cells, hcv core protein mostly localizes in the nucleus and specifically in the nucleolus where it is greatly enriched. in addition, we performed pull down assays, combined with mass spectrometry analysis, in order to identify host proteins associated with hcv core, which could be implicated in the functional effect previously observed to be induced by the presence of hcv-core in t cells. we found several proteins implicated in important functions that are associated with hcv core protein. thereby, our results shed light on the molecular mechanisms underlying the alterations in biological cell processes and the generation of adaptive regulatory-like cd + t cells in the periphery by the intracellular presence of a single hcv viral protein. human embryonic kidney (hek) lenti-x tm t cell line (clontech) and jurkat cell line (american type culture collection, manassas, va, usa) were maintained in dulbecco's modified eagle's medium (dmem tm ) supplemented with % (v/v) heat inactivated fetal bovine serum (fbs), mm l-glutamine, mm hepes, % (v/v) sodium pyruvate, µm -mercaptoethanol, u/ml penicillin and µg/ml streptomycin at • c, % co . human peripheral blood samples were obtained from healthy donors upon signature of an informed consent and following approval by the ethic sub-commission of the puerta del mar university hospital (dependent from the central quality commission), in accordance to spanish and european union regulations. peripheral blood mononuclear cells (pbmcs) were isolated by density gradient centrifugation using lymphocyte separation medium (eurobiotm, montpellier, france). cells were washed three times with pbs, subsequently stimulated with mg/ml phytohemagglutinin-p (pha) (sigmatm, saint louis, missouri, usa) and cultured in dmem supplemented with % (v/v) sodium pyruvate, non-essential aminoacids, vitamins, larginin, l-asparragin, folic acid, mm hepes, mm -mercaptoethanol, mg/ml streptomycin, u/ml penicillin (life technologies, carlsbad, ca, usa) and % heat-inactivated fbs (gibco) at • c, in a % co atmosphere. u/ml il- was added to the cultures every h, for a total of days to obtain blasts. hek lenti-x tm t cells (clontech) were used as packaging cell lines to produce lentiviral supernatant by co-transfecting plasmids pcmvdr . , coding for hiv- gag and pol proteins and pmd .g for the vesicular stomatitis virus g protein (vsvg) together with the transfer vector phrsincppt-sewhcv-core-gfp (hcv-core) coding for the first amino acids of the hcv polyprotein (serotype a) corresponding to hcv-core protein. transfer vector phrsincppt-sewgfp expressing only gfp (green fluorescent protein) was used as a control (dominguez-villar et al., ) . cells were transfected in optimem tm medium using cms diameter cell-culture dishes coated with collagen (collagen i, rat tail gibco r invitrogen cell culture), and polyethylenimine (pei), branched (sigma-aldrich) as cationic polymer. transfection efficiency was evaluated by facs analysis. supernatants were collected at and h, centrifuged to remove cells and debris and concentrated using lenti-x tm concentrator (clontech r ) to obtain a high-titer virus-containing pellet. pellets were frozen at − • c and stored until use. viral titer in supernatants was determined evaluating their efficiency in infecting jurkat cells. jurkat cells were transduced using hcvcore-gfp and gfp (as control) lentiviral supernatants, previously prepared, at a multiplicity of infection (moi) of . aliquots from the cultures were collected after h to determine the transduction efficiency. gfp expression was monitored by facs (cyanadp-mle tm ; dakocytomation tm ) presenting a transduction range > % in all experiments. untransduced, hcvcore-gfp and gfp transduced jurkat cells were collected h post-transduction, counted, washed with phosphate buffer saline (pbs) and fixed for h using % paraformaldehyde (pfa) in pbs, ph . . subsequently, cell pellets were pre-embedded in agarose x, taken out of the tube with a needle and sliced into approximately mm pieces. pre-embedding immunogold was performed. agarose pieces were washed in pbs, permeabilized using . % triton x− in pbs and incubated in aurion blocking solution containing normal goat serum (aurion r . ), for min at • c. sections were stained immunochemically with anti-gfp antibody (abcam ab ) diluted : , in pbs containing . % bovine serum albumin (bsa) at • c, overnight, and subsequently washed with the same buffer. for gold particle staining, sections were incubated with nm gold-labeled anti-rabbit igg (sigma-aldrich) diluted : in . % bsa/pbs at • c, overnight. agarose pieces were washed in . m caco-buffer ph . at room temperature. sections were post-fixed in . % osmium tetroxide and . m caco-buffer at room temperature, for h and washed with . m caco-buffer. fixed sections were dehydrated by sequential incubation in ethanol - % and propylene oxide, embedded in epoxy resin and polymerized at • c for h. semi ( nm) and ultra-thin ( - nm) sections, were obtained using a leica em uc ultramicrotome. uultra-thin sections were collected onto plastic-coated nickel grids and immunochemically contraststained with uranium salts (uranyl acetate) and lead citrate to reveal cell ultrastructure. finally, samples were analyzed using a transmission electron microscope em a fa.zeiss with the item soft imaging system software (olympus). untransduced jurkat cells and pbmcs cultured as was previously described, were collected, counted, washed with pbs and cells were incubated min with µl lysis buffer ( mm tris-hcl ph . , mm nacl, % nonidetp- , mm edta, mm phenylmethylsulfonyl fluoride, mm na vo , mm naf, on ice. cell debris was discarded by centrifugation at , g for min, • c, and soluble proteins were stored at − • c before further analysis. protein levels were measured using bradford protein assay kit (pierce), according to manufactures recommendations. two hundred microliter magnetic beads (dynabeads r myone tm streptavidin t invitrogen) were washed following manufacturer's recommendations and conjugated with hcv core genotype- b biotin-prospec (hcv- ) by incubating µl ( mg/ml) dynabeads with µl ( pmoles) hcv core protein in pbs, for h at • c, with gentle rotation. hcv core protein-coated beads were washed three times by resuspension in pbs containing . % twin- and decanting the supernatant while placed on a magnet. for mass spectrometry analysis, coated dynabeads (or uncoated as control for unspecific binding) were incubated overnight, at • c, with jurkat cells protein lysates, with gentle rotation, in the following ratio: µl of coated beads with µg of total protein lysate. for western blot experiments, coated dynabeads were incubated overnight, at • c, with protein lysates from pbmc blasts, with gentle rotation, in the following ratio: µl coated beads with mg protein sample. µl uncoated beads were incubated with mg protein lysate, in the same conditions, as a control of unspecific binding. samples were washed twice with pbs and once with ultrapure water and incubated with µl of mm dithiothreitol (dtt) in mm ammonium bicarbonate for min at • c. eluated proteins were quantified and µg were precipitated with acetone during h at − • c and centrifuged min at , g. subsequently, proteins were resuspended in urea m, reduced with mm dtt in mm ammonium bicarbonate at room temperature for min and alquilated with mm iodacetamide min, in the dark, subsequently trypsin digestion was performed with sequencing grade trypsin (promega, madison, wi, usa) at • c overnight (enzyme/substrate ratio : ). finally, the digestion was quenched adding % formic acid. the resulting peptides were transferred to a clean tube, dried in speed-vac and stored at − • c prior to analysis by mass spectrometry (ms). peptides were resolved by reverse phase chromatography using an eksigent ultra pump fitted with a µm id column cm length (acclaim pepmap c µm a). samples were initially loaded for desalting into a cm length µm id precolumn packed with the same chemistry as the resolving column prior to analytical chromatography. mobile phases used were % water . % formic acid (buffer a), % acetonitrile . % formic acid (buffer b). gradient was developed during min up to % b. column was equilibrated in % b for min and % b for min, using a constant flow of nl/min. peptides eluted from the column were analyzed using a sciex tripletof tm system. data dependent acquisition was collected upon a survey scan performed in a mass range from to , m/z in a scan time of ms. the top most intense precursors were selected for fragmentation with a transmission window width of . da. minimum accumulation time for ms/ms was set to ms giving a total cycle time of , ms. fragment ions were collected in a mass range from to , m/z in order to have a single q transmission window. precursors were excluded from further fragmentation during s. raw data files were processed using proteinpilot tm . software from sciex and mgf files were loaded onto proteinscape tm . software (bruker) for data grouping and protein identification via mascot program version . (matrix science ltd, london, uk). search parameters were: homo sapiens taxonomy. trypsin specificity was used and only one missed cleavage allowed, cysteine carbamidomethylation as fixed and oxidized methionine was used as variable modification. mass tolerance of precursor ion and fragment ions were ppm and . da, respectively. peptides charges of +, +, and + were selected and minimum peptide length was set at five residues. peptides and proteins were considered positively identified if their mascot score was higher than and , respectively. proteins were identified with the minimum of one peptide and a false discovery rate < %. proteins identified with one single peptide were only included if peptide scores were significant, and peptide spectra contained most "y" and/or "b" representative ions for the corresponding peptide sequence. examples are included in supplementary table . subsequently, we compared the identified proteins that bound to the dynabeads vs. the ones identified binding to hcv core protein using the "compare results" option from proteinscape software . . thus, proteins were found to specifically associate with hcv core protein. proteome profiling data has been deposited in the peptideatlas repository, identified as pass . samples obtained from the pull down assay were washed twice with pbs using the magnet, and incubated with x laemmli buffer at • c for min. subsequently, beads were removed from the proteins using a magnet and proteins resolved by sds-page and transferred to pvdf membranes (immobilon-fl). membranes were incubated with the corresponding primary antibody, followed by the appropriated secondary antibody, conjugated to hrp. primary antibodies used were pp gamma antibody (pa - ) from thermo fisher scientific r and mypt antibody (# ) from cell signaling technology r . reactive proteins were visualized using an ecl system and images were acquired in a chemidoc touch imaging system (bio-rad r laboratories, hercules, ca, usa). localization and network analysis of identified candidate proteins associated with hcv core protein string database version (search tool for the retrieval of interacting genes/proteins) (http://string-db.org) (szklarczyk et al., ) was applied to visualize detailed information about the subcellular localization and the main interactions of the identified proteins. ingenuity pathways analysis (ipa; ingenuity systems, redwood city, calif.) software was used for further protein characterization according to known and predicted associations into function canonical pathways and networks recorded in the ipa library. nuclear localization sequence (nolss) in hcv core protein prediction was obtained by entering a protein sequence in fasta format on the nod web server http://www.compbio.dundee.ac. uk/www-nod/ (scott et al., ) . nolss were predicted if the average score output by the artificial neural network ann of consecutive windows was at least . (scott et al., (scott et al., , . to analyze the localization of hcv core protein in t cells and to circumvent the lack of optimal anti-hcv core antibodies, we took advantage of a gfp-hcv core fusion construct that has been extensively used in our laboratory (dominguez-villar et al., , a fernandez-ponce et al., ) and an unfused gfp expressing construct as a control. jurkat cells were efficiently transduced with lentiviral vectors expressing hcvcore-gfp or gfp as a control. percentage of transduced cells analyzed by flow cytometry was > % in all cases (data not shown). jurkat cells transduced with gfp or hcv core gfp-expressing lentiviral construct or left untransduced were subsequently immunostained with anti-gfp antibody and a nm gold-labeled anti-rabbit igg and analyzed by transmission electron microscopy. as shown in figure , hcv core was mostly localized in the nucleus (figures b,d,e) and specifically in the nucleolus where it was greatly enriched (figures b-f) , although some immunostaining was observed in cytoplasm ( figure a) . hcv-core-gfp was detected in the nucleus in % out of hcv core gfp expressing jurkat cells analyzed, % showed the presence of gfp inside both nucleus and cytoplasm, % of the cells only in cytoplasm (figure a) , while % showed gfp immunolabelling in nucleus with enrichment in the nucleolus (figures b-f) . % of the cells were not stained. in hcv core expressing jurkat cells which showed core protein nucleolar localization, the total raw number of gold particles counted inside the nucleoli was , with an average of gold particles per cell and a standard deviation of . . regarding gfp transduced control cells, gfp was localized in the nucleus of % from gfp-expressing jurkat cells analyzed and in both nucleus and cytoplasm in %. there was no recognizable co-localizing with any organelle. nucleolus localization was not visible in any cell (figures g,h) . no immunogold staining was observed in untransduced jurkat cells (data not shown). thus, the study revealed specific immunolabeling of hcv core protein in the nucleolus of jurkat cells. based on the findings obtained with the electron microscopy assay, we decided to identify hcv core associated proteins that could mechanistically be correlated with hcv core protein nucleolar localization. thus, we performed pull down experiments using biotinilated hcv core protein as bait. further analysis with the string platform allowed us to classify the identified proteins depending on their localization (figure ) . thus, from the associated proteins identified (supplementary table ), have been previously described to localize in the nucleus (figure a) and in the nucleolus (figure b) , while most of the proteins described in the cytoplasm (figure c ) have also been identified in the nucleus and/or nucleolus. string also allowed us to determine potential protein functionalities according to previously published reports. interestingly, most of the identified hcv core associated proteins were found to participate in binding processes, as indicated in figure . regarding protein function, we mainly focused on the functional classification given by the ipa software, which, based on biomedical literature and integrated databases, allows to determine the most probable pathways and/or functions in which identified proteins are involved. thus, ipa core analysis of our dataset (figure ) revealed that the identified hcv core associated proteins were mainly described to participate in splicing and processing of rna, cell cycle progression, cell proliferation, apoptosis and rna virus infection. our findings support the concept that the subcellular localization of hcv core protein might have an impact on cd + t cells functions. in order to confirm, by an alternative method, some of the associations identified by mass spectrometry analysis, and most importantly to evaluate whether such associations were also present in primary t cells (pbmc), thus enhancing the relevance of our findings. postnuclear lysates from human pbmcs blasts (see methods), were subjected to hcv core protein pull down, using a biotinilated hcv core protein ( pmoles) bound to magnetic beads as a bait. uncoated magnetic beads were used as a control. experiments were first carried out in jurkat cells and inputs from jurkat and pbmc were loaded in parallel ( . % from the amount used per pull down), to evaluate the relative amount of each target protein (data not shown). serine/threonine-protein phosphatase was selected due to its known localization (it has a dynamic and predominantly nucleolar distribution) and to its function (it has been implicated in the regulation of several biological pathways previously described in t cells transduced with hcv core protein) (aggen et al., ; ceulemans and bollen, ; nie et al., ) . it was also selected based on the fact that both its catalytic subunit (pp γ) and its regulatory subunit a (mypt ), were identified by mass spectrometry, showing a despairing mascot score of . and . respectively. as shown in figure , western blot analysis of (pp γ) and (mypt ), confirmed the presence of both frontiers in microbiology | www.frontiersin.org figure | "actions" view, according to the "go cellular components" distribution option, in order to identify their subcellular localization. string analysis reported that proteins were nuclear (a) ( from ), nucleolar (b) ( from ), and cytoplasmic (c) ( from ). colored lines between proteins indicate the type of evidence for each interaction, with a minimum required confidence score of . . proteins in pbmcs lysates obtained from the pull down of hcv core protein coated magnetic beads (figure ) . in order to identify potential nucleolar localization sequences in hcv core protein, we used the nod web server (http://www. compbio.dundee.ac.uk/www-nod/), a program that predicts the presence of nolss in eukaryotic and viral proteins, based on a database of statistically analyzed human nucleolar localization sequences (scott et al., ) . two nolss were identified in hcv core protein ( several biological and immunological consequences of hcv core intracellular expression in cd + t cells have previously been described by us and others, (bergqvist and rice, ; bergqvist et al., ; dominguez-villar et al., , a doumba et al., ; fernandez-ponce et al., ) . these findings suggest an important role for the intracellular presence of hcv core in hcv pathogenesis and chronification. in this study, we have analyzed the ultrastructural localization of hcv core protein in cd + t cells. according to studies using cell lines unrelated to the immune system, hcv core protein has been shown to localize in the endoplasmic reticulum, mitochondrial outer membrane and nucleus of human embryonic kidney t cells (suzuki et al., ) ; associated to lipid droplets in cho, hepg and huh cells lines (barba et al., ; boulant et al., ; qiang and jhaveri, ) , in the cytoplasm, endoplasmic reticulum, in the proximity of the nuclear membrane, in the nucleus and nucleoli of hepatocytes isolated from chronically hcv infected patients (falcon et al., ) ; and in the cytoplasm and nucleus of non-parenchymal liver cells such as, lymphocyte-like cells, kupffer-like cells, polymorphonuclear-like cells, pit, endothelial, stellate, and fibroblast-like cells isolated from livers of chronically hcv infected patients (falcon et al., ) . in the present work, we found that in cd + t lymphocytes, hcv core protein mostly localizes in the nucleus and specifically in the nucleolus where it is greatly enriched and mainly organized in clusters (figure ) . regarding these findings, nuclear localization signals (nlss) described previously in hcv core protein, could be responsible for nuclear localization (chang et al., ; suzuki et al., suzuki et al., , , while hcv core protein traffic and residence in the nucleolus, could be explained by the presence of two nucleolar localization sequences (nolss), identified using the bioinformatic nucleolar localization sequence detector web server for eukaryotic and viral proteins (scott et al., ) . nolss are showed in figure . the statistical variation in core protein nucleolar localization shown by hcv-core expressing cells, could be due to differences in cellular cycle stage, as it has been described for other nucleolar resident proteins (chen and huang, ; stoldt et al., ; pirlot et al., ) . presence of nolss in cellular or viral proteins is a key factor in their dynamic traffic and residence within the nucleolus. presumably, nolss interact with nucleolar proteins and/or rna to mediate nucleolar targeting and retention. thus, in chimeric viral proteins with mutated nolss, trafficking to the nucleolus is abrogated, and heterologous nolss insertion restores their trafficking pattern (boyne and whitehouse, ; emmott et al., ) . interestingly, the function of hcv core protein inside the virus, can partly explain its subcellular localization as hcv core protein mainly interacts with hcv genomic rna, multimerizing around it and forming the capsid shell. while multimerizing inside the virus, we have not seen any multimerization in our experiments, which is in agreement with several studies showing that mammalian cell lines fail to produce capsid assembly (bukh et al., ; pietschmann et al., ; polyak et al., ; rouille et al., ; hourioux et al., ) , due to the lack of host cells factors that are essential for hcv core multimerization and assembly, or to the presence of inhibitory factors that induce the majority of hcv-core to be targeted away from the er (hope and mclauchlan, ; mclauchlan et al., ; polyak et al., ) . thus, un-multimerized hcv core protein traffics to alternate subcellular compartments. hcv core has been shown to bind rnas in addition to hcv genomic rna (kunkel et al., ; cristofari et al., ) , including ribosomal rna (santolini et al., ) and trna (kunkel et al., ) . since the nucleolus contains several copies of rrna genes and it could be a recruiting site for trnas (carmo-fonseca et al., ) it is likely that the nucleolar rna constitutes another important molecular target structure for hcv core protein. in agreement with our findings, several dna viruses, retroviruses and rna viruses, as well as viral proteins, have been described to traffic to the nucleolus and be associated with nucleolar proteins wurm et al., ; chen et al., ; dove et al., ; michienzi et al., ; cawood et al., ; hiscox, ; emmott et al., ; lam et al., ; jarboui et al., ) . the nucleolus is a dynamic nuclear organelle whose proteome is continuously changing. it seems to be a temporary storage or a sequestration site for a multiplicity of proteins (emmott and hiscox, ; hiscox et al., ) . functionally, there is extensive evidence that the nucleolus is implicated in ribosome biogenesis (stoykova et al., ; warner, ; scheer et al., ) , cell cycle regulation, cell growth, senescence, stress response signaling (andersen et al., ; emmott and hiscox, ; tsai and pederson, ; lam and trinkle-mulcahy, ) and the pathogenesis of several diseases such as cancer (james et al., ; orsolic et al., ; yang et al., ) , cardiovascular disease (hariharan and sussman, ) and neurodegenerative disorders (payao et al., ; lu et al., ; rieker et al., ; tsoi and chan, ; lee et al., a; parlato and liss, ; hernandez-ortega et al., ) . viral protein trafficking and localization into the nucleolus has shown implications in both viral life cycle and in host cell physiology and it has been narrowly related with the loss of essential nucleolar functions (hiscox, ) . in addition, it has been shown that accumulation of viral proteins in the nucleolus can cause volume exclusion and crowding effects, disrupting the nucleolar architecture (hancock, ; hiscox, ) . virus infection and some viral proteins from poliovirus, avian infectious bronchitis virus (ibv), coronavirus and human immunodeficiency virus- (hiv- ) induce disruption of the nucleolar architecture and changes on the subcellular distribution of nucleolar proteins or proteins that traffic to the nucleolus, such as nucleolin, p , b . (waggoner and sarnow, ; hiscox, ; dove et al., ) . these findings are closely related to the presence of perturbations in cell cycle, cytokinesis and apoptosis in the host cells (miyazaki et al., ; chen et al., ; galati et al., ; hiscox, ) . semliki forest virus nucleocapsid migrates to the nucleolus (jakob, ) and porcine reproductive and respiratory syndrome virus nucleocapsid specifically interacts with the small nucleolar rna (snorna)-associated protein, fibrillarin in virus infected cells (rowland et al., ; yoo et al., ) , while hepatitis b virus (hbv) core protein usually co-localizes with the nucleolar proteins, nucleolin and b (ning and shih, ) . in addition, it has been shown that coronavirus nucleocapsid protein localization in the nucleolus of infected cells and its association to the nucleolar protein b . is related to cell cycle stage and could be involved in cell cycle delay or arrest to promote virus replication wurm et al., ; cawood et al., ) . thus, trafficking to the nucleolus and nucleolar residency time of hcv core protein in cd + t cells could be narrowly related with previous findings from us and others showing that the intracellular presence of hcv core in cd + t cells induces decreasing cell proliferation, delay in cell cycle progression and a differential expression pattern of genes with relevant function, including anergy-associated genes, genes involved in cytoskeleton reorganization, vesicle trafficking, endocytosis, cytokines production, cell death, transcription, and translation (dominguez-villar et al., ) . in agreement with the described localization, we found hcv core protein association with several nuclear, nucleolar proteins or proteins described to traffic to the nucleolus, which showed mainly binding connections (figure ) . the wide range of proteins associated with hcv core, correlate with the findings obtained by dolan et al. who identified two computationally predicted molecular recognition features within the n-terminal intrinsically disordered region (idr) in the hcv core sequence. the identified molecular recognition features, mediate hcv core protein binding to hcv rna and to multiple host proteins, suggesting that hcv core protein exhibits hub protein properties (dolan et al., ) . interestingly, pathway and network-based analysis of proteins identified to be associated with hcv core protein, indicate that a wide range of these proteins are involved in several biological signaling pathways, such as, rna processing and splicing (figure a) , cell cycle progression, cell proliferation, apoptosis ( figure b ) and infection and replication of rna viruses, including hcv ( figure c) . with the integrated analysis of the present data, we confer a better description of the hcv core -human t lymphocyte relationship. concerning ribosomal biogenesis, cellular proliferation, cell cycle and apoptosis; several large (rpl) and small (rps) ribosomal proteins, and proteins involved in rna processing and splicing as dead-box rna helicases, precipitated with figure | predicted nucleolar localization sequences in hcv core protein. graph obtained from the nucleolar localization sequence detector web server (nod), displaying nols prediction score for each residue of hcv core protein. pink shaded regions represent the range of scores within which a -residues segment is predicted to be a nols. thus, pink shaded regions represent the nols candidate segment, which highlights scores above . . hcv core (figures a,b) (rocak and linder, ; xu et al., ) . interactions between viral and ribosomal proteins, splicing factors and dead-box rna helicases including ddx and ddx , have been previously described and suggested as a key mechanism for viral replication and production as well as for life cycle progression and survival (bortz et al., ; naji et al., ; yasuda-inoue et al., ; cervantes-salazar et al., ; klymenko et al., ; li et al., ) . implication of other identified proteins, such as protein red (ik) and filamin a (flna) (figure b ) in proliferation and cell cycle progression, have also been widely demonstrated (lee et al., b; sun et al., ) . thus, associations of hcv core protein in t cells could alter the function of the associated proteins, explaining some of the effects shown for hcv-core expression, including cell cycle delay and inhibition of cell proliferation (dominguez-villar et al., , a fernandez-ponce et al., ) . in addition, we found that many host proteins associated with hcv core, are involved in replication and infection of rna viruses including hcv ( figure c) . proteins as dexd/hbox helicases have been extensively studied in virus infection and have been described as proteins hijacked by viruses for their benefit. specifically, dhx is involved in virus replication, innate immunity response to viral dsrna and participation in the expression of ifn-stimulated genes (fullam and schroder, ) . rna binding proteins (rbps) such as the host poly(rc)-binding protein (pcbp) and different heterogeneous nuclear ribonucleoproteins (hnrps), are also interesting as they stabilize viral rnas, co-localize with the viral replicase complex and facilitate viral rna template selection (li and nagy, ) . in addition, interleukin enhancer-binding factor (ilf ) has been shown to be recruited to hcv replication complexes (li et al., ) and in other infections by rna viruses, ilf interaction with viral proteins has been described to affect virus replication among other virus life cycle stages (patino et al., ) . furthermore, the subcellular localization of hcv core and its association with nuclear and nucleolar proteins found in the present study, can aid in explaining the cd + t cell regulatory/exhausted phenotype described by us and others, in cd + t cells expressing hcv core protein (dominguez-villar et al., a; doumba et al., ; fernandez-ponce et al., ) . some such associations are with serine/threonine-protein phosphatase catalytic subunit (pp γ), protein phosphatase regulatory subunit a (mypt ) (nie et al., ) , interleukin enhancer-binding factor (ilf ) (shi et al., a,b) , complement component c q binding protein (c qbp) (kittlesen et al., ) and runt-related transcription factor (runx ) (klunker et al., ) . in conclusion, analysis of the association of hcv core with host proteins in cd + t cells and the study of its ultrastructural localization, open an extensive field of study poised to understand the mechanisms underlying functional findings previously described in cd + t cells expressing hcv core protein, that have demonstrated to be relevant for hcv immune system evasion and thus hcv chronification. conception, design and or interpretation of the work: fg-c, ea, and rl. wb (for localization studies). cb and md-r (for association studies). performed association studies: cf-p, md-r, and as-s. performed localization studies: cf-p, jm-m, and ma-e. run statistical analyses: rl and cf-p. wrote the paper: fg-c, cf-p, ea, rl, and md-r. performed confirmation co-ip experimentos: in-s and cf-p. all authors participated in critical revision and subsequently approved the manuscript. all authors agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. for ea. the funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication. regulation of protein phosphatase- nucleolar proteome dynamics hepatocellular carcinoma: role of hepatitis b and hepatitis c viruses proteins in hepatocarcinogenesis hepatitis c virus core protein shows a cytoplasmic localization and associates to cellular lipid storage droplets transcriptional activation of the interleukin- promoter by hepatitis c virus core protein the hepatitis c virus core protein modulates t cell responses by inducing spontaneous and altering t-cell receptor-triggered ca + oscillations the conserved 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localization of the truncated hepatitis c virus core protein with its hydrophobic c terminus deleted molecular determinants for subcellular localization of hepatitis c virus core protein string v : protein-protein interaction networks, integrated over the tree of life hepatitis c viruses genomes and molecular biology connecting the nucleolus to the cell cycle and human disease expression of expanded cag transcripts triggers nucleolar stress in huntington's disease hcv core and ns proteins manipulate human bloodderived dendritic cell development and promote th differentiation hcv core protein interaction with gc q receptor inhibits th differentiation of cd + t cells via suppression of dendritic cell il- production viral ribonucleoprotein complex formation and nucleolar-cytoplasmic relocalization of nucleolin in poliovirus-infected cells the nucleolus and ribosome formation localization to the nucleolus is a common feature of coronavirus nucleoproteins, and the protein may disrupt host cell division the role of ribosomal proteins in the regulation of cell proliferation, tumorigenesis, and genomic integrity nucleolar repression facilitates initiation and maintenance of senescence direct binding of hepatitis c virus core to gc qr on cd + and cd + t cells leads to impaired activation of lck and akt distinct ddx deadbox rna helicases cooperate to modulate the hiv- rev function colocalization and interaction of the porcine arterivirus nucleocapsid protein with the small nucleolar rna-associated protein fibrillarin interaction of avian influenza virus ns protein and nucleolar and coiledbody phosphoprotein we would like to thank christian hoffmann, evelyn janssen, beatrix martiny, jessicahausmann, mojgan ghilav and consuelo rivera for their excellent technical support, the plataforma andaluza de bioinformática (centro de supercomputación y bioinformática, university of málaga) for the use of the ingenuity pathways analysis (ipa) software and the core biomedical research facility of the university of cadiz for the use of core infrastructure. the supplementary material for this article can be found online at: https://www.frontiersin.org/articles/ . /fmicb. . /full#supplementary-material key: cord- -buzyani authors: prabakaran, ponraj; zhu, zhongyu; chen, weizao; gong, rui; feng, yang; streaker, emily; dimitrov, dimiter s. title: origin, diversity, and maturation of human antiviral antibodies analyzed by high-throughput sequencing date: - - journal: front microbiol doi: . /fmicb. . sha: doc_id: cord_uid: buzyani our understanding of how antibodies are generated and function could help develop effective vaccines and antibody-based therapeutics against viruses such as hiv- , sars coronavirus (sars cov), and hendra and nipah viruses (henipaviruses). although broadly neutralizing antibodies (bnabs) against the hiv- were observed in patients, elicitation of such bnabs remains a major challenge when compared to other viral targets. we previously hypothesized that hiv- could have evolved a strategy to evade the immune system due to absent or very weak binding of germline antibodies to the conserved epitopes that may not be sufficient to initiate and/or maintain an effective immune response. to further explore our hypothesis, we used the sequence analysis of a large naïve library of human igm antibodies which had been used for selecting antibodies against sars cov receptor-binding domain (rbd), and soluble g proteins (sg) of henipaviruses. we found that the human igm repertoires from the sequencing have diverse germline usages, recombination patterns, junction diversity, and a lower extent of somatic mutation. in this study, we identified antibody maturation intermediates that are related to bnabs against the hiv- and other viruses as observed in normal individuals, and compared their genetic diversity and somatic mutation level along with available structural and functional data. further computational analysis will provide framework for understanding the underlying genetic and molecular determinants related to maturation pathways of antiviral bnabs that could be useful for applying novel approaches to the design of effective vaccine immunogens and antibody-based therapeutics. broadly neutralizing antibodies (bnabs) against the hiv- are relatively rarely observed in patients; however, discovering hiv- vaccine candidates to elicit such bnabs remains a challenge due to the extensive genetic sequence variability and complex immune evasion strategies of the hiv- (burton, ; johnson and desrosiers, ; haynes and montefiori, ; prabakaran et al., ) . among the different factors thwarting the induction of bnabs, we previously found that all known hiv- bnabs are highly divergent from germline antibodies; germline antibodies of bnabs could not bind to the epitopes of respective mature antibodies, which led to a hypothesis that hiv- may have evolved to use the "holes" (absence of or weak binding to germline-lineaged bnabs) in the human germline b cell receptor repertoire (xiao et al., ) . consistent with our earlier hypothesis, we did not find any specific binders against the hiv- envelope glycoproteins (envs) but only identified binders against the sars cov receptorbinding domain (rbd), and soluble hendra virus g protein (sg) when combinatorial phage display libraries mimicking human antibody repertoire constructing from human igm libraries had been used for panning experiments . these findings had indicated that the major problem could be related to a high level of somatic mutations required for bnabs to accurately target the conserved structures on the hiv- envs. in this article, we have used high-throughput sequencing of a large naïve library of human igm antibodies to explore antibody repertoire landscape for finding germline usages, somatic mutations, intermediates, and phylogenetic relationships between the intermediates and corresponding antiviral-related bnabs including the hiv- , sars cov, and henipaviruses. this study helped to identify germline predecessors of bnabs observed in normal individuals, and find maturation pathways of antiviral bnabs. indeed, most of the known hiv- bnabs are highly divergent from their closest respective germlines as well as their intermediates as they undergo somatic mutations required for their neutralization function. the results corroborate that the hiv- may use a strategy to eliminate strong binding of germline antibodies due to the absence of closer anti-hiv antibody intermediates as an escape mechanism from adaptive immune responses, and finding of closer intermediates of bnabs from rare individuals might help designing the effective vaccines against the hiv- and other viral diseases. to amplify igm antibody sequences, cdna was prepared from peripheral blood b cells of healthy donors as received under the research donor program of frederick national laboratory for cancer research, usa, which we previously used to construct a naïve human fab phage display library for selecting antibodies against sars cov and henipaviruses. the complete set of primers used in the pcr amplification of igm-derived heavy and light chains were described in detail elsewhere ). for sequencing, primer combinations used to amplify cdna in separate reactions included the roche a and b adaptor sequences along with target amplification sequence for heavy and light chain variable domains. the gene fragments were amplified in cycles of pcr using the high fidelity pcr master from roche. more detailed description of sequencing can be found in our recent articles (prabakaran et al., . the standard roche gs titanium shotgun library protocol was adapted as found in the roche sequencing technical bulletin. for quality control of antibody sequences, we trimmed the sequence data and retained only sequences of length more than nucleotides (nt), covering the entire antibody variable domains consisting of the three complementarity determining regions (cdr) along with framework regions (fr). we used imgt/highv-quest (alamyar et al., ) , a high-throughput version for deep sequencing ngs data analysis resource for the immunogenetic analysis. the output results from the imgt/highv-quest analysis in csv files were stored at postgresql database, and structured query language (sql) was used to retrieve the data for the further analysis. heatmap generation and statistical calculations involving distributions of antibody hcdr lengths and mutations were carried out using sas jmp ® statistical software (sas institute, cary, nc). translated heavy and light chain variable sequences from the sequencing that shared the ighv genes of selected antiviral antibodies and associated immunogenetics data including the details of germlines, hcdr lengths, and mutations were retrieved from the database by using sql. sequence identities between the sequence data and germlines were calculated based on the pairwise alignment using local blast as implemented in bioedit v . . (hall, ) . phylogenetic analysis was carried out using the archaeopteryx software (han and zmasek, ). to analyze germline origin of antiviral antibodies against the hiv- , sars cov, and henipaviruses as expressed in the human igm repertoire, we performed sequencing of a non-immune library which was previously constructed from peripheral blood b cells of healthy donors and used to select antibodies against sars cov and henipaviruses (prabakaran et al., ; zhu et al., ) . a total of , sequences were obtained from which , sequences were found as unique with each had > nt in length. the total number of unique amino acid (aa) sequences for each v-gene subgroup in heavy and light chains that were found functionally productive as determined by imgt/highv-quest (alamyar et al., ) are shown in figures a,b , respectively. the read coverage or gene frequencies observed in the study suggested for biased germline usages and were comparable to the previous studies (glanville et al., ; prabakaran et al., ) but way far less than the theoretical diversity attainable by antibodies attributing to several factors such as library sampling, primer efficiency, and sequencing errors and limitations. nevertheless, we selected known bnabs against the viral targets including the hiv- , sars cov, and henipaviruses (table ) , and created sequence data sets related to those bnabs from the analysis as depicted in figures c,d showing the germline usage frequencies of ighv genes in the v h domains, igkv, and iglv genes in the v κ and v λ domains, respectively. we found that while all antiviral-related germlines were expressed in human igm repertoire, some preferential germline usages were noted, for example, hv - gene in ighv subgroups and kv - /lv - genes in igkv/iglv subgroups were overrepresented (figures c,d) . the role of heavy chains of antiviral antibodies in antigen recognition is found to be associated with longer hcdr s and extensive v h mutations ( table ) . most of the bnabs have longer hcdr s with aa lengths ranging from to , except for g , vrc and m . all of the v h genes of anti-hiv- antibodies have a high degree of somatic mutations when compared to non-hiv- antiviral bnabs. we analyzed hcdr length distributions and v h mutations preexisting in germline-lineaged precursor antiviral antibodies from the ighv genes of igm repertoires from which bnabs were generated. the box plots display the distributions of hcdr lengths and v h mutations, figures a,b , respectively, which indicates a high level hcdr length diversity and lesser extent of somatic mutations compared to bnabs ( table ) . to assess the vdj repertoire usage among different antiviral related ighv genes, we computed the frequencies of vdj recombination patterns as observed in the v h genes expressed in human igm repertoire involving those ighv genes of antiviral antibodies. the heatmap is shown in the figure depicting the figure | frequencies of vdj recombination types as observed in the human igm repertoire involving ighv genes related to the antiviral bnabs. the heatmap is colored according to the total number of unique vdj patterns existing in the corresponding ighv genes used in association with different ighd and ighj genes, and is shown on a blue-to-gray-to-red scale. the white-colored space represents the missed or absent vdj recombination types in the repertoire. august | volume | article | most (red) and least (blue) abundant vdj types existing in the germline-lineaged repertoire for the corresponding ighv genes used in association with different ighd and ighj genes. the ighv genes v - and v - were frequently found to recombine with ighj genes j and j , and ighd genes d and d . the intermediate antibodies corresponding to bnabs against the hiv- , sars cov, and henipaviruses were found by analyzing the human igm repertoire, and such intermediates with the closest similarities to the matured antiviral bnabs were selected for germline-linage analysis by using phylogenetic method. ighv germline gene alleles of bnabs were obtained from the imgt database. the mid-point phylogenetic neighbor-joining tree showing the evolutionary relationships of different antiviral antibodies with their corresponding germlines and intermediates is given in figure . we observed that some of the anti-hiv- antibodies ( g , ch , and vrc ) were found at distal nodes in the phylogenetic tree indicating high divergence from their corresponding germline and intermediate counterparts. in contrast, bnabs against sars cov, and henipaviruses, m and m , were found closer to their intermediates. we found unique ighv sequences from the v - gene family as intermediates of bnab b by using the sequence analysis of a human igm library. phylogenetic analysis of those intermediates revealed two major groups, one group consisting of germline related antibodies and the other having potential intermediates closer to the bnab b . we then constructed a phylogenetic sub-tree selecting only the potential intermediates and the v - * germline along with bnab . the tree was rooted at the known germline v - * of bnab b , and phylogram showed evolutionary relationship among the different intermediates ( figure a) . one of the intermediates, g jy , had the maximum of % sequence identity ( % sequence similarity) at aa level to the bnab b ( figure b) . however, the hcdr length of that intermediate was found to be aa long, which is aa shorter than that of b antibody. to find the closest hcdr to that of b , we scanned , unique hcdr sequences from the entire igm sequence data. we identified a hcdr with the same length ( aa) and % sequence identity to that of b (figure c) , which was found to be the most similar to the hcdr of b but the ighv gene associated with that hcdr was found to be v -b. we used the hiv- gp -b complex structure and mapped the v h somatic mutations, which showed the overlapping of three mutated residues of b (n from hcdr , y from hcdr , and w . from hcdr ) that contribute to the most of binding interactions with the gp as previously observed (zhou et al., ) (figure d ). in this study, we have described the sequence analysis of a large naïve library of human igm antibodies, and carried figure | the mid-point phylogenetic neighbor-joining tree shows the evolutionary relationships between different ighvs of (bnabs) with their corresponding germlines and intermediates. the ighv germline gene alleles follow the imgt nomenclature and the closest intermediates of bnabs as found from the human igm repertoire were designated with asterisks along with names of bnabs. some of the anti-hiv- antibodies ( g , ch , and vrc ) were found at distal nodes in the phylogenetic tree indicating high divergence from their corresponding germline and intermediate counterparts. out immunogenetic analysis to study the origin, diversity, and maturation of selected known bnabs against the hiv- , sars cov rbd, and henipaviruses sg proteins. we have found intermediates of antiviral related bnabs, of which most of those against the hiv- were highly diverged from their mature forms of bnabs as compared to other viral targets, sars cov, and henipaviruses. although antibodies are generated through various mechanisms involving vdj recombination, junctional modification, and hypermutations, the v-genes sculpt the most of the antigencombining sites, cdr and cdr , and support frameworks for the cdr . we found that antiviral antibodies targeting different env binding regions of the hiv- and other viruses utilized different germline v-genes as the origins (table ) . we noted that, among antiviral-related bnabs, the v - gene usage was dominated in the heavy chains while v - and v - genes of kappa and lambda were used with the highest frequencies in the light chains of human igm repertoire (figure ) . accordingly, four of the v h genes of bnabs ( e , x , m , and m ) originated the most similar hcdr sequence to that of bnab b from the igm repertoire was found to originate from v -b * gene and is shown in the pairwise alignment. (d) mapping of three of the somatically mutated residues n , y , and w . , as per imgt numbering scheme, from each of the hcdrs are shown as sticks using the complex crystal structure of hiv- gp -b (pdb code ny ). from the v - , and three of them paired with the kappa v - gene. one possible reason for dominance in the usage of those germline genes could be reflecting from the relatively higher frequencies of distributions observed in the expressed igm repertoire (figures a,b) . the hv gene was used in the three of the hiv- bnabs, g , pg , and ch . the structural data for most of the bnabs selected in this analysis were known and the heavy chains of these bnabs were dominantly used. the increased number of v h mutations and longer hcdr s are characteristics for the hvi- bnabs when compared to other antiviral bnabs (breden et al., ) . we analyzed the distribution of hcdr lengths and extent of somatic v h mutations in the human igm repertoire to compare with that of antiviralrelated bnabs (figure ) . the results showed that the longer hcdr s and low level of somatic v h mutations as compared to the hiv- bnabs existed in the intermediates as found from the sequencing. the somatic diversity through vdj recombination involving antiviral-related v-genes in the igm repertoire was found high; the most abundant vdj combination consisted of the hv - gene with certain d and j genes as depicted in gray and red (figure ) , which might be the reason for the preferential usage of that hv - in many other viral diseases (sui et al., ). further, bnabs against the sars cov and henipaviruses shared the heavy chain v-gene germline, hv - , with two of the hiv- bnabs, e , and x . all of these four bnabs were less divergent from their v-germlines and intermediates, when compared to other hiv- bnabs, and formed a single cluster at a mid-point rooted phylogenetic tree (figure ) . the gp membrane-proximal epitope region (mper) binding site bnabs, f , and m , were moderately divergent from their vgermlines and intermediates and formed distinct clusters. the v-gene of vrc bnab was the most divergent from its respective germline as well as the closest intermediate, and was placed at a distal branch of hv subgroup of bnabs. for the mid-point rooted phylogenetic analysis, we included the closest intermediates only; however, favored maturation pathways could involve other intermediates too. we created the germline-rooted phylogenetic tree as a use-case for the bnab b ( figure a ) and analyzed the maturation pathway along different v-gene intermediates from hv - gene family. the closest b intermediate, designated as g jy , had three mutations each at hcdr and hcdr compared to the germline, and were found similar though not identical to that of mature b ( figure b) . interestingly, we also identified a hcdr with the same length ( aa) and % sequence identity to that of b (figure c) , which was found to be the most similar to the hcdr of b but the ighv gene associated with that hcdr was found to be v -b. this might suggest for the possible maturation mechanism of bnabs which could be involving the vh replacement (chen et al., ) . these two mutated residues (n from hcdr and y from hcdr ) from the v-gene and a trp residue from the d-gene (w . from hcdr ) contributed to the most of binding interactions with the gp (figure d ) (zhou et al., ) . in summary, the sequence analysis of a large naïve human antibody repertoire corresponding to the selected antiviralrelated bnabs revealed the germline v-gene usage, vdj rearrangement, hcdr length diversity, and somatic mutations of potential intermediate antibodies of hiv- and other viruses such as sars cov and henipaviruses. thus, b cell germlinelineage analysis using the sequence data from different sources could help finding appropriate antibody intermediates, pathways, and mechanisms useful in the development of bnabs and vaccines against the hiv- and other viral diseases. quest: the imgt® web portal for immunoglobulin (ig) or antibody and t cell receptor (tr) analysis from ngs high throughput and deep sequencing comparison of antibody repertoires produced by hiv- infection, other chronic and acute infections, and systemic autoimmune disease antibodies, viruses and vaccines immunoglobulin heavy chain gene replacement: a mechanism of receptor editing and selection of monoclonal antibodies to viral envelope glycoproteins: implications for mechanisms of immune evasion and design of vaccine immunogens precise determination of the diversity of a combinatorial antibody library gives insight into the human immunoglobulin repertoire bioedit: a userfriendly biological sequence alignment editor and analysis program for windows / /nt phyloxml: xml for evolutionary biology and comparative genomics aiming to induce broadly reactive neutralizing antibody responses with hiv- vaccine candidates viral persistance: hiv's strategies of immune system evasion expressed antibody repertoires in human cord blood cells: sequencing and imgt/highv-quest analysis of germline gene usage, junctional diversity, and somatic mutations structure and function of the hiv envelope glycoprotein as entry mediator, vaccine immunogen, and target for inhibitors structure of severe acute respiratory syndrome coronavirus receptorbinding domain complexed with neutralizing antibody antibody sequencing -error characterization and correction structural and functional bases for broad-spectrum neutralization of avian and human influenza a viruses germline-like predecessors of broadly neutralizing antibodies lack measurable binding to hiv- envelope glycoproteins: implications for evasion of immune responses and design of vaccine immunogens structural definition of a conserved neutralization epitope on hiv- gp potent neutralization of hendra and nipah viruses by human monoclonal antibodies construction of a large naive human phage-displayed fab library through one-step cloning we thank the laboratory of molecular technology of saic-frederick inc. for providing roche sequencing service. we are grateful to eltaf alamyar and to the imgt® team for providing access to imgt/highv-quest. we thank ms. maria g. singarayan for constructing the postgresql database and java applications and helping with sql. this research was supported by the intramural research program of the nih, national cancer institute, center for cancer research, and by federal funds from the nih, national cancer institute, under contract no. no -co- . the content of this publication does not necessarily reflect the views or policies of the department of health and human services, nor does the mention of trade names, commercial products, or organizations imply endorsement by the us government. the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. key: cord- -i raopi authors: perley, casey c.; brocato, rebecca l.; wu, hua; bausch, christoph; karmali, priya p.; vega, jerel b.; cohen, melanie v.; somerville, brandon; kwilas, steven a.; principe, lucia m.; shamblin, joshua; chivukula, padmanabh; sullivan, eddie; hooper, jay w. title: anti-hfrs human igg produced in transchromosomic bovines has potent hantavirus neutralizing activity and is protective in animal models date: - - journal: front microbiol doi: . /fmicb. . sha: doc_id: cord_uid: i raopi we explored an emerging technology to produce anti-hantaan virus (htnv) and anti-puumala virus (puuv) neutralizing antibodies for use as pre- or post-exposure prophylactics. the technology involves hyperimmunization of transchomosomic bovines (tcb) engineered to express human polyclonal igg antibodies with htnv and puuv dna vaccines encoding g(n)g(c) glycoproteins. for the anti-htnv product, tcb was hyperimmunized with htnv dna plus adjuvant or htnv dna formulated using lipid nanoparticles (lnp). the lnp-formulated vaccine yielded fivefold higher neutralizing antibody titers using -fold less dna. human igg purified from the lnp-formulated animal (sab- ), had anti-htnv neutralizing antibody titers > , . sab- was capable of neutralizing pseudovirions with monoclonal antibody escape mutations in g(n) and g(c) demonstrating neutralization escape resistance. sab- protected hamsters from htnv infection when administered pre- or post-exposure, and limited htnv infection in a marmoset model. an lnp-formulated puuv dna vaccine generated purified anti-puuv igg, sab- p, with a neutralizing antibody titer > , . as little as . mg/kg of sab- p protected hamsters against puuv infection for pre-exposure and mg/kg sab- p protected puuv-infected hamsters post-exposure. these data demonstrate that dna vaccines combined with the tcb-based manufacturing platform can be used to rapidly produce potent, human, polyclonal, escape-resistant anti-htnv, and anti-puuv neutralizing antibodies that are protective in animal models. hantaan virus (htnv) and puumala virus (puuv) are zoonotic viruses, causing a disease known as hemorrhagic fever with renal syndrome (hfrs) (elliot et al., ) . hantaviruses, family hantaviridae, have a tripartite negative-sense rna genome, and infection with these viruses can result in hfrs characterized by varying degrees of capillary leakage, acute kidney injury, and hemorrhage (elliot et al., ) . each year tens of thousands of cases of hfrs are reported world-wide, with a case fatality rates between and % (elliot et al., ) . most hfrs cases caused by htnv and related viruses are in china and the korean peninsula, with puuv cases predominantly in scandinavia and western russia (jonsson et al., ; zhang et al., ; elliot et al., ) . there are currently no fda-licensed vaccines or therapeutics for disease caused by any hantavirus (schmaljohn, ; brocato and hooper, ) . the m-segment of hantaviruses encode viral envelope glycoproteins (g n and g c ) (cifuentes-munoz et al., ) . dna vaccines encoding the m-segment of numerous hantavirus species have demonstrated immunogenicity in a wide variety of animal species and humans (hooper et al., (hooper et al., , a (hooper et al., , (hooper et al., , (hooper et al., , b custer et al., ; boudreau et al., ; brocato et al., ; bounds et al., ; haese et al., ) . passive transfer of dna-vaccine derived antibodies was efficacious in preventing disease or infection against their cognate virus in hamster models of infection or disease (custer et al., ; hooper et al., hooper et al., , a brocato et al., ; haese et al., ) . for htnv specifically, passive transfer of sera from rhesus macaques vaccinated with a htnv dna m-segment vaccine protected hamsters against infection with htnv (custer et al., ) . transchromosomic bovines (tcb) produce antigen-specific, fully human, immunoglobulins (igg) in addition to chimeric antibodies (human γ heavy chain and bovine κ light chain) depending on the type of tcb (sano et al., ) . after vaccination against a particular target, fully human polyclonal igg against the cognate antigen can be purified from large volumes of plasma (hooper et al., a) . tcb have been used to produce polyclonal neutralizing antibody products against numerous viruses including zika virus, ebola virus (ebov), venezuelan equine encephalitis and middle east respiratory syndrome coronavirus, and two hantaviruses that cause hantavirus pulmonary syndrome, andes virus (andv) and sin nombre virus (snv) (hooper et al., a; bounds et al., ; dye et al., ; luke et al., ; gardner et al., ; stein et al., ) . for all of these viruses, passive transfer of tcb-derived igg immediately prior to or post challenge prevented disease, elicited partial protection, or rapidly reduced viral titer (hooper et al., a; bounds et al., ; dye et al., ; luke et al., ; stein et al., ; rosenke et al., ) . here, we demonstrate that it is possible to combine dna vaccine technology with the tcb platform to produce potent human polyclonal igg for use as a pre-or post-exposure prophylactic for hfrs caused by htnv and puuv infection. animal research was conducted under an iacuc approved protocol at sab biotherapeutics (usda registration number -r- ). animal research was conducted under an iacuc approved protocol at usamriid (usda registration number -f- & olaw assurance number a - ) in compliance with the animal welfare act and other federal statutes and regulations relating to animals and experiments involving animals. the facility where this research was conducted is fully accredited by the association for assessment and accreditation of laboratory animal care, international and adheres to principles stated in the guide for the care and use of laboratory animals, national research council, . the htnv m segment-based dna vaccine [pwrg/htn-m(co)] was previously described (hooper et al., a) . this htnv m segment dna vaccine sequence was used to generate the single and double escape mutants from the literature (wang et al., ; kikuchi et al., ) . the seoul virus (seov) m segment-based dna, pwrg/seo-m(opt ) is identical to the previously published pwrg/seo-m(x) vaccine (hooper et al., ) , however, the open reading frame was optimized for human codon usage and mrna stability (fath et al., ) . the puuv dna vaccine, pwrg/puu-m(s ), snv dna vaccine, pwrg/sn-m(opt), and andv dna vaccine, pwrg/and-m(opt ), have been published previously hooper et al., ; kwilas et al., ) . the choclo virus (chocv) dna vaccine, pwrg/chocv-m(opt) and dobrava virus (dobv) dna vaccine, pwrg/dob-m(opt), were constructed by cloning an optimized synthetic m gene open reading frame into the not -bglii site of pwrg . all m segments were generated at genewiz unless otherwise noted. research grade plasmids used to vaccinate tcb were manufactured by aldevron (fargo, nd, united states). the formulations for lipid-based delivery were prepared by mixing an arcturus-propriety combination of lipids dissolved in ethanol with an aqueous phase containing plasmid dna dissolved in citrate buffer (ph . ) using a nanoassemblr microfluidic device (precision nanosystems, vancouver, bc, canada). the molar percentage ratio for the constituent lipids is % atx (proprietary ionizable amino lipids), % dspc ( , distearoyl-sn-glycero- -phosphocholine) (avanti polar lipids), . % cholesterol (avanti polar lipids), and . % dmg-peg ( , -dimyristoylsn-glycerol, methoxypolyethylene glycol, peg chain molecular weight: , ) (nof america corporation). the solutions were combined in the microfluidic device at a flow ratio of : ethanol:aqueous phases. the total combined flow rate was ml/min, per microfluidics chip. the mixed material was then diluted with phosphate buffer after leaving the micromixer outlet. the diluted particles were purified by dialysis in hepes buffer (ph . ) containing sucrose using regenerated cellulose membranes (spectrapor tube-a-lyzers, spectrum laboratories). a total of diavolumes were exchanged, effectively removing ethanol and ensuring complete buffer exchange. the lnps were then concentrated using ultra-spin centrifugal filter units (amicon ultra centrifugal filter units, emd millipore). particle size was determined by dynamic light scattering (zen , malvern instruments). dna content and encapsulation efficiency were determined using ribogreen assay. encapsulation efficiency was calculated by determining unencapsulated plasmid dna content by measuring the fluorescence upon the addition of ribogreen (molecular probes) to the particles (fi) and comparing this value to the total plasmid dna content that is obtained upon lysis of the particles by % triton x- (ft), where % encapsulation = (ft − fi)/ft × . a potency assay was implemented to confirm the formulated material was potent enough for vaccination. briefly, a potency assay involves a -well plate of hek t cells plated at e cells/well h prior to assay. a non-formulated plasmid using ug of dna and fugene is used as the standard for transfection. the formulated material is run at ug. half-log dilutions are made of the fugene/unformulated plasmid mixture and the formulated material being tested as it is self-transfecting and does not require normal transfection setup steps. after - h, cells are fixed and stained using specific rabbit anti-sera. the well counted for potency has fluorescent focus units (ffu) between and green cells. this then is converted to ffu/ug. a plasmid must be at least ffu/ug to be deemed potent. the tcb used in this study (# and # ) are homozygous double knockouts in endogenous bovine heavy chain immunoglobulin genes (ighm −/− , ighml −/− ) and contain the human artificial chromosome (hac) vector ckslhacd (sano et al., ; matsushita et al., ) . the tcb # is homozygous triple knockouts in endogenous bovine immunoglobulin genes (ighm −/− , ighml −/− , and igl −/− ) and contain the human artificial chromosome (hac) vector kchacd (sano et al., ; matsushita et al., matsushita et al., , . the hac ckslhacd and kchacd contain a fragment of human chromosome , the human immunoglobulin heavy chain locus except the ighm constant region, which remains bovine, and human chromosome , the entire human immunoglobulin κ light chain locus (sano et al., ; matsushita et al., matsushita et al., , . tcb # was vaccinated four times intramuscularly with unformulated pwrg/htn-m(co) dna vaccine ( mg/vaccination) with sab proprietary adjuvant sabadj- . the dna vaccine was delivered using the pharmajet stratis device at four injection sites behind the left and right ear and on the left and right hind leg ( mg/injection, mg/injection site). sab-adj- was delivered as a . ml injection using needle and syringe adjacent ( - cm) to the pharmajet stratis dna-vaccination site. tcb # was vaccinated with a low-dose lipid nanoparticle-formulated pwrg/htn-m(co) dna vaccine ( . mg/vaccination), via the pharmajet stratis injection device at four injection sites behind the left and right ear and on the left and right hind leg ( . mg/injection, . mg/injection site). tcb # was vaccinated with an lipid nanoparticle-formulated pwrg/puu-m(s ) dna vaccine ( . mg/vaccination), via the pharmajet stratis injection device similar to tcb # vaccination. purification of human igg from tcb has been previously described previously (kuroiwa et al., ; hooper et al., a) . the purified anti-htnv tc human igg sab- used in neutralization and animal studies is derived from plasma drawn after the fourth vaccination and has a concentration of . mg/ml. the purified anti-puuv tc human igg sab- p used in neutralization and animal studies is derived from plasma drawn after the third, fourth, and fifth vaccinations and has a concentration of . mg/ml. as a negative control purified tc human igg from a non-immunized cow was provided at . mg/ml. all purified igg is in sterile liquid containing mm glutamic acid monosodium salt, mm d-sorbitol and . mg/ml tween (ph . ). hantaan virus strain - (lee et al., ) and puuv strain k (tkachenko et al., ) were propagated in vero e cells (vero c , atcc crl ) in t- flasks and collected from infected-monolayer supernatants. cells were maintained in eagle's minimum essential medium with earle's salts containing % fetal bovine serum (fbs), mm hepes (ph . ), × penicillin-streptomycin, amphotericin b ( . µg/ml) and gentamicin sulfate ( µg/ml) at • c in a % co incubator. cell debris was removed by low speed centrifugation in a table top centrifuge. htnv and puuv were twice plaque purified according to published methods (hooper et al., b) . virus stocks were aliquoted and stored at − • c or colder. virus identity has been confirmed by sequencing of the stocks. mabs d and hco were obtained from the joel dalrymple collection at bei resources. female syrian hamsters (mesocricetus auratus) - weeks of age (envigo, indianapolis, in), or adult marmosets (callithrix jacchus) weighing over g were anesthetized by inhalation of vaporized isoflurane using an impac veterinary anesthesia machine. once anesthetized, animals were injected with the indicated concentration of virus diluted in pbs. intramuscular (i.m.) (caudal thigh) injections consisted of . ml delivered with a ml syringe with a -gauge, / in needle. subcutaneous injections consisted of ml (hamster) or ml (marmoset) of anti-hfrs tc human igg diluted in pbs administered with a ml syringe with a -gauge, in needle at a single injection site. vena cava blood draws occurred under previously stated methods of anesthesia, and were limited to % of total blood volume per week. terminal blood collection was performed by heart stick at the time of euthanasia. all work involving animals was performed in an animal biosafety level (absl- ) laboratories. the enzyme-linked immunosorbent assay (elisa) used to detect n-specific antibodies (n-elisa) was described previously (elgh et al., ) . species-specific secondary antibodies were used at the following concentrations: peroxide-labeled antihamster ( : , ) (sera care, gaithersburg, md, united states), and alkaline phosphatase conjugated anti-monkey ( : , ) (milliporesigma, st. louis, mo, united states). assays using peroxide labeled antibodies were developed with tmb microwell peroxidase substrate at an absorbance of nm. a sample was considered positive if its peak optical density (od) value was greater than either . or the background value (the average of three negative control wells + times their standard deviation, whichever was higher. the specific od sum is the summation of all values greater than background and represents the area under the elisa titer curve. plaque reduction neutralization test (prnt) were performed as previously described with minor modifications (schmaljohn et al., ; chu et al., ; boudreau et al., ) . htnvinfected monolayers were fixed days post-infection by ml of % formalin per well. immunostaining was performed as previously described (hooper et al., b) . plaques were stained using d -conjugated to hrp ( : , ) (life sciences) eliminating the need for a secondary antibody. all sera samples were assayed in duplicate beginning at a : dilution. pseudovirions were produced using the htnv, seov, puuv, dobv, andv, snv, and chocv dna vaccine plasmids described above. hek t cells were seeded in t tissue culture flasks and transfected with the plasmid of interest using transporter (polysciences inc) at ∼ % confluency. after ∼ h the transfection media was removed and the cells were infected with vsv g * rluc at a multiplicity of infection of ∼ . for h at • c. the media was removed and fresh media was added, the flasks were then incubated at • c for h. the supernatant from infected cells was collected and clarified by high speed centrifugation, followed by a peg , precipitation with . % salt. the peg mixture is spun at k xg for min. the pellet was resuspended overnight in ml tne buffer, then filtered using a . µm filter, aliquoted and stored at • c. the psvna utilizes a replication-restricted, recombinant vesicular stomatitis virus (rvsv * g) expressing luciferase, which is pseudotyped with the hantavirus glycoproteins-ofinterest. htnv dna plasmids containing the h y, k q, and h y/k q double mutation were manufactured by genewiz. the psvna used to detect neutralizing antibodies in sera was described previously (hooper et al., a; kwilas et al., ) . first, heat-inactivated sera were diluted : , followed by five-fold serial dilutions that were mixed with an equal volume of eagle's minimum essential medium with earle's salts containing, % fetal bovine sera (gibco), % pen-strep (gibco), and % human complement (cedarlane) containing , fluorescent focus units of pseudovirions. this mixture was incubated overnight at • c. following this incubation, µl was inoculated onto vero cell monolayers in a clear bottom, black-walled -well plate in duplicate. plates were incubated at • c for - h. the media was discarded and cells were lysed according to the luciferase kit protocol (promega, madison, wi, united states). a tecan m pro was used to acquire luciferase data. the values were graphed using graphpad prism (version ) and used to calculate the percent neutralization normalized to cells alone and pseudovirions alone as the minimum and maximum signals, respectively. the percent neutralization values for duplicate serial dilutions were plotted. eighty percent psvna (psvna ) and % psvna (psvna ) titers were interpolated from -parameter curves, and geometric mean titers were calculated. nau for this study were calculated by multiplying the psvna /ml x volume (ml) used. details for each statistical test completed are described (supplementary table ) . briefly, unpaired t-tests, two-tailed were conducted for, n-elisa titers for comparisons of hamsters of the same viral challenge dose ( figure e) , and marmoset n-elisa specific od sum comparisons between treatment and control groups ( figure b ). ordinary one-way anova with multiple comparisons test was conducted on n-elisa titers on remaining figures (figures , ) . analyses were conducted using graphpad prism (version ). previous experiments to produce anti-andv and anti-snv tcb human igg have demonstrated that including an sab-adj- adjuvant at the injection sight increased immunogenicity of the andv and snv dna vaccines resulting in higher titer virusspecific neutralizing antibodies (hooper et al., a) . more recently we found that lnp formulation increased the efficiency of dna vaccine immunogenicity in multiple species, including tcb (manuscript submitted). here we initially compared the response elicited by a htnv m segment based dna vaccine using either sab-adj- adjuvant or lnp formulation. one tcb (# ) was vaccinated with mg dna using the pharmajet stratis r needle-free disposable syringe jet injection device. sab-adj- adjuvant was administered by needle at the site of dna injection. this is similar to the vaccination strategy used previously, except here adjuvant was delivered with each vaccination instead of just the final boost (hooper et al., a) . a second tcb (# ) was vaccinated with lnpformulated htnv dna vaccine at a lower dose ( . mg per vaccination), using the pharmajet stratis r injection device ( figure a) . serum samples collected throughout the vaccination series were analyzed for neutralizing antibody titers using a pseudovirion neutralization assay (psvna). both bovines developed neutralizing antibodies against htnv at the first blood collection timepoint, which was weeks after the first dose. while tcb # initially had higher neutralizing titers (psvna ∼ fold higher on day and ∼fourfold higher on day ), tcb # ultimately yielded higher titers beginning after the third vaccination (psvna ∼fourfold higher on day and ∼fivefold higher on day ). anti-htnv tc human igg (hence forth referred to as sab- ) was purified from plasma collected weeks after the fourth vaccination. with the marked increase in htnv titer using the lnp-formulated vaccine, a third tcb (# ) was vaccinated with an lnp-formulated puuv dna vaccine according to a -dose vaccination schedule ( figure a) . very high titer anti-puuv antibody was detected as early as day , the first timepoint tested. after the last vaccination the psvna titer was well over , . anti-puuv tc human igg (hence forth referred to as sab- p) was purified from plasma following the third, fourth, and fifth vaccination. sab- ( . mg/ml) and sab- p ( . mg/ml) were evaluated for anti-htnv and anti-puuv neutralizing activity by psvna. high levels of neutralizing activity were detected for both purified antibody products. psvna titers are plotted in figure b . the psvna and psvna titers of sab- were , and , , respectively. the psvna and psvna titers of sab- p were , , and , , respectively. based on the psvna data and protein concentrations of the purified material, the inhibitory concentration % (ic ) of sab- is ng/ml and the ic is ng/ml. the ic of sab- p is ng/ml and the ic is ng/ml. levels of crossneutralization against other hantaviruses associated with hfrs or hps were determined by psvna. the psvna values for all heterotypic hantaviruses tested were at least -fold lower than both the anti-htnv and anti-puuv titers ( figure b) . sab- was also monitored for stability. no significant change in functional activity, as measured by prnt, was detected after months at • c ( figure c) . to evaluate whether sab- was neutralizing htnv by binding multiple neutralizing epitopes versus one dominant neutralizing epitope, an experiment was conducted involving pseudovirions engineered to escape known monoclonal antibodies. neutralizing monoclonal antibody (mab) escape mutants of htnv g n and g c have previously been reported (wang et al., ; kikuchi et al., ) . two escape mutations were engineered into the htnv pseudovirions, individually and in combination. mutation h y in the g n protein escapes mab- d and mutation k q in g c escapes mab-hco . the h y, k q, and h y/k q double mutant pseudovirions were used in the psvna to evaluate the neutralization escape resistance of sab- (figure ) . each monoclonal antibody, alone and in a : cocktail, were evaluated using the wild-type and mutant pseudovirions (figure a) . as predicted, mab- d neutralized wild-type htnv pseudovirions and pseudovirions with the mab-hco escape mutation; however, this antibody did not neutralize pseudovirions with the h y mutation or the h y/k q double mutation where the ic were > ng/ml. similarly, mab-hco neutralized wild-type htnv pseudovirions and pseudovirions with the mab- d escape mutation; however, mab-hco failed to completely neutralize pseudovirions with the k q mutation or the h y/k q double mutation (i.e., > -fold difference in ic versus wild-type). the : mab cocktail neutralized the single escape mutants but only partially neutralized the double mutant. in contrast, sab- was capable of neutralizing wildtype and both single and double neutralization escape mutant pseudovirions ( figure b) . the positive control anti-htnv rabbit polyclonal antibody also neutralized all pseudovirions whereas, the negative control rabbit sera or normal tcb purified igg had no neutralizing activity ( figure b) . these data demonstrate that the potent neutralizing activity of sab- is targeting multiple epitopes involving both the g n and g c proteins and is resistant to mutations that allow escape from neutralizing monoclonal antibodies. hantaan virus is highly infectious in syrian hamsters (id = . pfu) but does not cause disease (perley et al., ). the hamster model was used to evaluate the capacity of sab- to protect against infection. first, sab- was injected subcutaneously at a dose of mg/kg and sera were collected over several weeks. mg/kg was equivalent to a dose of , neutralizing antibody units (nau)/kg where a nau/ml was defined as the psvna titer/ml. sera were evaluated for neutralizing activity by psvna ( figure a) . data from the -week psvna curve was used to calculate an antibody half-life of . days ( figure b ). to determine the dose of sab- required to protect against infection, hamsters were administered decreasing concentrations of neutralizing antibody ranging from . mg/kg to . mg/kg sab- subcutaneously day prior to a pfu htnv challenge. negative control hamsters were treated with . mg/kg normal tcb human igg via the same route. five weeks after challenge infection status was evaluated by anti-n elisa (figure c ). an anti-n response is indicative of a productive hantavirus infection whereas the absence of an anti-n response indicates the animals were protected from infection. hamsters receiving either . mg/kg or . mg/kg sab- were % protected from infection ( / hamsters seronegative, p < . , see supplementary table for experiment demonstrated that a dose as low as . mg/kg, which was equivalent to , nau/kg, could protect against infection in this model. next, we designed an experiment to determine how far in advance sab- could be administered and still protect against htnv challenge ( figure d) . beginning on day - and following every week thereafter, groups of hamsters were administered mg/kg sab- by the subcutaneous route. on day , hamsters were challenged with pfu htnv. all hamsters administered sab- on days - , - , - , and - were protected from infection (p < . for all groups). a single hamster administered sab- on day - was not protected from infection ( / animals seronegative, p < . ). of the hamsters administered sab- on days - and - , % and % were protected, respectively (p = . and p = . , respectively). a single negative control hamster was uninfected ( / animals infected). this experiment demonstrated that a dose of mg/kg of sab- administration several weeks prior to exposure to htnv was capable of protecting against infection. to determine if sab- could protect against higher doses of htnv, groups of hamsters each were administered mg/kg sab- or normal tcb-derived igg on day - and then challenged with increasing concentrations of htnv corresponding to id , id , or id (figure e) . administration of sab- protected at least / hamsters in all groups including the highest, id , challenge dose (p < . ). hamsters administered mg/kg of normal igg were not protected regardless of challenge dose ( / infected). these data demonstrated that sab- at mg/kg could protect against at least times the id . the anti-htnv purified human igg (sab- ) prediluted at : and control polyclonal antibodies were evaluated by psvna. the psvna titers were plotted. the positive control was anti-htnv rabbit (rab) sera; the negative control was normal rabbit sera; and the negative control was purified igg antibody from a naïve tcb (negative igg). *indicates that the mutation(s) in the gngc resulted in a > -fold reduction in psvna titer (escape). limit of quantitation is (gray shaded area). figures c,d , the level of neutralizing antibody in the sera at the time of challenge was measured by psvna. the psvna titers (n = ) combined with the protection status (yes or no), were analyzed by logistic regression to determine the fiducial limits yielding the estimated probability of protective levels of serum neutralizing antibody ( table ) . results of the logistic regression indicated that log titer is significantly associated with protection outcome. each unit log increase in pre-challenge htnv psvna titer was associated with an increase in odds of protection against htnv of . times (p < . , or % ci = . , . ). in general, a psvna titer of > is predictive of a > % chance of protection. like syrian hamsters, marmosets can be infected with htnv as measured by seroconversion but do not develop disease (perley et al., ) . specifically, an intramuscular injection of , pfu of htnv resulted in high anti-n by elisa and high neutralizing antibody titers days after challenge. to test if sab- can protect marmosets from , pfu of htnv, animals were administered either . mg/kg (i.e., , nau/kg) sab- or control human igg subcutaneously day prior to htnv intramuscular challenge ( figure a) . infection status was measured by n-elisa days after exposure. elisa o.d. values for the negative control animals increased from < . to . - . indicating that the animals were productively infected. similarly, the psvna titers for the negative control animals went from below detection on day - and day to > , on day . in contrast, all marmosets given sab- exhibited minimal change in the n-elisa o.d. days after challenge indicating infection had been affected by treatment (p = . ) (figure b) . the animals receiving sab- had psvna titers between and , on day representing input neutralizing antibody that was bioavailable ( figure c) . the psvna titers dropped by day indicating that the input antibody had waned and that there was no evidence of a productive endogenous immune response in the sab- treated animals. even a treatment dose of , nau/kg ( mg/kg) sab- remained bioavailable in marmosets days after intraperitoneal injection (supplementary figure ) . these data demonstrate that sab- successfully limited infection with htnv in marmosets. an initial experiment to test the efficacy of the candidate anti-puuv product, sab- p, was conducted with groups of hamsters each receiving descending dosages of sab- p ranging from . mg/kg to . mg/kg administered day prior to a , pfu puuv challenge. negative control hamsters were administered . mg/kg of normal tcb igg. neutralizing antibody levels in hamster sera on day (day of challenge) were measured by puuv psvna and infection status weeks after challenge was determined by anti-n elisa ( figure a ). all hamsters receiving . mg/kg sab- p were protected from infection, / hamsters receiving . mg/kg sab- p were protected, / hamsters receiving . mg/kg were protected and / hamsters receiving . mg/kg were protected resulting in statistically significant levels of protection ( . mg/kg sab- p treatment group, p = . ). eight of negative control hamsters were infected ( figure a ). next, we designed two experiments to determine if sab- and sab- p were protective if administered after virus exposure. sab- and sab- p, administered at mg/kg corresponding to treatment doses of , nau/kg and , nau/kg, respectively, were administered on days - , + , + , and + relative to either a pfu htnv ( figure b ) or , pfu puuv (figure c ) challenge. in both experiments, purified human igg administered on day + , but not + , resulted in a statistically significant level of protection from virus infection (sab- p = . , sab- p p = . ). the htnv and puuv dna vaccines used to immunize the tcb in this study are the same vaccine constructs that are currently under evaluation in phase and clinical trials (brocato and hooper, ) . the anticipated use of those vaccines is as a pretreatment to protect individuals living, traveling or working in areas where puuv and htnv are known to cause hfrs. for persons traveling or working in disease-endemic areas, such as military personnel deployed or training in those areas, there might not be sufficient lead time to receive the full vaccine regimen and develop protective immunity. in those cases, receiving passively transferred antibody as a form of instant, albeit relatively short-lived, protective immunity could be a prudent course of action. such a product would need to be potent enough to require a single injection and, like any pretreatment, would need to have a negligible safety risk. a potent neutralizing antibody product could also be used as a post-exposure prophylactic for person at very high risk. for example, persons sleeping in the same tent or performing the same work duties as an hfrs patient would be at high risk of exposure. treatment with a neutralizing antibody product also has the potential to lower viremia and might be effective as a therapeutic to reduce disease severity. in the present study we have demonstrated that the polyclonal neutralizing antibodies are effective when administered pre-and post-exposure but we do not have disease models to obtain post disease onset efficacy. in ongoing hfrs clinical trials, the dna vaccines are not lnp-formulated and no adjuvant is used. however, in tcb it is possible to use lnp-formulated dna or adjuvants approved for veterinary use to increase the immunogenicity of the vaccines. using lnp-formulated dna, we were able to produce purified human polyclonal antibody batches with very high neutralizing activity: sab- had an ic of ng/ml and sab- p had an ic of ng/ml, respectively ( figure b) . these ic values were lower than two protective monoclonal antibodies targeting andv where the ic of purified antibodies ranged from to , ng/ml andv (garrido et al., ) , and similar to mab (ic ng/ml), a potent anti-ebov monoclonal antibody currently in clinical trials as a monotherapy (corti et al., ; mulangu et al., ) . we used the psvna titer data to determine ic and ic of purified antibody preparations, and we chose psvna to determine nau. the ic values are used to compare relative potency for different lots of purified product when the antibody concentration is known. the nau/ml are used to compare the relative potency of serum, plasma, and purified antibody regardless of whether protein concentration is known. previously we had used prnt titers to determine nau (brocato et al., ) ; however, data generated to date indicate that the htnv psvna and prnt titers are acceptably similar. the speed of the psvna relative to prnt (i.e., days versus up to weeks for the prnt) and the advantage of running outside of high containment make the psvna the logical assay choice for antibody-based product potency testing. polyclonal antibodies are, by definition, broadly active against multiple epitopes decreasing the chances that the infectious agent will develop drug resistance. we used pseudovirions with known mab escape mutations to confirm that multiple epitopes spanning both the gn and gc proteins are targeted by the polyclonal antibody. while mapping the full repertoire of epitopes bound by the polyclonal antibodies, or the full repertoire of functional activities of the antibodies, is beyond the scope of the current study, it is clear that the purified sab- and sab- p antibodies exhibit extremely high titer neutralizing antibody levels and levels of neutralizing antibody in serum can predict protection against infection with htnv and puuv. batch to batch variability for polyclonal antibody products exist; however, the potency of the product, as measured by a functional assay such as the psvna, can be used to set acceptance criteria for the candidate product. currently, mab development involves identification of human survivors or vaccinees followed by screening for candidate molecules and the cloning of those molecules into appropriate expression systems. using only the virus target sequence (e.g., hantavirus m gene sequence) it is possible to produce a dna vaccine and vaccinate tcb to produce fully human anti-hantavirus antibodies. there is no need to isolate a target virus for vaccine generation and there is no need for patient involvement at any stage. the speed of vaccination, plasma collection, and antibody purification ( - months) and the relatively low costs (compared to monoclonal antibody development) make tcb-derived polyclonal antibodies a promising choice for emerging or rare infectious diseases. when a monoclonal antibody is used to counter an infectious agent, such as a virus, a drawback can be the unintentional selection for mutants capable of escaping the antibody. for example, palivizumab is a monoclonal antibody used in children less than years old to prevent lower respiratory tract infections caused by respiratory syncytial virus (rsv). palivizumab escape mutants have been isolated from approximately % of the patients where breakthrough disease occurred after monoclonal antibody treatment (zhu et al., ) . in this example, there is no evidence that monoclonal antibody escape in patients resulted in worsened disease or shedding of resistant virus; however, these possibilities cannot be ruled out. to counter this, monoclonal antibody drugs are often in the form of combinations of multiple monoclonal antibodies. two out of the three monoclonal antibody treatments for ebola in the palm clinical trial in the democratic republic of congo are combinations of three anti-ebov monoclonal antibodies (sivapalasingam et al., ; mulangu et al., ) . polyclonal antibodies such as the tcbderived anti-htnv and anti-puuv described in this report, are capable of binding multiple targets and theoretically would therefore be resistant to escape mutation. using historically identified monoclonal antibody escape mutants in htnv g n and g c , we demonstrated that sab- was, in fact, capable of neutralizing pseudovirions engineered to escape multiple neutralizing monoclonal antibodies where "escape" was defined as a -fold reduction in ic . if an anti-hantavirus monoclonal antibody-based product were to be developed it would likely need to either consist of a cocktail of multiple protective monoclonal antibodies binding different target sites or a single monoclonal antibody binding an epitope that cannot change. to date, an antihantavirus protective monoclonal antibody un-mutable epitope has not been identified. plasma infusion of confirmed hps cases in chile demonstrated a decrease in case-fatality rate with borderline statistical significance (vial et al., ) . that plasma trial demonstrated the promise of anti-hantavirus antibodies as treatment of hantavirus disease, but also highlighted some of the drawbacks of using human convalescent plasma or serum. for any human-derived product there is the chance of infectious agent transmission and of non-infectious risks. routine screening for blood borne pathogens and the infectious agent of interest mitigates but does not completely abolish these risks. non-infectious risks, such as transfusion-associated acute lung injury or allergy, must also be considered with plasma therapy (mora-rillo et al., ) . similarly, abo blood typing is required to prevent hemolytic transfusion reactions. the limited availability of convalescent serum or plasma has an impact on dose and repeated treatments. high volumes of a standardized anti-hantavirus igg product, such as a tcb-derived product, would eliminate the need to identify volunteers, match blood type, maintain a scalable source, and eliminate the inherent safety concerns associated with any human tissue-based product. as no disease models for htnv and puuv infections exist, infection models have been characterized and are used for testing vaccines and neutralizing antibodies, with sterile immunity used as a measure of protection from infection (hooper et al., (hooper et al., , a brocato et al., ; witkowski et al., ; perley et al., ) . administration of low levels of sab- and sab- p are sufficient to protect from infection, with serum neutralizing antibody titers at > psvna required to protect > % of hamsters from infection against low-dose htnv or puuv challenges. similarly, marmosets administered sab- had ∼ -fold reduced levels of anti-htnv antibody after htnv challenge. future studies will further refine the marmoset model to maximize its use as a primate infection model for htnv. passive transfer of negative control igg from unvaccinated tcb in both the hamster and the marmoset models did not have any effect on infection demonstrating the protective efficacy of the injected human igg is not a non-specific innate immune response to the heterologous species immunoglobulin. the data presented here from the sab- characterization experiments have implications for the use of tcb-derived igg products in humans. calculated from the bioavailability experiment ( figure a) , sab- has a half-life of approximately days in syrian hamsters ( figure b) . the same concentration of sab- administered days prior to htnv challenge was protective in hamsters ( figure c) . the half-life of igg in humans is approximately days. as the tcb-derived anti-mers cov human igg sab- has the similar half-life as endogenous human igg (beigel et al., ) . while the precise half-life of sab- will need to be calculated in humans, these data suggest that sab- administered at a dosage of mg/kg could be bioavailable and protective in humans for > days. until pragmatic animal models of hfrs disease are developed, it will be difficult to assess whether anti-htnv and/or anti-puuv can act as true therapeutics to cure disease. it is possible that, even if a disease model exists, antibody-based products alone will be insufficient to reverse disease after onset. for example, we have found that anti-andv antibodies produced in rabbits, non-human primates, or using tcb, can protect hamsters pre-and post-exposure to andv, but cannot protect after the animal has become viremic (custer et al., ; haese et al., ) . it is possible that, for human cases, treatment with potent antihantavirus neutralizing antibodies combined with supportive care, and possibly in combination with broad spectrum antiviral drugs (e.g., ribavirin), will result in clinical benefit when treatment starts after disease onset. as neutralizing antibody titers correlate with disease prognosis in humans (macneil et al., ) , there is a distinct advantage to early hantavirus disease treatment. we have demonstrated previously in the andv/hamster disease model that neutralizing antibodies administered prior to the onset of viremia protect hamsters from lethal hps (hooper et al., (hooper et al., , a brocato et al., ; haese et al., ) . the htnv/ and puuv/hamster infection models can be considered more rigorous models as the antiviral must prevent infection, as these viruses do not cause disease in hamsters. while viremia is not detected until day in the pfu htnv/hamster model and is undetectable in the , pfu puuv/hamster model (perley et al., ) , treatment with sab- or sab- p within days is necessary to prevent infection as measured by seroconversion (figures b,c) . this is similar to a previous study showing the mab hco is protective and can eliminate viremia in an hfrs model when administered days post infection (liang et al., ) . limiting viremia by antibody passive transfer has the potential to reduce disease severity, providing further justification for anti-hfrs antibody development. it also highlights the need for an early biomarker that can be used as a trigger-to-treat. thus far, this remains elusive. this is the first report to show that human igg products can be successfully generated, purified, and efficacious at preventing infection by viruses that cause hfrs in multiple animal models. the potency of these products compared to previous anti-hps human igg products highlights the utility of using lnp formulation for increasing vaccine immunogenicity. the proofof-concept research described in this report lays the groundwork for future ind-enabling studies aimed at advancing antihantavirus human igg products toward a phase i clinical trial. the datasets generated for this study are available on 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monoclonal antibodies targeting ebola virus glycoprotein in healthy adults: a randomised, first-in-human phase study human polyclonal antibodies produced in transchromosomal cattle prevent lethal zika virus infection and testicular atrophy in mice isolation in vero-e cells of hanta virus from clethrionomys glareolus captured in the bashkiria area of the u a non-randomized multicentre trial of human immune plasma for treatment of hantavirus cardiopulmonary syndrome caused by andes virus epitope mapping studies with neutralizing and non-neutralizing monoclonal antibodies to the g and g envelope glycoproteins of hantaan virus gastrointestinal tract as entry route for hantavirus infection hantavirus infections in humans and animals prevalence and significance of substitutions in the fusion protein of respiratory syncytial virus resulting in neutralization escape from antibody medi the animal study was reviewed and approved by the usamriid institutional animal care and use committee (iacuc). cp, rb, hw, pc, es, and jh conceptualized and designed the study. cp, rb, hw, cb, pk, jv, mc, bs, sk, lp, js, and jh executed the study. hw, cb, pk, and jv provided the novel reagents and/or analytical tools. cp, rb, hw, cb, pk, jv, sk, and jh analyzed the data. cp, rb, sk, and jh wrote the manuscript. all authors contributed to manuscript revision, read, and approved the submitted version. we thank the usamriid veterinary medical division for technical assistance. the supplementary material for this article can be found online at: https://www.frontiersin.org/articles/ . /fmicb. . /full#supplementary-material conflict of interest: pc, es, and jh are inventors on patents, or patents pending, related to technology used to develop proof-of-concept antibody-based products described in manuscript (i.e., lipid nanoparticles, tc bovine, and dna vaccines). hw, cb, and es were employed by sab biotherapeutics inc. pk, jv and pc were employed by arcturus therapeutics inc.the remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential key: cord- -db kb c authors: park, jun-gyu; Ávila-pérez, ginés; madere, ferralita; hilimire, thomas a.; nogales, aitor; almazán, fernando; martínez-sobrido, luis title: potent inhibition of zika virus replication by aurintricarboxylic acid date: - - journal: front microbiol doi: . /fmicb. . sha: doc_id: cord_uid: db kb c zika virus (zikv) is one of the recently emerging vector-borne viruses in humans and is responsible for severe congenital abnormalities such as microcephaly in the western hemisphere. currently, only a few vaccine candidates and therapeutic drugs are being developed for the treatment of zikv infections, and as of yet none are commercially available. the polyanionic aromatic compound aurintricarboxylic acid (ata) has been shown to have a broad-spectrum antimicrobial and antiviral activity. in this study, we evaluated ata as a potential antiviral drug against zikv replication. the antiviral activity of ata against zikv replication in vitro showed median inhibitory concentrations (ic( )) of . ± . μm and . ± . μm in vero and a cells, respectively; without showing any cytotoxic effect in both cell lines (median cytotoxic concentration (cc( )) > , μm). moreover, ata protected both cell types from zikv-induced cytopathic effect (cpe) and apoptosis in a time- and concentration-dependent manner. in addition, pre-treatment of vero cells with ata for up to h also resulted in effective suppression of zikv replication with similar ic( ). importantly, the inhibitory effect of ata on zikv infection was effective against strains of the african and asian/american lineages, indicating that this inhibitory effect was not strain dependent. overall, these results demonstrate that ata has potent inhibitory activity against zikv replication and may be considered as a potential anti-zikv therapy for future clinical evaluation. zika virus (zikv) belongs to the genus flavivirus within the flaviviridae family. zikv is an enveloped positive sense single-stranded rna virus with a genome size of ∼ . kb that encodes a single polyprotein, which is post-translationally processed by cellular and viral proteases into three structural (capsid, c; pre-membrane, prm; and envelope, e) and seven non-structural (ns , ns a, ns b, ns , ns a, ns b, and ns ) proteins avila-perez et al., ) . zika virus was initially isolated from uganda in and viral infections only occurred sporadically in africa and asia until . zikv appeared explosively as the first large-scale outbreak occurred in the yap island in and french polynesia in (weaver et al., ) . most recently, in , the first local transmission of zikv was found in territories of latin america and the caribbean, resulting in up to . million of zikv infection suspected cases (tang et al., ; tripathi et al., ) . like other members of the flaviviridae family, such as yellow fever virus (yfv), dengue virus (denv), japanese encephalitis virus (jev), and west nile virus (wnv), zikv is commonly transmitted by the bite of infected aedes mosquitos, but it can also be transmitted vertically from mother to child, through sexual contact, and in rare cases from blood transfusions (lessler et al., ; fink et al., ) . upon infection, zikv can be shed in blood, urine, semen, saliva, amniotic fluid, breast milk, and cerebrospinal fluid (nayak et al., ; colt et al., ; nazerai et al., ) . most people ( ∼ %) infected with zikv are asymptomatic or have mild symptoms such as fever, rash, joint pain, and conjunctivitis that can last for several days to a week (fink et al., ) . in rare cases, people with symptoms may have neurological guillain-barré syndrome complications (oehler et al., ; rivera-concepcion et al., ; nazerai et al., ) . in the case of pregnant women, zikv infection can lead to microcephaly and other fetal complications as occurred during the large-scale zikv outbreak in brazil in (lessler et al., ) . because of the significant outbreaks in south, central, and north america, zikv was declared a public health concern by the world health organization (who) in february (lazear and diamond, ; ramos da silva and gao, ; weaver et al., ; tripathi et al., ) . there are several vaccines and antiviral drugs currently under development for the prevention or treatment of zikv infection larocca et al., ; shan et al., ; fink et al., ) . dna-based larocca et al., ) , inactivated larocca et al., ; shan et al., ) , live-attenuated and mrna (richner et al., ) vaccines have been proposed for the prophylactic treatment of zikv infections. on the other hand, arbidol (arb) (fink et al., ; haviernik et al., ) , bortezomib, mycophenolic acid, daptomycin (barrows et al., ) , obatoclax, saliphenylhalamide, gemcitabine (kuivanen et al., ) , emetine (yang et al., ) , and sofosbuvir (bullard-feibelman et al., ) have been proposed for the therapeutic treatment of zikv infection. despite these tremendous efforts, there is currently no food and drug administration (fda)-approved vaccines and/or anti-viral drugs available for the treatment of zikv infection. since vaccination takes at least weeks to several months to show protective effects against zikv infection, vaccination is probably not the most appropriate prophylactic method for those who are traveling to areas where zikv is epidemic, endemic, or have already been infected. moreover, vaccination may cause an important issue, such as antibody-dependent enhancement (ade) priyamvada et al., ) . ade, which has been extensively described in denv (priyamvada et al., ) , is a phenomenon where preexisting antibodies facilitate binding and infection during subsequent exposure to infectious viruses, instead of neutralizing them, resulting in exacerbation of clinical signs priyamvada et al., ) . because of the structural similarities between denv and zikv, denv immunity-linked ade of zikv infection has also been reported priyamvada et al., ) . since vaccination for zikv could lead to denv ade, antivirals could represent a better choice for the control of zikv infection. aurintricarboxylic acid (ata), a polyanionic aromatic compound, has been shown to have inhibitory properties against several bacteria and viruses including, among others, yersinia pestis (liang et al., ) , cryptosporidium parvum (klein et al., ) , human immunodeficient virus (hiv) (mitra et al., ; de clercq, ) , hepatitis c virus (hcv) mukherjee et al., ; shadrick et al., ) , vaccinia virus (myskiw et al., ) , influenza virus (hung et al., ) , enterovirus (hung et al., ) and severe acute respiratory syndrome coronaviruses (sars-cov) (he et al., ) . mechanistic studies have suggested that ata has the ability to modulate various cellular enzymes such as activators of the janus kinase (jak ) and signal transducer and activator of transcription (stat ) families (rui et al., ) , inhibitors of nucleases (shadrick et al., ) , glucose- -phosphate dehydrogenase (bina-stein and tritton, ) , and topoisomerase ii proteins (catchpoole and stewart, ; benchokroun et al., ) as well as the enzymatic activity of the vaccinia virus ah l phosphatase (smee et al., ) . however, to date, the ability of ata to inhibit zikv infection has not been evaluated. herein, we investigated ata as a plausible prophylactic and therapeutic candidate against zikv infection. our results demonstrate that ata has a potent and effective antiviral activity against zikv in pre-and post-infection settings, including broadly antiviral activity against strains of the african and american/asian lineages with no toxicity up to , µm in cultured cells. these data support the feasibility of implementing ata for the treatment of zikv infection. african green monkey kidney epithelial vero (atcc ccl- ) and human adenocarcinoma alveolar basal epithelial a (atcc ccl- ) cells were maintained in dulbecco's modified eagle's medium (dmem; mediatech, inc.) supplemented with % fetal bovine serum (fbs) and % psg ( u/ml penicillin, µg/ml streptomycin, and mm l-glutamine) at • c in a % co atmosphere. paraiba/ zikv isolate was kindly provided by stephen dewhurst (department of microbiology and immunology, university of rochester). uganda/ (mr_ strain, catalog no. nr- ) and nigeria/ (ibh strain, catalog no. nr- ) zikv isolates were obtained from the biodefense and emerging infections research resources repository (bei resources). puerto rico/ (prvabc strain) and french polynesia/ zikv isolates were kindly provided from the centers for disease control and prevention (cdc). virus stocks were propagated in vero cells and titrated by plaque assay as previously described (marquez-jurado et al., ) . aurintricarboxylic acid (catalog no. a ) and arbidol (arb, catalog no. slm ) were purchased from sigma-aldrich, mo, united states. both compounds were prepared at mm stock solution dissolved in dimethyl sulfoxide (dmso) and kept at − • c until experimental use. each drug was diluted into infectious media (dmem % fbs, % psg) for the described experiments, where the maximum dmso concentration was . %. cell viability in vero and a cells was measured using the celltiter non-radioactive cell proliferation assay (promega) following the manufacturer's instructions. briefly, confluent vero or a cells ( -well plate format, × cells/well, triplicates) were treated with µl of dmem containing serially diluted (twofold dilutions, starting concentration of , µm) chemicals or . % dmso (vehicle control). plates were incubated at • c in a % co atmosphere for or h. samples were treated with µl of dye solution and incubated at • c in a % co atmosphere for h. next, cells were treated with µl of solubilization solution/stop mix and absorbance at nm was measured using a vmax kinetic microplate reader (molecular devices, waltham, ma, united states). viability of compound-treated cells was calculated as a percentage relative to values obtained with dmso-treated cells. non-linear regression curves and the median cytotoxic concentration (cc ) were calculated using graphpad prism software version . . confluent monolayers ( -plate format, × cells/well, triplicates) of vero cells were infected with plaque forming units (pfu)/well of paraiba/ , uganda/ , nigeria/ , puerto rico/ , and french polynesia/ at • c in infection media. after h of adsorption, virus inoculum was removed and cells were washed three times with infection media before adding fresh infection media containing % microcrystalline cellulose (avicel, sigma-aldrich) and the indicated concentration of compounds, or . % dmso as vehicle control. in case of pre-treatment experiments, the cell monolayers were treated with the indicated concentration of compound, or . % dmso, for the indicated times before zikv infection. infected cells were incubated at • c for - h, depending on virus strains. for immunostaining, cells were fixed with % paraformaldehyde for h, washed three times with phosphate buffered saline (pbs) and permeabilized with . % triton x- for min at room temperature. then, the plates were blocked with . % bovine serum albumin (bsa) in pbs (blocking solution) for h at room temperature, followed by incubation with µg/ml of the pan-flavivirus envelop (e) protein monoclonal antibody g (atcc, catalog no. vr- ) diluted in blocking solution for h at • c. after incubation with the primary antibody, cells were washed three times with pbs and developed with the vectastain abc kit and the dab peroxidase substrate kit (vector laboratory, inc., ca, united states) according to the manufacturers' instructions. stained plaques were analyzed using the ctl immunospot plate reader and counting software (cellular technology limited, cleveland, oh, united states). virus titers were calculated as pfu/ml (nogales et al., ) . non-linear regression curves and the median inhibitory concentration (ic ) were determined as described above. confluent monolayers ( -well plate format, . × cells/well, triplicates) of vero or a cells were infected (multiplicity of infection, moi, . ) with paraiba/ diluted in infection media for h at room temperature. after viral absorption, cells were incubated with infection media containing the indicated concentrations ( , , . , and µm) of ata. at , , , and h post-infection (h p.i.), tissue culture supernatants were collected and titrated on vero cells by immunostaining as described previously (marquez-jurado et al., ) . levels of apoptosis were measured using the caspase-glo r / assay (promega, wi, united states) following the manufacturer's instruction. briefly, vero and a cells ( -well plate format, . × cells/well, triplicates) were infected with zikv paraiba/ (moi of . ) and, at the indicated times post-infection, cells and tissue culture supernatants were collected and centrifuged. twenty five microliters of supernatants were mixed with µl of caspase- / reagent using a plate shaker, incubated at room temperature for h, and luminescence at nm was measured using a spectramax id (molecular devices, waltham, ma, united states) following the manufacturer's instructions. two-way anova was used to evaluate significant differences. data are expressed as the mean ± standard deviation (sd) of at least three independent experiments in triplicates using microsoft excel software. value were considered statistically significant when * p < . , * * p < . , * * * p < . , * * * * p < . . all data were analyzed with prism software version . (graphpad software, ca, united states). cc and ic were determined using sigmoidal dose response curves (graphpad software, ca, united states). the selective index (si) of each compound was calculated by dividing the cc with the ic . before examining the inhibitory effect of ata (figure ) against zikv infection, we first determined the cc of ata on vero and a cells (figure ) . for this, we treated both cell lines with serial (twofold) dilutions of ata and measured cell viability at and h post-treatment. as an internal control for these studies, we used arb, a drug that has been previously described to have antiviral activity against zikv in vero (haviernik et al., ) and a (fink et al., ) cells. we did not observe any toxicity with ata in vero (figure a ) or a ( figure b ) cells at or h post-treatment, even at the highest concentration tested ( , µm), while arb showed cc values of . ± . or . ± . µm in vero ( figure c ) and . ± . or . ± . µm in a ( figure d ) cells (table ) at or h post-treatment, respectively. to determine the ic of ata, vero and a cells were infected with pfu/well of paraiba/ and after h of viral absorption, virus inoculum was replaced with infection media with twofold serial dilutions (starting concentration of , µm) of ata or arb (figure ) and the ic calculated as described in the section "materials and methods." although the ic of ata ( figure a) and arb ( figure c) in vero cells were similar ( . ± . µm and . ± . µm, respectively), the selective index (si, cc /ic ) of ata (> . ) was significantly higher than that of arb ( . ) ( table ) . likewise, the ic of ata ( figure b ) and arb ( figure d ) in a cells were similar but with clearly different si values (> . for ata and . for arb) ( table ) . notably the cc , ic , and si of arb were similar to those previously described in the literature in these cell lines (fink et al., ; haviernik et al., ) . these data suggest that ata exhibited an effective inhibition of zikv infection with limited toxicity and si values better than those previously described for arb. we also observed that zikv replication was completely inhibited at a concentration of µm of ata in vero ( figure a ) and a ( figure b ) cells while . µm and µm concentrations of ata showed partial viral inhibition in vero and a cells (figures a,b) , respectively, demonstrating a dose-dependent inhibition of viral replication in both cell lines. we next evaluated the ability of ata to protect cells from the cytopathic effect (cpe) induced during zikv infection ( figure ) . to that end, vero and a cells were infected (moi . ) with paraiba/ and, after h of viral absorption, cells were treated with , . , , and µm of ata. at h p.i., cells were observed under a light microscope for evaluation of their morphology and cpe ( figure a ). as expected from our previous results, µm and more clearly µm of ata were able to prevent zikv-induced cpe in both cell lines (figure a) . to quantify the ability of ata to prevent zikv-induced apoptosis, tissue culture supernatants from zikv-infected vero and a cells were harvested at , , and h p.i. to measure the level of apoptotic signal as determined by caspase and activities ( figure b) . zikv-infected cells showed figure | aurintricarboxylic acid inhibition of zikv replication: vero (a) and a (b) cells ( -well plate format, . × cells/well, triplicates) were infected (moi . ) with paraiba/ . tissue culture supernatants were collected at , , and h p.i., and viral titer were calculated by immunostaining (fluorescent forming units, ffu/ml). dotted line indicates the limit of detection ( ffu/ml). data was expressed as mean and standard deviations (sd) from three independent experiments conducted in triplicates. statistical analysis was conducted by two-way anova, * p < . , * * p < . , * * * p < . , * * * * p < . , or no significance (n.s.). figure | aurintricarboxylic acid protects vero and a cells from zikv-induced cell death: vero and a cells ( -well plate format, . × cells/well, triplicates) were infected (moi . ) with paraiba/ . after h viral adsorption, cells were treated with the indicated concentrations ( , , . , and µm) of ata. at h p.i., cells were observed and imaged under an optical microscope. scale bar = µm. (a) caspase / levels were measured in the tissue culture supernatants at , , and h p.i. (b) data of each time point was compared to mock-infected control cells and expressed as mean of relative percentage and sd from three independent experiments conducted in triplicates. statistical analyses were conducted by two-way anova, * p < . , * * p < . , * * * p < . , * * * * p < . , or no significance (n.s.). increased caspase and levels up to eightfolds in vero cells and up to . -folds in a cells compared to mock-infected cells ( figure b ). levels of caspase and activation were dose-dependently reduced by ata with µm of ata showed only . -and . -fold induction as compared to mock-infected vero and a cells, respectively ( figure b ). we next determined whether ata is able to inhibit both ancestor african (uganda/ and nigeria/ ) and contemporary asian/american (puerto rico/ and french polynesia/ ) zikv lineage strains using our microplaque reduction assay (figure ) . we observed similar ic values of ata with uganda/ ( figure a , ic = . ± . µm), nigeria/ ( figure b , ic = . ± . µm), puerto rico/ ( figure c , ic = . ± . µm), and french polynesia/ ( figure d , ic = . ± . µm), compared to those observed with paraiba/ (figure and table ), demonstrating the broad antiviral activity of ata against different zikv strains, regardless of the year and place of isolation. to demonstrate the feasibility of using ata for the prevention of zikv infection, important for travelers to regions where zikv is endemic, we next evaluated whether pre-treatment with ata results in inhibition of zikv replication (figure ) . to that end, vero cells were pre-treated with ata for ( figure a) , ( figure b) , (figure c) , or ( figure d ) h prior to infection (moi . ) with paraiba/ . pre-treatment with ata for - h before zikv infection resulted in similar ic values ( . ± . µm, . ± . µm, . ± . µm, and . ± . µm; respectively) demonstrating that ata is stable and able to prevent zikv infection even when administered days previous to viral infection (figure and table ). the recent outbreak of zikv accompanied with severe pathology, including microcephaly in newborns, prompted many researchers to develop prophylactic vaccines and to identify therapeutic drugs against zikv infection larocca et al., ; lessler et al., ; shan et al., ; fink et al., ) . currently, there are no commercially available vaccines and/or antiviral therapies for the treatment of zikv infection. therefore, there is an urgent medical need for the development of effective counter measurements to control zikv infection. in this study, we demonstrated that ata (figure ) has limited toxicity (figure ) and an effective and dose-dependent antiviral activity against zikv infection (figures , ) in both monkey kidney epithelial vero and human alveolar a cells. notably, ata can prevent zikv-induced cpe and apoptosis in both cell lines ( figure ) and has broad anti-viral activity against representative zikv strains from the african (uganda/ and nigeria/ ) and the asian/american (puerto rico/ and french polynesia/ ) lineages (figure ) . moreover, ata can also prevent zikv infection even when administered days before infection (figure ) . aurintricarboxylic acid is a polyanionic aromatic compound that structurally relates to suramin (balzarini et al., ) and is believed to influence over host and viral enzymes (shadrick et al., ) . although the exact mechanism by which ata inhibits zikv infection was not identified in this study, there are several plausible mechanisms on zikv inhibition mediated by ata, including the targeting of viral and cellular proteins. in terms of inhibiting viral proteins, ata could bind to zikv ns helicase and prevent its binding to either atp or nucleic acids, as previously described for hcv (mukherjee et al., ; shadrick et al., ) . likewise, ata could inhibit the zikv rna-dependent rna polymerase (rdrp) ns protein, as described for hcv mukherjee et al., ; shadrick et al., ) and enterovirus (hung et al., ) . similarly, ata could inhibit the methyltransferase activity of ns involved in mrna capping processes, as previously described for other flaviviruses (denv and yfv) (milani et al., ; garcia et al., ) . because of the structural similarities between denv and zikv ns proteins, it is feasible that, similar to denv, ata binds to ns to inhibit zikv infection . moreover, it is possible that ata targets and has inhibitory activities against one or more of the viral proteins described above. in terms of targeting cellular proteins important for the efficient replication of zikv, it has been previously described that ata has anti-apoptotic properties in a variety of cells (chen et al., ) . it is possible that the anti-apoptotic activity of ata protects against zikv-induced cell death, as demonstrated in this study (figure ) . notably, it has been recently shown that zikv infection induced apoptosis through caspase and in a cells and through caspase in neonatal mice brain (huang et al., ; frumence et al., ) . these results suggest that inhibition of zikv replication results in a decrease in the level of apoptotic cells and that the anti-apoptotic effect of ata affects zikv replication. further research is guaranteed to yield a better understanding of the antiviral activity of ata on zikv infection, and other viruses, before the use of ata as an antiviral drug. during january to february , a total of residents from states in the united states were diagnosed with zikv infection (armstrong et al., ) . out of patients, ( %) traveled to areas of active zikv transmission before the infection and five ( %) did not travel but reported sexual contact with a traveler who had a symptomatic illness (armstrong et al., ) . for these reasons, preventive efforts are required prior to travel to areas of active zikv transmission. in this study, cells pretreated with ata for up to h prior to infection with zikv showed similar ic than those in post-treatment settings, potentially suggesting that ata might target a cellular protein required for zikv replication or that the concentration and stability of ata in pre-treated cells is sufficient to inhibit zikv infection, or both. nevertheless, these results demonstrate the feasibility of using ata for the prophylactic treatment of viral infection, including those traveling to areas where zikv is endemic. moreover, due the broad inhibition effect of ata against others viruses and parasites (liang et al., ; he et al., ; de clercq, ; myskiw et al., ; klein et al., ; chen et al., ; hung et al., hung et al., , mukherjee et al., ; shadrick et al., ) that are present in zikv endemic areas, treatment with ata could be used for the broad prevention of denv, yfv (milani et al., ; shadrick et al., ; garcia et al., ) , hcv mukherjee et al., ; shadrick et al., ) , and parasitic infestation (cryptosporidium parvum) (klein et al., ) for people traveling to these endemic regions. moreover, the broad spectrum antiviral activity of ata against different african and asian/american zikv strains further guarantees the feasibility of implementing ata to prevent zikv infection to travelers around the world. although ata has been amply evaluated in vitro, only few studies have assessed the activity of ata in vivo, including its use as a curative agent against thrombosis (strony et al., ) , apoptosis (roberts-lewis et al., ; heiduschka and thanos, ) , parasite infestations (klein et al., ) , bacterial (y. pestis) (liang et al., ) , and vaccinia virus (smee et al., ) infections. in the case of vaccinia virus, ata did not protect mice from a lethal challenge at a dose of mg/kg/day (smee et al., ) . further studies are needed to evaluate the anti-viral activity of ata in vivo for the treatment of viral infections, including zikv. our studies show limited toxicity, if any, of ata in cultured cells, including human a cells. the lack of knowledge about the use of ata in pregnant women requires future additional safety tests, including studies using validated animal models of zikv infection, before using ata for the treatment of zikv infection during pregnancy. based on the effectiveness of ata against zikv infection (si = . in vero cells and . in a cells) as compared to other previously described drugs, including emetine (si = . in snb- cells and . in env+ cells) (yang et al., ) , obatoclax [si = in human retinal pigment epithelial (rpe) cells] (kuivanen et al., ) , saliphenylhalamide (si > in rpe cells) (kuivanen et al., ) , gemcitabine (si > , in rpe cells) (kuivanen et al., ) , sofosbuvir (si > . in huh- cells and . in jar cells) (bullard-feibelman et al., ) and arb (si = . in vero cells and . in a cells) (fink et al., ; haviernik et al., ) and this study (ata, in vero cells, and . in a cells), it is possible that ata represents one of the most reasonable options of the treatment of zikv infection. protective efficacy of multiple vaccine platforms against zika virus challenge in rhesus monkeys travel-associated zika virus disease cases among u.s. residents-united states reverse genetic approaches for the generation of recombinant zika virus aurintricarboxylic acid and evans blue represent two different classes of anionic compounds which selectively inhibit the cytopathogenicity of human t-cell lymphotropic virus type iii/lymphadenopathy-associated virus enhancement of zika virus pathogenesis by preexisting antiflavivirus immunity a screen of fda-approved drugs for inhibitors of zika virus infection aurintricarboxylic acid, a putative inhibitor of apoptosis, is a potent inhibitor of dna topoisomerase ii in vitro and in chinese hamster fibrosarcoma cells aurintricarboxylic acid is a nonspecific enzyme inhibitor the fda-approved drug sofosbuvir inhibits zika virus infection inhibition of topoisomerase ii by aurintricarboxylic acid: implications for mechanisms of apoptosis inhibition of cytokine-induced jak-stat signalling pathways by an endonuclease inhibitor aurintricarboxylic acid characterization of aurintricarboxylic acid as a potent hepatitis c virus replicase inhibitor transmission of zika virus through breast milk and other breastfeeding-related bodily-fluids: a systematic review emerging anti-hiv drugs the antiviral drug arbidol inhibits zika virus the south pacific epidemic strain of zika virus replicates efficiently in human epithelial a cells leading to ifn-beta production and apoptosis induction inhibitors compounds of the flavivirus replication process arbidol (umifenovir): a broad-spectrum antiviral drug that inhibits medically important arthropod-borne flaviviruses potent and selective inhibition of sars coronavirus replication by aurintricarboxylic acid aurintricarboxylic acid promotes survival and regeneration of axotomised retinal ganglion cells in vivo zika virus infection during the period of maximal brain growth causes microcephaly and corticospinal neuron apoptosis in wild type mice inhibition of enterovirus replication and the viral d polymerase by aurintricarboxylic acid aurintricarboxylic acid inhibits influenza virus neuraminidase in vitro and in vivo activity of aurintricarboxylic acid preparations against cryptosporidium parvum obatoclax, saliphenylhalamide and gemcitabine inhibit zika virus infection in vitro and differentially affect cellular signaling, transcription and metabolism vaccine protection against zika virus from brazil zika virus: new clinical syndromes and its emergence in the western hemisphere assessing the global threat from zika virus aurintricarboxylic acid blocks in vitro and in vivo activity of yoph, an essential virulent factor of yersinia pestis, the agent of plague an alanine-to-valine substitution in the residue of zika virus ns a protein affects viral rna synthesis and attenuates the virus in vivo flaviviral methyltransferase/rna interaction: structural basis for enzyme inhibition hiv- upregulates fas ligand expression in cd + t cells in vitro and in vivo: association with fas-mediated apoptosis and modulation by aurintricarboxylic acid identification and analysis of hepatitis c virus ns helicase inhibitors using nucleic acid binding assays aurintricarboxylic acid inhibits the early stage of vaccinia virus replication by targeting both cellular and viral factors pathogenesis and molecular mechanisms of zika virus a 'furrytale' of zika virus infection: what have we learned from animal models? replication-competent influenza a viruses expressing a red fluorescent protein zika virus infection complicated by guillain-barre syndrome-case report, french polynesia humoral immune responses against zika virus infection and the importance of preexisting flavivirus immunity zika virus: an update on epidemiology, pathology, molecular biology, and animal model modified mrna vaccines protect against zika virus infection the zika virus: an association to guillain-barre syndrome in the united states -a case report aurintricarboxylic acid protects hippocampal neurons from nmda-and ischemia-induced toxicity in vivo activation of the jak -stat signaling pathway in nb lymphoma cells by an anti-apoptotic agent, aurintricarboxylic acid aurintricarboxylic acid modulates the affinity of hepatitis c virus ns helicase for both nucleic acid and atp a live-attenuated zika virus vaccine candidate induces sterilizing immunity in mouse models lack of efficacy of aurintricarboxylic acid and ethacrynic acid against vaccinia virus respiratory infections in mice aurintricarboxylic acid in a canine model of coronary artery thrombosis zika virus infects human cortical neural progenitors and attenuates their growth a novel zika virus mouse model reveals strain specific differences in virus pathogenesis and host inflammatory immune responses zika virus: history, emergence, biology, and prospects for control emetine inhibits zika and ebola virus infections through two molecular mechanisms: inhibiting viral replication and decreasing viral entry key: cord- -f rto t authors: loens, katherine; ieven, margareta title: mycoplasma pneumoniae: current knowledge on nucleic acid amplification techniques and serological diagnostics date: - - journal: front microbiol doi: . /fmicb. . sha: doc_id: cord_uid: f rto t mycoplasma pneumoniae (m. pneumoniae) belongs to the class mollicutes and has been recognized as a common cause of respiratory tract infections (rtis), including community-acquired pneumonia (cap), that occur worldwide and in all age groups. in addition, m. pneumoniae can simultaneously or sequentially lead to damage in the nervous system and has been associated with a wide variety of other acute and chronic diseases. during the past years, the proportion of lrti in children and adults, associated with m. pneumoniae infection has ranged from to more than %. this variation is due to the age and the geographic location of the population examined but also due to the diagnostic methods used. the true role of m. pneumoniae in rtis remains a challenge given the many limitations and lack of standardization of the applied diagnostic tool in most cases, with resultant wide variations in data from different studies. correct and rapid diagnosis and/or management of m. pneumoniae infections is, however, critical to initiate appropriate antibiotic treatment and is nowadays usually done by pcr and/or serology. several recent reviews, have summarized current methods for the detection and identification of m. pneumoniae. this review will therefore provide a look at the general principles, advantages, diagnostic value, and limitations of the most currently used detection techniques for the etiological diagnosis of a m. pneumoniae infection as they evolve from research to daily practice. days or even several weeks and are therefore not relevant for the management of acute illness. alternative diagnostic procedures were developed: detection of igm and/or igg by elisa, antigen detection by immunochromatography, and nucleic acid amplification techniques (naats), mainly pcr, although also isothermal amplification techniques such as lamp (loop-mediated isothermal amplification method) have been developed. the utility of culture for m. pneumoniae was assessed by comparing it to pcr and igm serology in a large study (she et al., ) . given the extremely low yield of culture and the wide availability of naat and serology, the authors concluded that culture for m. pneumoniae should be discontinued. nowadays, most studies are serology and/or pcr-based. different clinical specimens can be used as described in the review by loens et al. ( ) for the latter. pcr is accepted as a rapid diagnostic test. few of the currently available naats have been extensively validated against culture. the sensitivity of naats is almost always superior to that of traditional procedures and they are more and more considered as the "new gold standard." an increasing body of literature describing the use of inhouse naats for detection of m. pneumoniae dna or rna in various diseases is available with a great variation of methods used from study to study, including variability of target (p adhesin gene, s rrna, atpase gene, protease gene, cards toxin gene), naat (conventional, nested, real-time; monoplex vs. multiplex; pcr vs. isothermal amplification technologies), detection formats, and different platforms. an overview of the literature on the use of naats to detect m. pneumoniae since is given in two reviews (loens et al., (loens et al., , a . lately, efforts have been mainly emphasized on the development of multiplex assays (nummi et al., ; shen et al., ) and on the evaluation of commercially available assays. respiratory viruses and other so called "atypical bacteria" are all responsible for rtis that may produce clinically similar manifestations. in order to reduce costs and hands-on-time, multiplex naats for the simultaneous detection of , , or up to more than different respiratory pathogens in one tube with a mixture of primers have been developed by some groups. however, comparison between mono-and multiplex assays has been rarely performed. findings and conclusions result frequently in contradictory and conflicting data concerning the sensitivity and specificity of the multiplex naats compared to the mono naats. this is not unexpected since the presence of several pairs of primers may increase the probability of mispairing resulting in non-specific amplification products and the formation of primer-dimers. furthermore, enzymes, primers, and salt concentrations as well as temperature cyclings required for each target may be slightly different. the results of the proficiency panels (loens et al., b (loens et al., , described previously seem to confirm that multiplex assays are somewhat less sensitive than monoplex assays but until the number of organisms present in clinical specimens of diseased individuals is known, it is impossible to state whether the degree of sensitivity attained is clinically acceptable. since the previous review (ieven and loens, ) new naats became commercially available such as the illumigene (meridian bioscience, usa) kit. it has been proposed that industryproduced assays in kit form result in better standardization. the analytical sensitivity of the illumigene assay was evaluated by using frozen stock cultures of m. pneumoniae reference strains, and a collection of other microorganisms and human dna. (ratliff et al., ) . serial dilutions of cultures with a known cfu/ml defined the analytical sensitivity at ≤ cfu/ml. based on the results obtained with archived respiratory specimens, previously cultured for m. pneumoniae, the clinical sensitivity and specificity were found to be and %, respectively, after resolving discrepancies by pcr and sequencing. a second example of a test approved for the detection of a number of respiratory viruses by the us food and drug administration is the filmarray respiratory panel (biomérieux, france). the filmarray is a small desktop closed single-piece flow real-time pcr system. it includes automation of nucleic acid extraction, an initial reverse transcription and multiplex pcr, followed by singleplex second stage pcr reactions for the detection of viral agents including adenovirus, coronavirus hku , coronavirus nl , human metapneumovirus, rhinovirus/enterovirus, influenza a/b, influenza a h , ah , a h , parainfluenza - , and respiratory syncytial virus (poritz et al., ) . in may , the us food and drug administration expanded the use for the filmarray respiratory panel with the addition of b. pertussis, m. pneumonia, and c. pneumoniae. the expanded panel detects now a total of viruses and three bacteria. the test requires min hands-on-time and min instrumentation time. in , a new version of the filmarray (version . ) was released (doern et al., ) . the argene respiratory mwsr-gene concept allows the detection of numerous pathogens (influenza a/b, respiratory syncytial virus/human metapneumovirus, rhinovirus/enterovirus, adenovirus/bocavirus, chlamydia/ mycoplasma pneumoniae, human coronavirus/parainfluenza virus, bordetella, bordetella parapertussis) in the same run. in addition, the diagnostic strategy can be adapted to the season: searching for the most likely pathogens can be considered in st stage, the remaining pathogens being searched for systematically in a nd stage. pillet et al. ( ) compared six commercially available multiplex assays for the diagnosis of respiratory pathogens. two out of six were also capable of detecting m. pneumoniae: the respifinder smart (pathofinder, the netherlands) and the seeplex rv onestep ace detection and pneumobacter ace detection (seegene inc, south korea). sensitivities and specificities were calculated against the argenechla/myco pneumo assay (biomérieux, france). sensitivity and specificity were . and %, respectively, for the respifinder assay and . and . % for the seegene assay. dumke et al. compared four commercially available real-time pcr assays recommended for use with the roche lightcycler . and . instruments [diagenode mycoplasma/chlamydophila pneumoniae real-time pcr (diagenode, belgium), geneproof m. pneumoniae (geneproof, czech republic), bactoreal m. pneumoniae (ingenetix, austria), lightmix kit m. pneumoniae (tib molbiol, germany)] for the detection of m. pneumoniae to results obtained with an in-house approach (dumke and jacobs, ) by using serial dilutions of a cultured m. pneumoniae strain tested in eight parallel runs and clinical specimens, previously found to be m. pneumoniae positive by the in-house assay. all naats detected colony forming units (cfu)/ µl sample. only the in-house-test (repmp -based approach) was able to detect . cfu/ µl sample. / , / , / , / m. pneumoniae positive clinical specimens were confirmed by the diagenode test, the ingenetix and lightmix assay, and the geneproof assay respectively. an overview of commercially available naats for the detection of m. pneumoniae is presented in table . since the calculation of the sensitivities of the commercial multiplex assays was mainly dependent on dna copy number, further evaluation and standardization using an extended number of clinical specimens that may have a low bacterial load are needed. the use of an international standard developed by the who for harmonization of mycoplasma naat (nübling et al., ) or the yearly participation in the external quality assessment (eqa) panel for m. pneumoniae and chlamydophila pneumoniae available from quality control for molecular diagnostics (qcmd, united kingdom) should be considered. so far, it is unclear whether asymptomatic carriage of m. pneumoniae in adults and children exists and if colonization could be differentiated from infection by the current diagnostic methods. there are only few data on the relation between the bacterial load and the severity of infection. asymptomatic children and children with a rti were enrolled in a cross-sectional study (spuesens et al., ) . nasopharyngeal washings and pharyngeal swabs were investigated by culture and quantitative real-time pcr (qpcr). serum was collected for igm and igg elisa. neither qpcr, serology nor culture was capable of differentiating colonization from infection. in . and . % of the asymptomatic and symptomatic children, m. pneumoniae dna was detected. in addition, persistence of m. pneumoniae in the upper respiratory tract was shown for up to months by longitudinal sampling. a retrospective study investigated the clinical significance of the m. pneumoniae bacterial load in children with a m. pneumoniae pneumonia (jiang et al., ) . the authors concluded that a high bacterial load was indicative for a m. pneumoniae infection, whereas for a low bacterial load the etiologic role of m. pneumoniae remains to be determined. edin et al. developed a qpcr with duplex reactions targeting eight bacteria, including m. pneumoniae, and six viruses (edin et al., ) . clinical specimens from the upper and lower respiratory tract were used to compare the qpcr assay with standard microbiological methods. the use of the qpcr assay resulted in positive identifications in respiratory specimens compared with by using standard diagnostics. the authors conclude that in parallel qpcr detection of the targeted respiratory bacteria and viruses is feasible since a good technical performance of the assay in clinical specimens was obtained. in contrast to the above mentioned studies, jain et al. ( ) examined specimens from hospitalized children with community-acquired pneumonia and asymptomatic controls for the detection of a variety of respiratory pathogens. m. pneumoniae was detected in %, and in % or less of controls. another trend is the simultaneous detection of m. pneumoniae and mutations associated with macrolide resistance directly in clinical specimens liu et al., ; nummi et al., ; zhao et al., ) . serological methods, in particular enzyme-linked immunosorbent assays (elisa), are most widely used to diagnose a m. pneumoniae infection. the complement fixation test (cft) has been replaced by assays which allow for quantification of igm, iga, or igg. however, the most convincing evidence of an ongoing infection is a significant increase in igg or an igg seroconversion in paired sera, collected - weeks apart (nir-paz et al., ) . although igm antibodies appear earlier than igg antibodies, and are thus an attractive alternative for diagnosis of a m. pneumoniae infection, one should realize that igm is not often produced in very young children, in a proportion of primary infections and during re-infections (waites et al., ; loens et al., a) . ten serological assays for the diagnosis of a m. pneumoniae infection were recently evaluated by using sera from patients (busson et al., ) : seromp igm and igg (savyon diagnostics), seromp recombinant igm, iga and igg (savyon diagnostics), liaison m. pneumoniae igm and igg (biotrin international ltd), m. pneumoniae igm, iga and igg medac (medac gmbh). a low igm specificity and cross-reactivity was noticed for the seromp recombinant and liaison assay. for iga, the medac assay tended to be less specific than the seromp recombinant assay. all four tests showed discrepancies in the igg measurements confirming results of previous studies beersma et al., ) . in conclusion, serology remains a diagnostic tool of choice but improvement and standardization of the assays are still needed, especially for the determination of igg. the clinical significance of a serologic test, both for igm and igg, should be defined by studies of patients with a documented infection and for whom detailed information concerning the time lapses between onset of disease and the collection of the serum specimens are known. a promising blotting technique improving the performance of the m. pneumoniae serological assays has been described (dumke et al., ) . data from recent studies using pcr based methods and serology published during the last decade in different patient populations from around the world are summarized in the recent reviews published by ieven and loens (loens et al., a; ieven and loens, ) and updated in table . the availability of the very sensitive naats has in recent years also put the often used serological tests in their right perspective and allow a better interpretation of the serological test results and their limitations such as the low sensitivity of igm antibodies in acute phase specimens and importance of the delay between two serum samples. studies in which also naat's are used on respiratory specimens should allow a better interpretation of the serological test results. a rapid response report from the canadian agency for drugs and technologies in health (canadian agency for drugs and technologies in health, ) presents the results of a literature search in order to identify the diagnostic test accuracy, clinical effectiveness, and cost-effectiveness of serum igm and molecular tests for the detection of m. pneumoniae in patients with a respiratory infection . six relevant studies were identified, but no evidence regarding the clinical effectiveness or cost-effectiveness of a serum igm test compared with molecular tests was identified. zhang et al. conducted a systematic review and meta-analysis on the diagnosis of m. pneumoniae by pcr and serology (zhang et al., ) and reported a significant heterogeneity between the studies and inconsistent results as well. two studies compared the application of real-time pcr and serology in children with pneumonia. in , / children were found to be positive by pcr (chang et al., ) , / were m. pneumoniae igm positive. . % of patients were found to be m. pneumoniae positive by both tests at the same time. using pcr as gold standard, a sensitivity and specificity of resp. . and . % were obtained. the specificity could be increased to . % by increasing the cut-off without changing the sensitivity of the igm assay. a study conducted by medjo et al. ( ) applied pcr, culture, igm and igg in paired sera for the detection of a m. pneumoniae infection in children. using igg serology as gold standard, the sensitivity of igm, pcr, and culture was found to be equal ( . %), specificity was found to be , . , and % respectively. it was concluded that during the acute phase of disease, detection of igm antibodies in combination with pcr allowed for a precise and reliable m. pneumoniae diagnosis. a prospective study in children with community-acquired cap (kakuya et al., ) compared loop-mediated isothermal amplification, (lamp), culture and serology at first visit. patients were defined positive if positive by culture and/or sero-conversion or a four-fold increase in igg in paired sera. / patients met the criteria. thirteen were positive by culture and serology, on culture only, and one by serology only. a positive lamp result was obtained for all patients that were culture positive. the sensitivity and specificity for lamp, eia, and the particle agglutination test, were . , . , . , and %, . and . %, respectively. when establishing the etiology in adult cap-patients in norway, were found to be m. serology, seven and one by pcr applied to a nasopharyngeal flocked swab and an oropharyngeal flocked swab, respectively (holter et al., ) . newer technologies such as microfluidics and the application of nanotechnology offer the potential to an even more rapid detection of important pathogens allowing even near-patient testing. since these technologies, as naats, do not require viable organisms, and thus avoid any adverse effect of longer specimen transport, they can be successfully applied to both the inand outpatient settings. several companies currently possess the technical expertise and research infrastructure to bring a useful diagnostic testing approach to the clinical trial stage shortly. li et al. ( ) developed a colloidal gold-based immunechromatographic assay by using a pair of monoclonal antibodies targeting a region of the p gene. when applied to clinical specimens from children suspected with a m. pneumoniae infection, the sensitivity and specificity against real-time pcr were and . %. this is in contrast to the results obtained with a commercially available rapid antigen test targeting the ribosomal protein l /l (ribotest mycoplasma). compared to real-time pcr, a sensitivity and specificity of respectively . and . % were obtained when applied to clinical specimens (miyashita et al., ) . based on these results, the authors concluded that treatment decisions should not be taken based on the ribotest results alone. other amplification-free detection methodologies are currently being developed as biosensing detection strategies: a proto-type of an enzyme-free electrochemical genosensor on nanostructured screen-printed gold electrodes (garcia-gonzalez et al., ) ; a silver nanorod array-surface enhanced raman spectroscopy biosensing platform was successfully applied for the detection of m. pneumoniae in simulated and clinical throat swabs (henderson et al., (henderson et al., , . with the use of tools such as naats a greater understanding of the etiology and epidemiology of m. pneumoniae is possible. taken into account the results obtained in recent studies, there is more evidence that real-time naats are superior to other m. pneumoniae detection strategies during the early phase of infection. naats, however, cannot completely replace serology. in epidemiological studies, serology is certainly more useful than for the management of individual patients with lrti or even cap since results are often delayed by the need for paired sera to detect a seroconversion or a significant rise in titer; early in the course of an infection, false-negative results often occur. in case a specific igm test is used, serology should not completely be abolished despite the fact that igm serology shows a moderate sensitivity. nowadays, a combination of the detection of igm antibodies and pcr may be the most optimal approach for early diagnosis of a m. pneumoniae infection, especially in children. the implementation of quantitative tests could shed further light on the relation between bacterial load and the seriousness of the disease, produce useful prognostic information and help in the differentiation between colonization and infection. more information could be gathered on the length of the post infection carrier state as well as on the importance of subclinical infections and how prone these are for spreading infection. it remains important to recognize the urgent need for the adoption of a more unified and consistent diagnostic approach for current and future investigations. therefore, a common set of recommendations should be developed. kl drafted the manuscript. gi revised and approved the final manuscript. kl is supported through the belgian 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does not comply with these terms. key: cord- -n ezxnjc authors: bertasio, cristina; giacomini, enrico; lazzaro, massimiliano; perulli, simona; papetti, alice; lavazza, antonio; lelli, davide; alborali, giovanni; boniotti, maria b. title: porcine epidemic diarrhea virus shedding and antibody response in swine farms: a longitudinal study date: - - journal: front microbiol doi: . /fmicb. . sha: doc_id: cord_uid: n ezxnjc the porcine epidemic diarrhea virus (pedv) causes an acute and highly contagious enteric disease characterized by severe enteritis, vomiting, watery diarrhea, and a high mortality rate in seronegative neonatal piglets. in the last few years, ped had a large economic impact on the swine industries in asia and the us, and in , the pedv also re-emerged in europe. two main pedv variants circulate worldwide but only the s indel variant, considered a mild strain, is spreading in europe. to gain insights into the pathogenicity of this variant, its viral load and temporal shedding pattern were evaluated in piglets from infected farms. quantitative real-time pcr (qpcr) targeting the spike gene, was validated according to the minimum information for quantitative real-time pcr experiments guidelines. the qpcr was applied to longitudinal studies conducted in four swine farms naturally infected with the pedv s indel variant. clinical data, fecal swabs, and blood samples were collected from piglets at – -day intervals for – months. on all four farms, diarrhea was observed in sows during gestation and in farrowing units, and the mortality rates of piglets were , , , and %. different clinical pictures ( - % of diarrhea positivity), viral titer levels (mean . - . log( ) genome copies/ml), and antibody conditions ( - % of positivity) were registered among sows on the four farms. the percentage of qpcr positive piglets varied greatly from the beginning ( – %) to the end ( %) of the infection course. clinical signs were present in % of the qpcr positive animals. viral loads ranged from . log( ) to log( ) genome copies/ml in suckling pigs at – days of age and were not statistically different among farms, despite the different patterns observed in sows. after – weeks, only a few piglets still showed detectable viral levels and clinical signs, and they developed antibody responses. moreover, co-infections with other pathogens and biosecurity procedures limiting the circulation of the virus could have influenced the severity of ped infection. qpcr and clinical data were useful in understanding the dynamics of pedv infections and, therefore, in implementing appropriate control measures. porcine epidemic diarrhea virus (pedv) causes an acute and highly contagious enteric disease, which is characterized by severe enteritis, vomiting, watery diarrhea and a high mortality rate in neonatal piglets. belonging to the family coronaviridae, genus alphacoronavirus, pedv has a single-stranded, positive-sense rna genome of ∼ kb that encodes four structural proteins, spike (s), envelope, membrane, and nucleocapsid, and three nonstructural proteins (kocherhans et al., ) . in particular, the s protein is important in regulating interactions with specific cell receptor glycoproteins to mediate viral entry, inducing neutralizing antibodies (bosch et al., ) , growth adaptation in vitro and in virulence attenuation in vivo (sato et al., ) . since , ped has caused large economic losses in the swine industries of asia and north america (usa and canada) (stevenson et al., ; kochhar, ) . based on the nucleotide sequence of the s spike gene (lee, ) , two main genetic variants have been detected: a "highly virulent" strain, called "non-s indel (insertions and deletions)" (vlasova et al., ; sun et al., ) , and a "mild" strain, called "s indel", identified in pigs with mild clinical signs and no mortality (vlasova et al., ; wang et al., ) . since , in europe, the non-s indel pedv strain has been detected only in the ukraine (dastjerdi et al., ) , while the s indel strain has spread throughout many countries including germany, france, belgium, portugal and italy (grasland et al., ; hanke et al., ; mesquita et al., ; theuns et al., ; boniotti et al., ) . all of the s indel pedv european strains share % nucleotide identity with s indel pedv oh , but in contrast with field observation in the usa , outbreaks with high mortality rates in suckling piglets have been reported in germany and portugal (mesquita et al., ) . in italy, ped has been documented since , with a few cases appearing per year. however, between and , a severe pedv epidemic occurred in italy (martelli et al., ) , which was characterized by mortality rates in neonatal piglets of up to %, whereas it was very low in adults. during - , only sporadic confirmed clinical cases of ped were reported (boniotti et al., ) . during this period, two swine coronavirus clades were identified. the first resembled the oldest global pedv strain cv (pedv/italy/ / ) and the second resembled a new transmissible gastroenteritis coronavirus/pedv recombinant variant (secov/italy/ / ). during summer , animals on two farms displaying mild clinical signs were detected as positive for pedv by pcr (boniotti et al., ) , and at the beginning of a new severe epidemic wave occurred (efsa ahaw panel, ) . recently, the virulence of the s-indel strain has been evaluated and compared to the non-s indel strain through experimental infections (lin et al., ; yamamoto et al., ; chen et al., ) . in one study, -day-old piglets inoculated with an s indel pedv strain (usa/il/ / ) did not develop clinical signs and had only mild histopathological lesions (chen et al., ) . lin et al. ( ) showed that the virulence of the s indel pedv strain was lower than the non-s indel us pedv based on a longer incubation time, a shorter duration of diarrhea, a lower percentage of infected enterocytes and a lower piglet mortality rate. however, the severity of clinical signs and mortality rates ( - %) varied greatly among litters inoculated with the s indel strain. these variations were associated with piglet birth weights and the sow's health and lactation status (lin et al., ) . to evaluate differences in morbidity and the virulence of the s indel strains, field data are extremely important, as many concurrent factors, not reproducible in an experimental infection, can determine the infection severity. presently, the limited available field observations mesquita et al., ; stadler et al., ) indicated that the virulence level of s indel pedv varied greatly. the different immunological states of infected piglets, due to lactogenic immunity, is a likely explanation of such variations. viral load is an important indicator in evaluating the virulence of a strain and the susceptibility of infected animals, and in understanding the mechanisms of viral transmission and circulation within the different farm units. at the moment, very limited data have been published on the pedv viral load during an outbreak under field conditions (bjustrom-kraft et al., ) . in this study, we describe acute outbreaks of ped in three farrow-to-finish and one farrow-to-wean farms in northern italy. we conducted a longitudinal study by sampling the feces and blood of piglet groups from each farm at fixed intervals during a - months period, and then we determined pedv shedding and the antibody presence. in particular, the quantification of pedv in fecal samples was used to better describe the infection dynamics under field conditions. three farrow-to-finish and one farrow-to-wean farms located in the north of italy were chosen for this study, which occurred from january to may . farms were selected based on the following criteria: location in the high density swine production area of the po valley, a short distance from the diagnostic laboratory to facilitate the conservation of samples and quick delivery, sudden onset of enteric clinical signs, mortality in newborn piglets clearly referable to ped and an absence of an anamnesis of ped on the farm. in table , data on the four farms are summarized. the ped clinical and epidemiological characteristics, as well as the disease course, for the four farms are recorded in supplementary table . at each sampling time, clinical evaluations of the farms were determined by vets from our institute, recording the percentages, in four ranges, low ( - %), medium ( - %), high ( - %), and very high (> %), of diarrheic pigs among the different farm units (supplementary table ). fecal consistency was visually evaluated and scored using the following criteria: = normal, = formed and soft, = semi-solid, = watery, and = presence of mucus and blood. the maximum observed fecal score attributed to each farm unit was recorded at each sampling time and reported in supplementary table . at - days from the first appearance of clinical signs on the four farms (f −f ), sows per farm, and , , , and newborn piglets from f , f , f , and f , respectively, were selected from symptomatic litters, identified by ear tag and sampled every weeks (samplings - ), − weeks (samplings − ) and after weeks (sampling ). the number of sampled animals decreased as the study continued due to mortality and animal sales (supplementary table ). the sows were sampled only once at the beginning of the study. the presence of diarrheic piglets was recorded at each sampling time (supplementary table ). fecal and blood samples were collected from sows, whereas blood and rectal swabs were collected from piglets. feces were diluted : (w/v) in minimum essential media (mem). rectal swabs were suspended in ml of mem, vortexed and incubated at • c for min to allow the release of the feces from the cotton. fecal suspensions were clarified by centrifugation for min at , × g to eliminate fecal debris. viral rna was extracted from µl of sample using a commercial kit (nucleomag r vet kit, macherey-nagel, düren, germany), according to the manufacturer's instructions. an exogenous internal control rna (ic) (qiagen, hilden, germany), was added to specimens prior to rna extraction to verify the success of the procedure and the absence of inhibitors. the extraction was carried out on the biosprint instrument (qiagen) using the nucleomag vet protocol. nucleic acids were eluted into µl of elution buffer and immediately subjected to rt-pcr or stored at − • c until used. extracted viral rna was subjected to a "one-step" rt-pcr assay using the commercial quantifast pathogen rt-pcr kit (qiagen) with primers and probe targeting the s gene of pedv that were previously developed at the university of minnesota veterinary diagnostic laboratory ( ), and are reported in table . the optimum concentrations of primers and probe were deduced by titration experiments and are reported in table . pcr reactions were performed on µl of extracted viral rna in a final volume of µl, which also contained µl of × pcr-master mix, . µl of a × internal control assay, . µl of each forward and reverse primer (final concentration nm), . µl of ped_s probe (final concentration nm), and . µl of enzyme mix. the pedv rna was reverse transcribed at • c for min, followed by cycle of taq polymerase activation at • c for min. amplification consisted of cycles at • c for s and • c for s. amplification were performed on a cfx touch real-time pcr detection system (bio-rad laboratories) and data were analyzed with the software bio-rad cfx manager . , using the single threshold method for the cq determination. an experiment was accepted when the cq of the "no template control" (ntc) was > and the ic of a negative control was < . we cloned a fragment of the s gene (nucleotide positions - ; genbank dq . ) into a pcr r . -topo r vector (topo ta cloning r kit, invitrogen) according to the manufacturer's instructions. plasmid dna was linearized by restriction enzyme digestion and then subjected to transcription using the ribomax large scale rna production system (promega) to produce an rna transcript. the rna concentration was determined using an infinite r nanoquant spectrophotometer (tecan). a -fold serial dilution in nuclease-free water of ssrna transcripts ( × - × copies/µl) was used to generate a standard curve and to quantify pedv rna in the samples. triplicates of each dilution were run in each assay. the following equation: where x represents the genome copies/µl, was used to transform the samples' cq values into estimates of genome copies of pedv rna per ml of fecal homogenate. the qpcr assay used to quantify pedv rna was validated according to the minimum information for quantitative realtime pcr experiments guidelines (bustin et al., ) . validation results are summarized in table . to ascertain the specificity of the qpcr used in this study, we tested eight samples that were negative for pedv and positive for different viral agents, including the limit of detection (lod) and limit of quantification (loq) were determined by testing replicates of a -fold serial dilution of ssrna transcript, from an initial concentration of inhibition assay . cycles r , linear correlation index; lod, limit of detection; loq, limit of quantification. × to a final concentration of × − genome copies/µl (supplementary table ). the lod was calculated as the lowest concentration at which % of the positive samples are detected. the loq was defined as the lowest concentration of viral rna that can be determined with acceptable precision (relative standard deviation ≤ %) under the stated conditions of the test. the linear correlation index (r ) and the slope of the calibration curve were calculated using mean values from three replicates of four different runs. the reaction efficiency (e) under our experimental conditions was determined by the following formula: the dynamic range was determined by testing three replicates of a -fold serial dilution of ssrna transcript, from an initial concentration of × to a final concentration of × genome copies/µl (supplementary figure ) . repeatability was evaluated by analyzing three pedv positive samples in triplicate in the same extraction and qpcr run (supplementary table ). intra-assay variance was expressed as the range of the relative standard deviation (rsd%) associated with copy number/µl. reproducibility was determined by testing three pedv-positive samples having different concentration levels. samples were extracted and quantified on four different days (supplementary table ). inter-assay variance was expressed as mean rsd% with minimum and maximum values of copy numbers at each concentration level, calculated for the different concentration levels. to verify the absence of contaminants that can cause the inhibition of qpcr assays after the rna extraction procedure, the extracted rnas of specimens were diluted : (v/v) in water and subjected to qrt-pcr. inhibition was evaluated by calculating the differences in mean cq values between diluted and undiluted samples. the s gene sequences of pedv positive samples on farm , were obtained from one sow and one piglet at the first sampling and from three piglets at days of age, as described by boniotti et al. ( ) (accession number: ky -ky ). an in-house pedv whole virus elisa was used. in brief, a pedv strain cv -based elisa was developed and validated at izsler based on the previously described double-antibody sandwich elisa protocol (sozzi et al., ) . the elisa microplates were coated with the f capture monoclonal antibody (mab). serum samples diluted : or : were mixed with equal volumes of whole pedv inactivated with ß-propiolactone and pre-incubated in an auxiliary microplate for h at • c. then, µl of the pre-incubated mixtures were transferred into the f mab-coated plate and the conjugated horseradish peroxidase mab c was added. following a further h incubation at • c, the plate was washed. the colorimetric reaction was performed, and optical densities (od) were measured at nm using an elisa plate reader. results were calculated by determining the absorbance value reduction, expressed as percentage of inhibition (pi) using the control wells as the reference. the antibody-blocking reaction was considered positive if the pi was ≥ %. for differential diagnoses and to investigate concomitant infections, fecal and serum samples were further investigated to detect transmissible gastroenteritis coronavirus, porcine deltacoronavirus, swine rotavirus a, b, c, and h, porcine reproductive and respiratory syndrome virus; influenza a virus, escherichia coli, clostridium perfringens, and salmonella typhimurium, as previously described (kim et al., ; marthaler et al., a,b) . statistical analyses were performed on quantitative data from fecal specimens of the first sampling both in sows and piglets, using the kruskal−wallis test and dunn's multiple comparisons test (graphpad instat prism . software). at the beginning of the s indel pedv epidemic wave in northern italy (january ), four farms with symptoms referable to ped were selected for a longitudinal study. farm production type, onset of outbreak, mortality rate in suckling pigs and sampled animals for each farm, were described in table table ). watery diarrhea with presence of mucus and blood was observed in piglets of the four farms, but it was also present in gestation and farrowing sows. growing and fattening animals from f , f , and f also showed severe clinical signs including watery diarrhea and anorexia. vomiting was observed in fattening animals on f . dehydration was observed in litters on f and f and cachexia in litters on f and f . agalactia was present in the sows from f , f , and f . for differential diagnoses and to investigate concomitant infections, which potentially could impact the evolution of clinical signs and the course of the disease, fecal and serum samples were further investigated to detect other viral and bacterial pathogens. rotavirus a was present in both the sows and piglets of f , e. coli, expressing the virulence factor f , in f and f , c. perfringens in f and f , and s. typhimurium in f . however, no clinical signs, referable to a specific enteric disease, were present before the beginning of the longitudinal study. symptoms at the first sampling, watery diarrhea or soft diarrhea was observed in % ( / ), % ( / ), % ( / ), and % ( / ) of the sows from f , f , f , and f , respectively (table ) . at the same time high percentages ( - %) of - -day-old piglets were observed to have diarrhea on all of the farms ( table ) . clinical symptoms in piglets progressively disappeared at the following sampling times. however, a second outbreak of diarrhea was observed in out of animals ( %) at two months of age on f . samples were only taken from sows at the initial sampling time, which occurred at , , , and days after the onset of symptoms on f , f , f , and f , respectively. pedv rna was detected in / sows on f , / sows on f , / on f and / on f ( table ). the highest fecal pedv rna shedding titers were detected on f and f ( . and . log genome copies per ml of fecal homogenate), with a mean titer among shedding animals of . and . log copies/ml, respectively ( figure a) . mean lower titers were observed in f and f animals ( . and . log copies/ml, respectively) where the highest titers were . and . log copies/ml, respectively. the fecal viral shedding in piglets from the four farms is summarized in table and figure b . all of the farms had a similar virus-shedding pattern, with high percentages of pedv pcr positive - -day-old animals (sampling ) that decreased in - -day-old animals (sampling ) and was no longer present in - -day-old animals (sampling ) to the end of the study period (table ) . furthermore, on f , a second peak of viral shedding was detected in weaning piglets at days of age (sampling ). two weaning piglets did not show detectable viral shedding in the previous samplings. interestingly, the presence of different strains was assessed by sequencing the s gene of positive samples collected at the first and forth samplings. two genetic variants with a single amino acid substitution ( e > g) were detected at samplings and . on f , . % of -day-old piglets were pcr positive, four of them ( . %) were positive at days of age (sampling ) and only one animal was still positive at -days old (sampling ). moreover, one pig showed intermittent pedv shedding, being pcr positive at the first sampling ( days after birth), negative at -and -days old (samplings and , respectively) and again positive at -days old (sampling ) (supplementary table ). on f , all of the day-old piglets were pcr positive using rectal swab samples but only three of them remained positive at -days old (sampling ). at the following sampling times, when they were -and days old, they were all pcr negative. on f , . % of -day-old piglets were pcr positive but viral shedding was not detected in -day-old piglets through the end of the study. the highest fecal pedv rna shedding titer was observed in - day-old piglets with mean values (among shedding animals) of . , . , . , and . log copies/ml on f , f , f , and f , respectively ( figure b; supplementary table ). no significant differences were statistically evident among the farms. the titer values observed in - -day-old piglets were . (one animal), . (mean value of two animals), . (mean value of three animals) log copies/ml on f , f , and f , respectively (supplementary table ). pedv-antibodies were detected in / , / , / , and / sows on f , f , f , and f , respectively ( table ). in newborn piglets, pedv-antibodies were detected in , , , and % of animals from f , f , f , and f , respectively ( table ). in total, % of the sows had anti-pedv antibodies at delivery but only a few piglets ( %) showed detectable antibodies and a lack of clinical signs at - days of age. most of the piglets on the four farms developed antibody responses within weeks of age, and they remained stable until the end of the study ( - days of age). in the last few years, ped had a large economic impact on the swine industries in asia and the us, and in , the pedv +, presence/positive; −, absence/negative; na, not available (missing sample). *animals died or were not available (na). the number of sampled animals decreased as the study continued due to mortality, earmarks lost (na a ), and animal sales (na b ). also re-emerged in europe. two main pedv variants circulate worldwide but only the s indel variant, considered a mild strain, is spreading in europe. during summer , animals with mild clinical signs on two farms were detected as pcr positive for pedv in northern italy (boniotti et al., ) . thereafter, a new severe ped epidemic wave occurred. to gain insights into the pathogenicity of this variant, we described the results of a longitudinal study conducted during acute outbreaks of ped in three farrow-to-finish (f −f ) and one farrow-to-wean farm (f ), occurred in the beginning of . on the four farms, the mortality rates of suckling piglets were high ( , , and % on f , f , f and f , respectively) ( table ) . however, they did not reach the percentages observed in the us, caused by the original pedv strain (> %) (alvarez et al., ) . moreover, the four farms showed high percentages ( − , > %) of diarrheic animals, in particular, in all the units of the f , in piglets of f and in sows and piglets of f and f , but differences among farms were also observed. the course of ped was particularly severe on f , where we observed a high percentage of animals with diarrhea in suckling, weaning and fattening animals (supplementary table ). moreover, the virus appeared to circulate longer on this farm since positive animals were detected even and days after the first pedv detection within the farm. in particular, at days of age a second peak of viral shedding was observed in three animals. the s gene sequences of the strain identified in these animals showed a the different patterns and severity of clinical signs observed on the four farms may have resulted from the concurrent effects of co-infections with other pathogens. in particular, rotavirus, with a well-known pathogenic aptitude, which is mainly dosedependent, could have had a synergistic effect with pedv on f , causing a slight change in the course of the disease in sows and litters after the first week, and predisposing animals to secondary infections, such as those caused by e. coli f , c. perfrigens, and s. typhimurium. in this study, we determined the fecal pedv shedding in sows and in their piglets from to days to − days of age. in particular, we selected groups of sows and - piglets on each farm. none of the farms had reported pedv infections prior to this study, and thus, we can surmise that the sows had been recently infected at the time of their enrollment in the study, while piglets were infected after birth, through contact with infected sows. more than % of the sows showed detectable levels of pedv at the first sampling but only the % showed diarrhea. the severity of clinical signs caused by pedv is agedependent (jung et al., ) . in adult animals, clinical signs are usually milder or completely absent. moreover, in our study, pedv exposure dose and the gestational stage of the sows could have influenced their health status at the moment of delivery in terms of clinical signs, viral loads and pedv antibody level. on f and f , where the onset of pedv symptoms occurred in the fattening unit, and out of sows, respectively, had pedv antibodies. on f , where the onset of pedv symptoms occurred during the gestation period, out of sows had diarrhea but only had already developed pedv antibodies. on this farm, all of the sows showed high mean viral loads ( . log copies/ml) as determined by pedv rna. sows on f probably had less time to develop antibodies and transmit them to their litters through the colostrum. in fact, all of the piglets were infected and showed high titers of pedv rna in fecal materials. similarly, on f , where the onset of the outbreak took place in the delivery room, out of sows showed detectable levels of pedv antibodies. at the first sampling, a high percentage ( - %) of the - -day-old piglets from all of the farms were positive by qpcr. despite the different immunity levels among the sows on the four farms, and the different proportions of infected piglets, the quantitative pedv rna results in piglets were not statistically different among farms. as proposed by other authors, we assumed that once the pigs were infected and viral replication began, the initial viral dose appeared to have little impact, at the group level, on the average amount of fecal shedding (thomas et al., ) . at weeks of age, the proportion of pcr positive piglets decreased to - . %, and at month of age only one animal was positive. the pedv rna titer also decreased with time, confirming that ped is characterized by high pedv fecal shedding titers a few days post infection and the titers tend to decrease after week. intermittent shedding can be observed until days post infection, as shown in f and f , and such variations in excretion levels and viral loads could be determined by poor management and a lack of biosecurity measures, but it could also be due to a new introduction of the virus, as likely occurred on f . for immunity responses, % of the animals on f showed detectable levels of pedv antibodies up to the end of the study ( days of age). on f , f , and f , the percentage of seropositive animals decreased slightly starting from days of age. the longer and biphasic circulation of the virus on f might have favored subsequent exposures of the animals to the virus, and thus, a more durable immunity. the likely outcome of a massive outbreak of pedv within one herd is a diffuse and long-lasting immunity. indeed, especially in farrow-to-finish farms, such herd immunity and, particularly, the maintenance of seropositive sows could be an important tool to prevent severe cases of ped in newborn piglets by transferring passive maternal immunity with colostrum. goede et al. ( ) reported that durable lactogenic immunity was present in sows previously exposed ( months) to a s indel strain of pedv and that this immunity induced cross-protection to an original virulent pedv. cumbersomely, in this study we did not plan to investigate the presence of maternal antibodies (igg and iga) in the colostrum or to register the piglets' birth weight, two important factors influencing the protective maternal immunity. however, considering the high percentage of diarrheic and pedv positive piglets, and the presence of clinical signs in animals of different ages, including sows, we can suppose that these farms were originally naïve with regard to pedv infection, and thus the lactogenic antibodies were not present at all, or anyway sufficient to effectively protect piglets from infection. thus, future management of pedv infection in farrow-to-finish or farrow-to-wean farms cannot disregard to periodically check and evaluate the iga and igg titers in sow sera and in the colostrum. determining the viral loads and shedding rates of pedv in real field situations during outbreaks is important in evaluating the virulence of a strain and in predicting the susceptibility of infected animals, at different ages and in the various farm units, within a herd. at the moment, very limited data have been published on the pedv viral load during an outbreak under field conditions (bjustrom-kraft et al., ) . the qpcr assay used in this study was validated according to the minimum information for quantitative realtime pcr experiments guidelines (bustin et al., ; table ; supplementary table ). the method validation is an important requirement to allow the comparison of quantitative results from different studies, which would be otherwise difficult to achieve. moreover, understanding the mechanisms of viral transmission and circulation within the farm can be useful in implementing appropriate control measures on the farm to limit the infections spread. considering the ability of pedv to be dispersed rapidly through fecal contamination, particular attention should be paid to biosecurity and hygiene measures, such as the proper disinfection of equipment and sites, manure disposal, suitable procedures for the movements of animals within the farm, and the use of disposable clothes and shoes for personnel, staff and visitors. longitudinal field studies examining natural infections are comparatively uncommon amongst reports of pedv in comparison to the several experimental studies already performed. in fact, many unforeseen events can adversely affect the success of this kind of study: dead of animals, lost of the earmark, selling or moving of the animals due to unexpected need of the farmer. these practical hitches could negatively influence the sampling procedures (e.g., respect of time points) and data registration. therefore, by planning these "observational" studies, so tight inclusion criteria cannot be established and limited field data could be successfully registered. on the other hand, the longitudinal studies, directly conducted in natural outbreaks, are inclusive of the several "farm factors and field effects" which are very difficult to reproduce in experimental trials, and thus, the obtained results, being more reliable and adherent to real conditions could be effectively used in risk analysis and for defining control strategies. in conclusion, longitudinal studies conducted under field conditions during and after a ped outbreak could be useful in determining the level of immunity acquired at the herd level and may be integrated with the data acquired from experimental infections. they provide an added value, in the possibility to study and evaluate the effects of cofactors, such as other infectious agents, and management and environmental conditions, on the evolution and epidemiology of the disease. the study was exempt of ethical approval procedures because animal samplings were performed during the routinely 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real-time rt-pcr for detection and differentiation of virulent and variant strains of porcine epidemic diarrhea viruses from the united states isolation and experimental inoculation of an s indel strain of porcine epidemic diarrhea virus in japan we thank anna mangeli for the skilled technical assistance and federico scali for the statistical analysis. thanks also to science docs for the english language editing of this manuscript. the supplementary material for this article can be found online at: http://journal.frontiersin.org/article/ . /fmicb. . /full#supplementary-material key: cord- -x hq b authors: versluys, anne birgitta; boelens, jaap jan title: morbidity and mortality associated with respiratory virus infections in allogeneic hematopoietic cell transplant: too little defense or harmful immunity? date: - - journal: front microbiol doi: . /fmicb. . sha: doc_id: cord_uid: x hq b the impact on morbidity and mortality of community acquired respiratory virus (carv) infections in patients undergoing allogeneic hematopoietic cell transplant (hct) is widely studied. here we give an overview of the current literature on the incidence and chance of progression to severe disease in this highly immune compromised population. we discuss the issue whether it is predominantly direct viral damage that causes clinical deterioration, or that it is in fact the allogeneic immuneresponse to the virus that is most important. this is an important question as it will guide therapeutic decision making. it asks for further collaborative studies focusing on sensitive surveillance with pcr techniques and relating clinical data with parameters of immune reconstitution. community acquired respiratory virus infections (carv) include a variety of viruses such as rhinovirus, coronavirus, respiratory syncytial virus (rsv), influenza virus, para influenza virus, and metapneumo virus. carv infections range from asymptomatic carriership to significant respiratory disease. prevalence of carv largely depends on season, detection mode, age of patient and immune status (shah et al., ; hirsch et al., ; green, ) . influenza and rsv have significant seasonal variation, whereas para influenza or rhinovirus cause disease year round (green, ) . highly sensitive diagnostic techniques like polymerase chain reaction (pcr) for the detection of viral dna and rna reveal a high prevalence of carv in the normal population. in healthy children attending day care prevalence is as high as % (moe et al., ) . in children admitted to hospital for respiratory disease - % are tested pcr positive, almost twice as high as in adults admitted to hospital for respiratory disease (ching et al., ) . in the immune compromised pediatric population the prevalence of carv is around - % (fazekas et al., ) with mostly mild symptoms at time of detection. there are many reports on carv prior to, or early after, hematopoietic cell transplantation (hct). recently a large multicenter retrospective analysis in , pediatric hct recipients showed an incidence of . % symptomatic carv infections within year after transplant (fisher et al., ) . surveillance studies in the same population on nasopharyngeal aspirates (npa) routinely performed prior to transplant showed an incidence of % (versluys et al., ) . most studies discuss the risk of progression to viral pneumonia, for various types of carv. only few groups looked at long term outcome. results are conflicting, reported risk of progression is - % (bredius et al., ; hirsch et al., ; chemaly et al., b; fisher et al., ; green, ) . hct is a curative treatment for several malignant and non-malignant childhood diseases. its success is limited by infections, alloimmunity and toxic events. respiratory viruses contribute to post-transplantation morbidity and mortality in different ways. here we provide an overview of recent literature on carv in the hct setting, focusing on the risk of progressive viral lung disease, the role of viruses in the lung microbiome and the potential viral trigger for allo-immunity after hct. reported incidence of rsv in hct-recipients varies between and % (shah et al., ; robinson et al., ; fisher et al., ) , depending largely on season, patients' age and detection methods. progression to lrti occurs in - % (shah et al., ; kim et al., ; fisher et al., ) , with risk factors for progression related to age, donor source, use of steroids, immune status, and concomitant infections (renaud et al., ; chemaly et al., b; kim et al., ; shah et al., ) . mortality rates are around % (chemaly et al., a; robinson et al., ; fisher et al., ) it is important to notice the in most pediatric studies, rsv-positive patients were treated with the antiviral drug ribavirin (molinos-quintana et al., ) , with anti-rsv monoclonal antibodies (palivizumab) or with nonspecific intravenous immunoglobulins (ivig) (el-bietar et al., ) , or with a combination (chávez-bueno et al., ) . aerosolized ribavirin and ivig is recommended for adult hct recipients with rsv lrti (dignan et al., ; waghmare et al., ) . para influenza virus (piv) in hct recipients is systematically reviewed by shah et al. ( a) . the incidence of piv in hct recipients is % (range . - %), % progressing to lrti. significant predictors of lrti progression were infection within days after hct, lymphocytopenia/neutropenia at the onset of infection, use of corticosteroids, younger age, and respiratory co-infections. reported overall mortality is % ( - %), in piv-lrti %. there is currently no licensed therapy for piv pneumonia. influenza is diagnosed in approximately - % of hct recipients, and in up to % of patients with respiratory symptoms in the flu-season. progression to viral pneumonia occurs in - % of patients, and is associated with mortality in - % (fisher et al., ; green, ) . these numbers are strongly influenced by seasonal outbreaks and the subtype of the influenza virus. risk factors for progressive influenza disease are lymphocytpenia/neutropenia and steroid use (kmeid et al., ) . in contrast to many other respiratory viruses, influenza can be treated with neuraminidase inhibitors (waghmare et al., ; green, ) . human rhino virus (hrv)is the most common cause of respiratory virus infections in both immunocompetent and immunocompromised individuals. reported incidence in hct recipients is - % (versluys et al., ; shah et al., ; campbell et al., ; fisher et al., ) . for a long time there was uncertainty about the ability of hrv to cause lower respiratory tract disease. recent studies however, suggest that hrv may be a clinically significant pathogen with the potential to cause serious pulmonary disease in hct recipients (campbell et al., ; seo et al., seo et al., , versluys et al., ) with risk of progression to lrti of - % (shah et al., ; campbell et al., ; fisher et al., ) and hrv related mortality of - % (shah et al., ; campbell et al., ; fisher et al., ) . there is increasing interest in the pathogenecity of human metapneumo virus (hmpv). in a systematic review (shah et al., b) shah et al. summarized all published data on hmpv in patients with hematologic malignancies or undergoing hct. about one third of described cases were children. they report an overall incidence of % (range - %), with a risk for progression to lrti of % (range - %) and a mortality rate of % ( - %) in the total hmpv positive group, and % in the hmpv-lrti group. other respiratory viruses like bocavirus (bov) and coronavirus (cov) in hct setting, are scarcely studied. prevalence of either bov or cov is % in the adult hct population with respiratory symptoms. a significant proportion of the cov infected patients required hospitalization, and some progressed to lrti. in contrast, bov detection was rare and almost always related to co-pathogens (pinana et al., ) . a surveillance study on bov in children found % of children with rti to be pcr positive, as well as % of the healthy controls; thus showing a high prevalence of the virus without necessarily causing disease. progression to lower infection however was associated with higher bov load and viremia, suggesting a pathogenic role in a subgroup of patients (christensen et al., ) . figure shows the incidence of carv in mainly adult hct population, and the impact of carv on lrti and mortality. profound immunosuppression in patients undergoing hct obviously leads to a greater risk of infection, with prolonged shedding of the virus, a higher chance of transmission of disease and a greater risk of progression to severe lower respiratory tract disease. as antiviral therapy is only effective in the minority of the respiratory viruses, and vaccines are not widely available, prevention is very important. the working group of the fourth european conference on infections in leukemia (ecil- ) reviewed the literature on carv in leukemic patients and patients after hct (hirsch et al., ) . for prevention they recommend infection control measures like good personal hygiene, avoiding contact with individuals with a respiratory tract infection and restricting young children from visiting patients. administration if ivig preparations in patients with hypogammaglobulinemia (igg < g/l), and the use of intravenous monoclonal antibody specific for the rsf-f protein (palivizumab) during rsv outbreaks may be considered. they do not advocate routine screening for carv. deferral of chemotherapy or conditioning should be considered, figure | incidence of common respiratory viral infections (crv) and associated progression to lower respiratory tract infections (lrti) and mortality in hct recipients. updated from shah et al. ( ) , based on (versluys et al., ; shah et al., ; chemaly et al., b; campbell et al., ; robinson et al., ; fisher et al., ; green, ) . as well as treatment for rsv and hpiv only in the hct setting. in a joint working group in the uk (dignan et al., ) has reviewed the available literature and made recommendations for the diagnosis and management of respiratory viral infections in patients with hematological malignancies or those undergoing hematopoietic stem cell transplantation. as far as prevention is concerned they come to the same conclusions as ecil- , and add the recommendation for influenza vaccinations in household contacts and medical staff, and post-exposure prophylaxis with oseltamivir in hct patients who have been in contact with influenza. in a recent large prospective study, including adults and children undergoing allogeneic hct, clinical outcomes associated with respiratory viruses (rv) detected prior to hct were analyzed (campbell et al., ) . multiplex pcr testing for rv was done on nasal washes or nasopharyngeal swabs. in % of patients a rv was detected, % of them being asymptomatic. in the pediatric subgroup, defined as aged < years, the prevalence of rv was higher ( %), with a larger proportion being without symptoms ( %). the rv positive patients were significantly younger and had higher risk underlying disease with lower lymphocyte count. overall mortality at day was significantly higher in rv-patients than in non-rv patients ( . vs. . %; hr . ; % ci . - . ; p = . ). in % of the deceased patients the cause of death was thought to be directly related to the pre-hct rv, no data are given about the cause of death in the other patients. patients with rhinovirus performed worse compared to the other rv. this may be partly explained by the fact that most patients with rsv or influenza where treated with antiviral therapy or had their transplant delayed. data on longer follow up are lacking (campbell et al., ) . hutspardol et al. retrospectively studied treatment related mortality (trm) and long-term pulmonary complications in children who had respiratory symptoms and a rv detected within days after allogeneic hct. the overall frequency of documented rv infections was . %, half of the patients presented with signs of a lrti and mortality rate at day was %. cause of death was pneumonitis/ards in all, with symptoms occurring on day - after hct. during follow up ( . years, range . - . ) no chronic pulmonary complications nor allo-immune lung syndrome was observed (hutspardol et al., ) . with regard to long term pulmonary function chien et al. studied , adult hct recipients by performing routine pulmonary function tests years after hct. airflow obstruction, defined as an annualized decline in fev of more than %, occurred in % of patients and had impact on overall mortality. higher age at transplant, gvhd category, pulmonary function pre-transplant and the occurrence of a respiratory virus infection within the first days after hct were significant risk factors for airflow obstruction (chien et al., ) . erard et al. further studied the association of rv and airflow decline, and found that this was particularly true in patients after lrti caused by parainfluenza virus or respiratory syncytial virus (erard et al., ) . in a retrospective study among , pediatric hct recipients in us centers . % acquired symptomatic rv within the first year after hct (fisher et al., ) . in line with others, rhinovirus was the most common virus, followed by rsv and piv. rv was detected after a median of ( - ) days after hct. most children had urti only, in patients with hmpv there was significantly more lrti. during months follow up % required mechanical ventilation and % had significant pulmonary sequelae like bronchiolitis obliterans, subacute pulmonary problems and other not specified pulmonary complications. all cause mortality among rv positive patients was %, compared to % in the non-carv group. recent steroid exposure and rv detection within days after hct were poor prognostic factors for morbidity and death. at least % of death were not attributable to carv infection. the timing of the events is also remarkable, as % of deaths occurred more than days after diagnosing carv infection, which is at least months after hct for most. the widespread use of pcr diagnostics has led to an increase in the detection of carv in patients undergoing hct. many of these patients become symptomatic and a significant proportion develops lrti. there is a clear increased risk for mortality in carv positive patients. hence, prevention and development of anti-viral drugs are of great importance. however, one could debate about the reason for severe morbidity and mortality in carv positive patients. how do you diagnose progressive viral infection? the carv will not be cleared for months because of the immunocompromised state of the host after hct, so finding positive pcrs is not convincing enough. timing of (progression of) symptoms in relation to immune reconstitution might be helpful in answering the question if it is progression of viral damage or if the donor derived immunity actually is targeting the lung. in last decade more and more evidence has emerged that "triggered" alloreactivity may play a crucial role in toxicity and mortality. this holds true for hct, but is also recognized in solid organ transplantation. in the context of lung transplantation several studies have examined the role of rv in the development of chronic lung allograft dysfunction (clad), a form of chronic rejection of the lung (kumar et al., (kumar et al., , fisher et al., ) . many, but not all, reported an association between rv and clad. pooled analyses of studies on rv and clad (vu et al., ) did not confirm the association, mainly due to the heterogeneity of studies and limitations in design, diagnostic techniques and definitions. fisher et al. tried to overcome these limitations by studying a more homogenous cohort of lung transplant recipients, using modern molecular assays to detect rv and applying consensus definitions of clad (fisher et al., ) . in patients, ( %) developed clad at a median of weeks (interquartile range (iqr): - weeks). in patients ( . %) a respiratory viral episode was seen, after a median of weeks post lung transplantation (iqr: - weeks). in multivariate analysis rv was associated with clad (hr . , % ci . - . ; p = . ). this association was stronger the more proximate the rv occurred after lung transplantation. our group studied the role of respiratory viruses (rv) in immune mediated lung disease after hct, analogous to this phenomenon as described after lung transplantation (versluys et al., (versluys et al., , . the host-vs.-graft chronic allograft rejection in lung transplantation is in many ways comparable to the graft-vs.-host inflammation in hematopoietic cell transplantation. in a cohort of children undergoing allogeneic hct routine npa and bal sampling for the presence of rv was done prior to transplant. rv was found in % ( % in bal/npa, % in npa-only). rhinovirus was the most frequently detected rv ( %). allo-immune lung syndrome (allo-ls), defined as bronchiolitis obliterans syndrome (bos) or idiopathic pneumonia syndrome (ips), occurred in %, after a median of . weeks ( - weeks). rv-positivity in bal was a predictor for allo-ls (hr . , % ci . - . ; p = . ). no other predictors were found. the hypothesis is that rv causes epithelial damage and triggers an allogeneic immune response leading to severe lung disease. so the lung disease does not occur primarily from progressive viral infection during the period of low immunity, but from allo-immune mediated damage weeks after hct. this inflammatory aspect of disease might explain the reported benefit of steroid use on the risk of mechanical ventilation among hct recipients with influenza (choi et al., ) , and the controversy on risks of steroid use in case of carv after hct (waghmare et al., ) . more and more is known about the role of microbiota in human health. so far research has largely focused on the gut microbiome, also in the context of gvhd. but the microbial ecosystem at other body sites, including the respiratory tract is attracting growing attention. host and environmental factors influencing the respiratory microbiota include genetics, microbial exposure (birth mode, feeding type, day care), vaccination, infections and antibiotics (man et al., ) . viral infection interacts with the microbiome by disrupting the airway epithelial barrier facilitating bacterial adhesion, liberating host derived nutrients and decreasing muco-ciliary clearance. in addition respiratory viruses can modulate innate and adaptive immune responses promoting bacterial colonization (man et al., ) . moreover, it is becoming clear that the virome should be seen as a part of the microbiome, that affects the function of the host immune system (cadwell, ) . the role of a disturbed respiratory microbiome/virome in lung disease is postulated for asthma and chronic obstructive pulmonary disease (copd) (zou et al., ) . impact of carv on outcomes after hct is an intriguing topic where pathogenesis is not completely understood. is the poor immune system associated with progressive infection? does alloimmunity play a crucial role in lung toxicity? most studies describe data on symptomatic patients where carv is detected at time of symptoms. only few studies report on pre-hct sampling, although we know a large proportion of our patients is carv positive with only mild symptoms. time of (worsening) of symptoms, warranting viral diagnostics and thus detecting the carv, is often weeks after hct. are these nosocomial acquired viruses, or were these virus already present and giving symptoms after a certain period of time? are the viruses acquired after discharge? but then immunity is usually restored to a certain degree. from various infectious diseases, like rsv bronchiolitis (fonseca et al., ) , immune reconstitution inflammatory syndrome (iris) in hiv patients (with cryptococcal meningitis, cmv retinitis or bcg-itis) (walker et al., ) or iris in non-hiv immunesuppressed patients with immune recovery (followed by worsening of treated tuberculosis, idiopatic pneumonia or hepatitis) (sueki et al., ) , we know the harmful effect of immune response on the patient. the debate about inappropriate immune response on infectious triggers is especially intriguing in the allogeneic setting. the definition criteria for alloimmune mediated lung syndromes (panoskaltsis-mortari et al., ; jagasia et al., ) describe the clinical, radiologic and functional aspects of lung pathology, with exclusion of other evident causes of this phenotype, like heart failure and infection, including respiratory virus infection. one can argue if this holds true for respiratory viruses detected by pcr. the detection modes have become much more sensitive over time, so the impact of positive findings on the disease criteria should be reevaluated. in the hct population with its high prevalence of rv, these viruses will have a long persistence making them detectable for weeks after initial infection, with uncertain meaning for their role in pathology. an interesting paper in this matter was recently published by seo et al. ( ) . in patients with ips, they went back to bal samples at time of diagnosis, and applied more sensitive diagnostics for microbial pathogens. in % of patients an occult pathogen was found, % being a respiratory virus. all patients were treated with steroids because of ips. overall mortality was higher in the group of patients with an occult pathogen, than in the group without. the authors conclude that these patients had had to be excluded as ips patients, that they had infectious pneumonia and that steroid treatment had adversely influenced their outcome. however, as we are not informed about rv status pre-hct, this could also be persisting rv after hct, triggering immune-mediated lung disease (ips). in that situation steroids are beneficial in the treatment at the moment of clinical deterioration. in conclusion, despite the growing awareness of carv infections in hct patients, well-designed studies are lacking that systematically evaluate diagnostic and therapeutic strategies of carv. only then we will be able to better understand the direct viral impact and the indirect alloimmune pathology, both largely influencing clinical outcome of patients. detailed longitudinal studies, combining data from microbioma/virioma surveillance with data on immunerecovery after hct and clinical outcome are needed to better understand pathogeneic mechanisms involved in lung disease and carv after hct. this insight should largely influence the therapeutic decision of delaying transplant, treating rv and most important increasing or decreasing immune suppression after transplant. prospective study of respiratory viral infections in pediatric hemopoietic stem cell transplantation patients the virome in host health and disease clinical outcomes associated with respiratory virus detection before allogeneic hematopoietic stem cell transplant intravenous palivizumab and ribavirin combination for respiratory syncytial virus disease in high-risk pediatric patients respiratory syncytial 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patients research on the human virome: where are we and what is next all authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication. the reviewer pv declared a past co-authorship with one of the authors jb to the handling editor.the remaining author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © versluys and boelens. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- -ji njeha authors: saleh, maged; rüschenbaum, sabrina; welsch, christoph; zeuzem, stefan; moradpour, darius; gouttenoire, jérôme; lange, christian m. title: glycogen synthase kinase β enhances hepatitis c virus replication by supporting mir- date: - - journal: front microbiol doi: . /fmicb. . sha: doc_id: cord_uid: ji njeha hepatitis c virus (hcv) infection is associated with alterations in host lipid and insulin signaling cascades, which are partially explained by a dependence of the hcv life cycle on key molecules in these metabolic pathways. yet, little is known on the role in the hcv life cycle of glycogen synthase kinase (gsk ), one of the most important kinases in cellular metabolism. therefore, the impact of gsk on the hcv life cycle was assessed in human hepatoma cell lines harboring subgenomic genotype b and a replicons or producing cell culture-derived hcv genotype a by exposure to synthetic gsk inhibitors, gsk gene silencing, overexpression of gsk constructs and immunofluorescence analyses. in addition, the role of gsk in hepatitis e virus (hev) replication was investigated to assess virus specificity of the observed findings. we found that both inhibition of gsk function by synthetic inhibitors as well as silencing of gsk β gene expression resulted in a decrease of hcv replication and infectious particle production, whereas silencing of the gsk α isoform had no relevant effect on the hcv life cycle. conversely, overexpression of gsk β resulted in enhanced hcv replication. in contrast, gsk β had no effect on replication of subgenomic hev replicon. the pro-viral effect of gsk β on hcv replication was mediated by supporting expression of microrna- (mir- ), a micro-rna which is mandatory for wild-type hcv replication, as gsk inhibitors suppressed mir- levels and as inhibitors of gsk had no antiviral effect on a mir- -independent hcv mutant. in conclusion, we have identified gsk β is a novel host factor supporting hcv replication by maintaining high levels of hepatic mir- expression. hepatitis c virus is a member of the flaviviridae family which has a positive-sense single-stranded rna genome (lange et al., b) . the hcv genome encodes the viral structural and nonstructural proteins, which are required for the hcv life cycle (lange et al., b) . in addition to hcv proteins, a number of host factors have been discovered on which hcv critically depends (lange et al., b) . though most of these host factors are cellular proteins, a liver-specific micro-rna (mir- ) has been identified which is mandatory for hcv replication and which is (at least partially) responsible for the hepatotropism of this virus (lange et al., b) . chronic infection with hcv is not only a major cause of liver cirrhosis and hepatocellular carcinoma, but also associated with metabolic traits including insulin resistance and dyslipidemia (kawaguchi et al., ; shintani et al., ; bose et al., ) . while the effects of hcv infection on glucose metabolism are clearly documented in vivo and in vitro, the benefit of these alterations for the virus remains largely unclear (petta et al., ; negro, ) , although insulin resistance was identified as a negative predictor of outcome of interferon-based therapies (romero-gómez et al., ; dai et al., ; grasso et al., ; moucari et al., ; khattab et al., ; fattovich et al., ) . gglycogen synthase kinase is a serine/threonine protein kinase that exists in two isoforms, gsk α and gsk β, which are encoded by two different genes. approximately substrates of gsk have been proposed, highlighting a central role for gsk in numerous cellular processes such as proliferation, migration, apoptosis, immune modulation and -importantlyglucose metabolism (jope et al., ) . of note, substrates of gsk α and gsk β are overlapping only partially. the activity of gsk is modulated by inhibitory phosphorylation at ser for gsk α and ser for gsk β by upstream kinases, where the phosphorylated n-terminal tail acts as a pseudosubstrate that prevents incoming substrates from entering the catalytic center (cohen and frame, ) . the importance of gsk is further highlighted by its role in the pathogenesis of relevant diseases including diabetes, cancer and inflammation (beurel et al., ) . accordingly, several gsk inhibitors are in preclinical and clinical development, for example for the treatment of alzheimer's disease or diabetes (del ser et al., ; lovestone et al., ) . of note, gsk has also been identified as a host factor required for replication of influenza and sars viruses (wu et al., ; könig et al., ). yet, the role of gsk in the hcv life cycle remains to be characterized. in view of the close relationship between hcv infection and metabolic alterations, the present study aimed at investigating a possible role of gsk in the hcv life cycle. cell culture, subgenomic replicons, cell culture-derived hcv, plasmids huh- . human hepatoma cell line was provided by charles m. rice (the rockefeller university, new york, ny) and cultured in dmem (life technologies, carlsbad, ca) containing % heat-inactivated fcs. hcv subgenomic replicon construct pcon /sg-neo(i)/aflii (con strain, genotype b) (blight et al., ) was provided by charles m. rice. full-length hcv construct pfk-jfh j c- _dg (jc virus, genotype a) (pietschmann et al., ) was provided by ralf bartenschlager (university of heidelberg, germany). hcv u virus, a genotype a full-length strain resistant to mir- inhibition was provided by jens bukh (university of copenhagen, denmark) (li et al., ) . huh- . cells were electroporated with in vitro transcribed full-length hcv rna and h later the virus infectivity in the supernatant was assessed by foci forming unit (ffu) determination using anti-hcv core monoclonal antibody (mab) c - (moradpour et al., ) , as described (zhong et al., ) . the s - cell line harboring a hev genotype replicon derived from kernow-c p strain (provided by suzanne u. emerson (national institutes of health, md) was used as previously described (dao thi et al., ) . sofosbuvir (alsachim sas, illkirch-graffenstaden, france), hcv ns b inhibitor, was resolved in dmso and used at a final concentration of µm. gsk inhibitors lithium chloride (licl) and chir (sigma-aldrich, st. louis, mo, united states) were resolved in sterile water and dmso, respectively. cytotoxicity was assessed using the wst- assay (sigma-aldrich). haemagglutinin-tagged gsk β wild-type plasmid (ha-gsk β wt pcdna ) was a gift from jim woodgett (lunenfeld-tanenbaum research institute, mount sinai hospital, toronto, on, canada) (addgene plasmid # ) (he et al., ) . transfection of gsk constructs for overexpression was performed with a final dna concentration of . µg/ml using x-tremegene hp dna transfection reagent (roche diagnostics, mannheim, germany). immunoblotting was performed as described previously (lange et al., a) . antibodies against total gsk α/β ( s), phosphorylated-gsk α/β (ser / ) ( s), and β-catenin ( s) were purchased from cell signaling technologies (danvers, ma, united states). mouse mab e against hcv ns a was provided by charles m. rice (lindenbach, ) and mab b against hcv ns b was described earlier (moradpour et al., ) . ecl donkey hrp-conjugated anti-rabbit (na v) and hrp-conjugated anti-mouse (sc- ) secondary antibodies were from ge healthcare (little chalfont, united kingdom) and santa cruz biotechnology (dallas, tx, united states), respectively. total rna extraction was done using rneasy mini kits and mirneasy mini kit for mrna and micro-rnas, respectively (qiagen, hilden, germany). reverse transcription of mrna and micro-rna was performed using the primescript rt reagent kit (takara bio, otsu, japan) and the taqman microrna reverse transcription kit (applied biosystems, foster city, ca, united states), respectively. for real-time pcr, steponeplus real-time pcr system (applied biosystems) was used. for hcv and gapdh, experiments were carried out using taqman gene expression master mix (applied biosystems). for micro-rnas, taqman universal master mix ii (applied biosystems) was used for quantification of the mirna cdnas. the following primers were used for hcv amplification: forward, -acgcagaaagcgtctagccat- , reverse, figure | gsk inhibition suppresses hcv replication. (a) confirmation of gsk inhibition by chir . immunoblot analysis was carried out for total and phosphorylated-gsk α/β (ser / ), β-catenin, phosphorylated glycogen synthase (gs), eukaryotic initiation factor bε, and β-actin after treatment of huh- . cells harboring con replicon with µm chir for , , and h, as indicated. (b) suppression of subgenomic hcv replication by gsk inhibition. huh- . cells harboring con replicon were treated with different concentrations of chir for and h. hcv rna levels normalized to gapdh mrna are expressed relative to untreated cells. (c) assessment of gsk inhibitors' cytotoxicity. wst- cytotoxicity assay was carried out for cells stimulated for and h with different concentrations of chir at seeding densities of , and , cells per well in -well plates. absorbance readings (a -a nm) of treated cells were compared to negative control cells treated with the dimethyl sulfoxide vehicle. data are presented as mean ± sem of six independent experiments. asterisks denote statistically significant differences ( * p-value ≤ . ; * * p-value ≤ . ; * * * p-value ≤ . ). frontiers in microbiology | www.frontiersin.org figure | gsk β is required for hcv replication, not gsk α. (a) gsk β silencing suppresses hcv replication. huh- . cells harboring subgenomic con replicon (left panel) or replicating the full-length jc clone were transfected with sirnas of gsk α (sirna # ), β (sirna # ) or both for h. hcv rna levels normalized to gapdh mrna are expressed relative to untreated cells. data are presented as mean ± sem of two experiments performed in triplicate. asterisks denote statistically significant differences ( * p-value ≤ . ; * * p-value ≤ . ; * * * p-value ≤ . ). immunoblot analysis was carried out for gsk α/β to confirm gene silencing efficiency, and for hcv ns b to assess suppression of hcv replication on the protein level (a, lower panel). β-actin was detected as a loading control. ns b signal intensities were normalized to β-actin for quantification and were presented as mean ± sem of signal intensities of duplicate bands. (b) gsk β overexpression promotes hcv expression. huh- . cells harboring con replicon were transfected with pcdna plasmid expressing ha-tagged wild-type gsk β (ha-gsk β) for and h. immunoblot analysis was carried out for gsk α/β to confirm transfection efficiency. the overexpression was confirmed by visualizing ha-gsk β distinct bands with a slightly higher molecular weight than that of the endogenous gsk β. hcv ns b protein was detected to assess hcv replication level and β-actin was detected as a loading control. ns b signal intensities were normalized to β-actin for quantification and were presented as mean ± sem of signal intensities of duplicate bands. frontiers in microbiology | www.frontiersin.org -tactca ccggttccgcaga- , hcv taqman probe, -tcctggaggctgcacgacactca- . taqman gene expression assay containing the pre-designed primers and taqman probe from applied biosystems was used for gapdh. for mir- and mir- , pre-designed primers (assay id no. and , respectively) from thermo fisher scientific were employed. for hev, the procedure and primers have been previously described (dao thi et al., ) . results were calculated using the − ct method and shown as fold change compared to control. gene silencing was performed as described previously (lange et al., a) using predesigned sirnas from life technologies. single sirnas for gsk α (s and s ) and β (s and s ) as well as a non-targeting control sirna were transfected at a final concentration of nm using lipofectamine rnaimax (life technologies). chir is a highly selective small molecule inhibitor of gsk α and gsk β that inhibits gsk by competition with atp in the atp-binding site of the kinase meijer et al., ) . to prove efficacy of gsk inhibition by chir , immunoblot analyses of β-catenin, phosphorylated glycogen synthase (gs), and phosphorylated eif b ε as downstream targets of gsk were performed. as shown in figure a , β-catenin was upregulated and phosphorylated gs and eif b ε were downregulated by chir , as expected. we next exposed huh- . cells harboring the subgenomic replicon (con strain) to different concentrations of chir for and h and quantified hcv rna by pcr. as shown in figure b , treatment with chir led to an inhibition of hcv rna replication. of note, no relevant effect on cell viability was observed as a consequence of gsk inhibition, as assessed by wst- assay ( figure c) . comparable results were observed for treatment of huh- . cells harboring the subgenomic con replicon with lithium chloride, another inhibitor of gsk (beurel et al., ) (figure ) . next, huh- . cells electroporated with full-length hcv genotype a jc rna were treated with chir for h to assess the effects of gsk inhibition on infectious hcv. as shown in figure a , gsk inhibition resulted in a profound inhibition of replication of the infectious hcv clone, as well as of particle production ( figure b ). to assess whether both gsk isoforms are required for hcv replication, we silenced gsk α and / or gsk β gene expression by transfecting sirnas and quantified hcv rna in huh- . cells harboring hcv subgenomic replicon (con ) or infectious hcv (jc ). as shown in figure a (upper panel), silencing of gsk β gene expression resulted in a substantial decrease of hcv rna levels of both the subgenomic replicon and the full-length hcv construct, whereas silencing of gsk α gene expression had only a minor effect on hcv replication. comparable results were observed for a second set of sirnas directed against gsk α or gsk β (supplementary figure ) . the suppressive effect of gsk β gene silencing on hcv replication was further confirmed on the protein level by quantifying hcv ns b protein (figure a lower panel) . furthermore, the enhancing effect of gsk β on hcv rna replication was confirmed by overexpression of a functional gsk β construct, which increased hcv rna replication ( figure b ). to test whether the pro-viral effects of gsk β are specific for hcv, we assessed the impact of gsk inhibition on replication of hev, another hepatotropic positive-strand rna virus. as shown in figure , neither silencing of gsk α and / or gsk β gene figure | the pro-viral effects of gskβ are limited to hcv in comparison to hev. no effects of gsk inhibition on hev replication. huh- -derived cells (s - ) harboring a selectable hev replicon construct (hev kernow-c strain) were treated with µm chir for h or transfected with sirnas to silence gsk α and β gene expression for h. hev rna levels normalized to gapdh mrna are expressed relative to negative control sirna-transfected cells. data are presented as mean ± sem of two experiments performed in triplicate and compared by wilcoxon m whitney u-test (n.s., not significant) with the control group. (b) gsk inhibition has no effects on mir- -resistant hcv. huh- . cells, pre-treated for h with . % dimethyl sulfoxide (dmso), µm sofosbuvir (sof) or µm chir , were infected with either jc or mir- -resistant u virus (moi = . ) and treated in the same conditions for h before harvesting. hcv rna levels normalized to gapdh mrna level are expressed relative to dmso-control. data are presented as mean ± sem of two experiments performed with six samples each. (c) gsk inhibition has no effects on mir- -resistant hcv infectious particles production. infectivity was determined by foci forming units (ffu) assay in cell supernatants harvested h post-infection of jc or u rna and treated with either µm sof or µm chir . results were shown as infectivity level normalized to control samples cultured in presence of . % dimethyl sulfoxide vehicle. for all experiments, data are presented as mean ± sem of two experiments performed with six samples each. asterisks denote statistically significant differences ( * p-value ≤ . ; * * p-value ≤ . ; * * * p-value ≤ . ). expression nor treatment with the gsk inhibitor chir had a relevant effect on hev rna levels, suggesting that the pro-viral effect of gsk β is specific to hcv. given the well-known dependency of hcv on mir- (jopling et al., (jopling et al., , machlin et al., ) and the regulatory circuit between insulin-like growth factor receptor, gsk β, and mir- identified previously (zeng et al., ) , we assessed the effect of gsk inhibition on mir- expression in huh- . cells harboring a subgenomic hcv replicon. as shown in figure a , treatment with chir resulted in downregulation of mir- levels, in a similar magnitude compared to hcv replication inhibition. for further validation, huh- . cells transfected with hcv jc or u full-length rna, the latter being a hcv construct replicating independently from mir- , were treated with chir . as shown in figure b , chir inhibited the replication of the full-length hcv jc genome, the replication of which depends on the presence of mir- . in contrast, chir had no inhibitory effect on replication, nor entry and particle production, of the hcv u . in line with these observations, the production of jc infectious viral particles was strongly inhibited by chir whereas this inhibitor had no effect on infectious particle production of the u virus ( figure c) , suggesting that gsk β inhibition mainly impacts hcv rna replication. in view of remarkable associations between hcv and metabolic traits, we have investigated the role in the hcv life cycle of gsk , a key kinase in glucose metabolism and numerous other cellular processes. our study shows that gsk β, but not gsk α, serves as an important host factor of viral rna replication. the proviral effects of gsk β appeared to be mediated, at least in part, by maintaining hepatocellular levels of the key microrna mir- . microrna- is an important microrna predominantly expressed in hepatocytes, where it accounts for approximately - % of the entire microrna pool. the high abundancy of mir- in liver cells underlines key functions in liver development, growth, differentiation and homeostasis (bandiera et al., ) . it is not surprising, therefore, that repression of mir- expression promotes the development of hepatocellular carcinoma (hcc). in addition, mir- is involved in important metabolic functions of the liver including cholesterol and fatty acid synthesis and iron homeostasis (esau et al., ; castoldi et al., ) . a unique feature of the hcv life cycle is its dependence on the presence of mir- , which stabilizes the hcv genome and promotes viral replication through binding to the -untranslated region ( -utr) of the viral genome (jopling et al., (jopling et al., , machlin et al., ) . given the liver-specific expression of mir- , this micro-rna is also considered to be a key determinant of the hepatotropism of hcv. the strong dependence of hcv on mir- is evidenced by profound suppression of viral loads by therapeutic silencing of mir- in vivo (lanford et al., ; janssen et al., ) . given the tumor suppressor function of mir- , these therapeutic silencing strategies may be associated with an increased risk of hcc development. in addition, a recent study has shown that hcv functionally regulates (sequesters) mir- within hepatocytes, which results in a de-repression of mir- target genes in the presence of hcv and which may partially explain the oncogenic potential of chronic hepatitis c (luna et al., ) . our finding that specifically gsk β (and not gsk α) is required to maintain mir- levels in hcv infected hepatocytes adds another facet to the complex reciprocal relationship between hcv, mir- and the implications of chronic hcv infection on metabolic and oncogenic traits. yet, it is a limitation of our study that we cannot completely exclude that targets of gsk other than mir- are additionally involved in the suppression of hcv replication by gsk inhibition. however, it appears plausible that depletion of mir- plays a key role in mediating suppression of hcv replication by gsk -inhibition because mir- is one of the most important host factors on which hcv replication critically depends. a further confirmation of this notion is the important finding that the mir- -independent hcv mutant is resistant to gsk inhibition. in addition, gsk inhibition had no suppressive effect on replication of hev, a virus which does -in contrast to hcv -not depend on mir- . our results confirm a study by zeng et al., which has described a regulatory circuit between gsk , mir- and insulin-like growth-factor receptor in hepatocytes (zeng et al., ) . gsk is a multifunctional protein with an estimated different substrates, thereby regulating numerous cellular processes and pathways beyond glucose metabolism. hence, inappropriate gsk signaling appears to be involved in diverse diseases such as diabetes mellitus, cancer or alzheimeŕs disease. though a number of gsk inhibitors are in preclinical and clinical development, there is a concern of serious side effects due to the pleiotropic effects of gsk . however, some of the most significant undesirable effects of gsk inhibition, in particular β-catenin accumulation with potential deleterious carcinogenic effects, develop as a result of combined inhibition of gsk α and gsk β (rayasam et al., ) . in this regard, the finding that only gsk β but not gsk α affects mir- levels is important because it may serve as a proof-of-principle that selectively targeting gsk isoforms may be a suitable approach to manipulate specific functions of gsk . our data further illustrate the need for dissecting the role of gsk isoforms separately instead of non-specifically referring to gsk inhibition. in view of the pleiotropic cellular functions of gsk it is not surprising that viruses engage this important kinase to complete their life cycle. it has been shown that replication of the sars coronavirus depends on gsk -mediated phosphorylation of the nucleocapsid (n) protein which is a necessary process for viral replication (wu et al., ). in addition, it was shown that gsk β promotes the life cycle of influenza virus through facilitating the viral entry step (könig et al., ) . the here reported role of gsk β in the hcv life cycle adds another facet to the dependence of viruses on this central kinase. furthermore, the role of gsk in the hcv life cycle may be not restricted to hcv replication, as a recent report has shown that gsk supports hcv assembly by its involvement in lipoprotein production (sarhan et al., ) . while we did not make this observation in our experimental settings, our data indicate that it likely remains marginal compared to the role of gsk in supporting hcv rna replication. yet, the importance of these and our findings may rather consist in its implications for the understanding of the pathogenesis of chronic hepatitis c than in the identification of a novel target of antiviral therapy. gsk is a downstream target of insulin which is inhibited by insulin receptor signaling (welsh and proud, ) . reciprocally, gsk inhibits pivotal downstream targets of the insulin receptor under nonstimulated conditions, such as glycogen synthase (woodgett and cohen, ; roach, ) and irs- (eldar-finkelman and krebs, ; liberman and eldar-finkelman, ) . as a result, increased gsk activity has been observed in diabetic tissues (eldar-finkelman et al., ; nikoulina et al., ) and inhibition of gsk activity improves insulin resistance (cline et al., ; henriksen et al., ; nikoulina et al., ) . consequently, one may speculate that the dependence of hcv on active gsk could explain a benefit for hcv of insulin resistance, which is induced by hepatitis c and which had been associated with lower chances of treatment-induced viral clearance in the era of interferon therapy (romero-gómez et al., ) . mir- -a key factor and therapeutic target in liver disease glycogen synthase kinase- (gsk ): regulation, actions, and diseases efficient initiation of hcv rna replication in cell culture hepatitis c virus activates the mtor/s k signaling pathway in inhibiting irs- function for insulin resistance the liver-specific microrna mir- controls systemic iron homeostasis in mice effects of a novel glycogen synthase kinase- inhibitor on insulin-stimulated glucose metabolism in zucker diabetic fatty (fa/fa) rats the renaissance of gsk insulin resistance predicts response to peginterferon-alpha/ribavirin combination therapy in chronic hepatitis c patients sofosbuvir inhibits hepatitis e virus replication in vitro and results in an additive effect when combined with ribavirin * treatment of alzheimer's disease with the gsk- inhibitor tideglusib: a pilot study phosphorylation of insulin receptor substrate by glycogen synthase kinase impairs insulin action increased glycogen synthase kinase- activity in diabetes-and obesity-prone c bl/ j mice mir- regulation of lipid metabolism revealed by in vivo antisense targeting the homeostasis model assessment of the insulin resistance score is not predictive of a sustained virological response in chronic hepatitis c patients insulin resistance predicts rapid virological response in nondiabetic, non-cirrhotic genotype hcv patients treated with peginterferon alpha- b plus ribavirin glycogen synthase kinase- and dorsoventral patterning in xenopus embryos modulation of muscle insulin resistance by selective inhibition of gsk- in zucker diabetic fatty rats treatment of hcv infection by targeting microrna glycogen synthase kinase- (gsk ): inflammation, diseases, and therapeutics position-dependent function for a tandem microrna mir- -binding site located in the hepatitis c virus rna genome modulation of hepatitis c virus rna abundance by a liver-specific microrna hepatitis c virus down-regulates insulin receptor substrates and through up-regulation of suppressor of cytokine signaling insulin resistance predicts rapid virologic response to peginterferon/ribavirin combination therapy in hepatitis c genotype patients human host factors required for influenza virus replication therapeutic silencing of microrna- in primates with chronic hepatitis c virus infection vitamin d receptor and jak-stat signaling crosstalk results in calcitriol-mediated increase of hepatocellular response to ifn emerging therapies for the treatment of hepatitis c microrna- antagonism against hepatitis c virus genotypes - and reduced efficacy by host rna insertion or mutations in the hcv ' utr serine phosphorylation of insulin receptor substrate- by glycogen synthase kinase- attenuates insulin signaling complete replication of hepatitis c virus in cell culture a phase ii trial of tideglusib in alzheimer's disease hepatitis c virus rna functionally sequesters mir- masking the ' terminal nucleotides of the hepatitis c virus genome by an unconventional micrornatarget rna complex pharmacological inhibitors of glycogen synthase kinase functional properties of a monoclonal antibody inhibiting the hepatitis c virus rna-dependent rna polymerase characterization of three novel monoclonal antibodies against hepatitis c virus core protein insulin resistance and geographical origin: major predictors of liver fibrosis and response to peginterferon and ribavirin in hcv- facts and fictions of hcv and comorbidities: steatosis, diabetes mellitus, and cardiovascular diseases inhibition of glycogen synthase kinase improves insulin action and glucose metabolism in human skeletal muscle potential role of glycogen synthase kinase- in skeletal muscle insulin resistance of type diabetes visceral adiposity index is associated with histological findings and high viral load in patients with chronic hepatitis c due to genotype construction and characterization of infectious intragenotypic and intergenotypic hepatitis c virus chimeras glycogen synthase kinase : more than a namesake selective glycogen synthase kinase inhibitors potentiate insulin activation of glucose transport and utilization in vitro and in vivo multisite and hierarchal protein phosphorylation insulin resistance impairs sustained response rate to peginterferon plus ribavirin in chronic hepatitis c patients glycogen synthase kinase β inhibitors prevent hepatitis c virus release/assembly through perturbation of lipid metabolism hepatitis c virus infection and diabetes: direct involvement of the virus in the development of insulin resistance glycogen synthase kinase- is rapidly inactivated in response to insulin and phosphorylates eukaryotic initiation factor eif- b multisite phosphorylation of glycogen synthase. molecular basis for the substrate specificity of glycogen synthase kinase- and casein kinase-ii (glycogen synthase kinase- ) glycogen synthase kinase- regulates the phosphorylation of severe acute respiratory syndrome coronavirus nucleocapsid protein and viral replication a novel gsk- beta-c/ebp alpha-mir- -insulin-like growth factor receptor regulatory circuitry in human hepatocellular carcinoma robust hepatitis c virus infection in vitro ms, sr, cw, jg, and cl collected the data. ms, sr, sz, dm, jg, and cl analyzed the data. all authors have contributed to the manuscript by planning the study, and preparing as well as revising the manuscript. this study was supported by the else kröner-fresenius-stiftung ( -a to cl), the deutsche forschungsgemeinschaft (la / - and la / - ), and the swiss national science foundation ( a_ and a_ to dm). the authors express their gratitude to yolanda martinez for expert technical assistance, rolf marschalek for helpful discussions as well as ralf bartenschlager, jens bukh, suzanne u. emerson, charles m. rice, and jim woodgett for reagents. parts of this work have been presented as poster presentations at the international liver congress in paris, france, and at the annual meeting of the german association for the study of liver diseases in hamburg, germany. the supplementary material for this article can be found online at: https://www.frontiersin.org/articles/ . /fmicb. . /full#supplementary-material key: cord- -w x dkds authors: zhao, xuesen; li, jiarui; winkler, cheryl a.; an, ping; guo, ju-tao title: ifitm genes, variants, and their roles in the control and pathogenesis of viral infections date: - - journal: front microbiol doi: . /fmicb. . sha: doc_id: cord_uid: w x dkds interferon-induced transmembrane proteins (ifitms) are a family of small proteins that localize in the plasma and endolysosomal membranes. ifitms not only inhibit viral entry into host cells by interrupting the membrane fusion between viral envelope and cellular membranes, but also reduce the production of infectious virions or infectivity of progeny virions. not surprisingly, some viruses can evade the restriction of ifitms and even hijack the antiviral proteins to facilitate their infectious entry into host cells or promote the assembly of virions, presumably by modulating membrane fusion. similar to many other host defense genes that evolve under the selective pressure of microorganism infection, ifitm genes evolved in an accelerated speed in vertebrates and many single-nucleotide polymorphisms (snps) have been identified in the human population, some of which have been associated with severity and prognosis of viral infection (e.g., influenza a virus). here, we review the function and potential impact of genetic variation for ifitm restriction of viral infections. continuing research efforts are required to decipher the molecular mechanism underlying the complicated interaction among ifitms and viruses in an effort to determine their pathobiological roles in the context of viral infections in vivo. interferon-induced transmembrane proteins (ifitms) are a family of small proteins that localize in the plasma and endolysosomal membranes. ifitms not only inhibit viral entry into host cells by interrupting the membrane fusion between viral envelope and cellular membranes, but also reduce the production of infectious virions or infectivity of progeny virions. not surprisingly, some viruses can evade the restriction of ifitms and even hijack the antiviral proteins to facilitate their infectious entry into host cells or promote the assembly of virions, presumably by modulating membrane fusion. similar to many other host defense genes that evolve under the selective pressure of microorganism infection, ifitm genes evolved in an accelerated speed in vertebrates and many single-nucleotide polymorphisms (snps) have been identified in the human population, some of which have been associated with severity and prognosis of viral infection (e.g., influenza a virus). here, we review the function and potential impact of genetic variation for ifitm restriction of viral infections. continuing research efforts are required to decipher the molecular mechanism underlying the complicated interaction among ifitms and viruses in an effort to determine their pathobiological roles in the context of viral infections in vivo. keywords: host susceptibility, interferon-induced transmembrane proteins, ifitm, single nucleotide polymorphisms, viral infection interferon-induced transmembrane proteins (ifitms) are a family of small proteins that can be found in single cell organisms and are evolutionally conserved across vertebrates (siegrist et al., ; zhang et al., ) . the human ifitm family comprises five members, including immune-related ifitm , ifitm , and ifitm , as well as ifitm and ifitm with no known role in immunity. as key host defense genes, ifitms evolved under the selective pressure of microorganism infection (compton et al., ) . ifitm proteins are involved in many aspects of virus-host interaction and play important roles in viral pathogenesis. among the number of single-nucleotide polymorphisms (snps) in ifitm gene that have been identified in human populations, several are associated with disease severity and prognosis of influenza a virus (iav) and other viral infections (everitt et al., ; zhang et al., ; xu-yang et al., ; allen et al., ) . mechanistically, these snps either alter the expression of ifitm or result in expression of n-terminally truncated ifitm isoform, -ifitm , with reduced antiviral activity against different viruses (everitt et al., ; allen et al., ) . in this review, we will summarize the findings on human ifitm structural features related with antiviral activity, and impact of genetic variation on ifitm antiviral function in the control and pathogenesis of viral infections in humans. to date, ifitms have been shown to inhibit the infection of enveloped rna viruses from viral families , non-enveloped rna viruses, e.g., reovirus (anafu et al., ) and foot-and-mouth disease virus , and several dna viruses (li et al., ) . ifitms efficiently inhibit a number of medically important human pathogenic viruses, including iav (brass et al., ; bailey et al., ) , dengue virus (denv) (brass et al., ; jiang et al., ) , west nile virus (wnv) (brass et al., ; jiang et al., ) , zika virus (zikv) (savidis et al., ) , ebola virus (ebov) (huang et al., ; wrensch et al., ) , marburg virus (marv) (huang et al., ) , severe acute respiratory syndrome coronavirus (sars-cov) (huang et al., ) , rift valley fever virus (rvfv) (mudhasani et al., ) , hantaan virus (htnv) (mudhasani et al., ; xu-yang et al., ) , hepatitis c virus (hcv) (wilkins et al., ) , and human immunodeficiency virus (hiv) (lu et al., ; compton et al., ; yu et al., ; compton et al., ; foster et al., ; chesarino et al., ; tartour et al., ; wang et al., ) . in vivo studies in ifitm knockout mice demonstrate the critical role of ifitm in restricting infection and reducing disease severity of infection by iav (bailey et al., ; everitt et al., ) , wnv , chikungunya virus and venezuelan equine encephalitis virus , and respiratory syncytial virus . ifitm in mice not only protects lung epithelia cells from iav infection, but it was also shown to restricts iav infection of lung dendritic cells, which traffic to lymph nodes to prime cd + t cell anti-viral response (infusini et al., ) . moreover, lung resident memory cd + t cells in mice were programmed to retain ifitm expression, facilitating their survival and protection from viral infection during subsequent exposures (wakim et al., ) . ifitm proteins localize at the plasma membrane as well as the membranes of endocytic vesicles and lysosomes (bailey et al., ) . ifitms can also be incorporated into envelope membranes of many viruses (yu et al., ; tartour et al., ) . emerging evidence suggests that ifitm proteins on both viral and cellular membranes can restrict the infectious entry of diverse envelope viruses by inhibiting viral fusion at cell plasma or endolysosomal membranes, but ifitm does not impede endocytosis of virions into cells li et al., ; perreira et al., ; compton et al., ; desai et al., ) . as extensively discussed in a previous review , the potency of ifitm restriction of viral cell entry is generally correlated to the co-localization of ifitm proteins at the sites of viral fusion. for instance, ifitm more efficiently restricts the viruses that enter the cytoplasm via direct fusion with plasma membrane or via rab- positive early endosomes, whereas ifitm more efficiently inhibits viruses that enter via rab -positive late endosomes or lysosomes. this rule is highlighted by the finding that mutation of ifitm endocytic signal results in its cell surface accumulation and gains a function to restrict the infection of human parainfluenza virus (hpiv- ), which enters cells via direct fusion with the plasma membrane (rabbani et al., ; zhao et al., ) . another example is that the sensitivity of iavs to ifitm appears to depend on the ph value at which the iav hemagglutinin triggers membrane fusion and thus the endocytic compartments where the membrane fusion take place (gerlach et al., ) . however, exceptions of this rule do exist. for instance, it remains to know how moloney leukemia virus (mlv) and sendai virus that fuse at cell plasma membrane (brass et al., ; hach et al., ) as well as lassa fever virus (lasv) and lymphocytic choriomeningitis virus (lcmv) that fuse at rab -positive late endosomes (brass et al., ; mudhasani et al., ) to escape ifitm and ifitm restriction, respectively. the mechanism of ifitm inhibition of viral fusion and cell entry is not yet resolved. one study reported that ifitm inhibited jaagsiekte sheep retrovirus envelope and iav hemagglutinin fusion of viral envelope to cellular membranes prior to coalescence of lipid-bilayers, a process known as hemifusion . however, another study using a direct viruscell fusion assay in viable cells to investigate iav entry found that overexpression of ifitm protein in late endosomes did not alter lipid mixing, but rather inhibited the release of viral contents into the cytoplasm, suggesting that ifitm inhibits the transition from hemifusion to full fusion of the respective lipid membranes (desai et al., ) . others studies suggested that ifitm multimerization decreases membrane flexibility by altering membrane curvature, with the consequence of interruption of the virus-cell membrane fusion (john et al., ; lin et al., ) . another group reported that ifitm expression increased cholesterol content in the endosome or lysosome via an interaction with vesicle-associated membrane protein-associated protein a (vapa), which abrogated endolysosomal fusion with the viral envelop (amini-bavil-olyaee et al., ). however, this later observation was not confirmed by other studies (desai et al., ; wrensch et al., ) . in addition, ifitms were reported to affect the trafficking of vacuolar atpase (v-atpase), implying that ifitms may indirectly restrict viral entry by modulating endosomal acidity (wee et al., ) . it is also well documented that the antiviral potency of ifitm proteins varies among different cell types (huang et al., ; zhao et al., ) , suggesting that ifitms work together with other cellular proteins to modulate viral fusion. in support of this notion, zinc metallopeptidase ste (zmpste ), a transmembrane metalloprotease localized in the inner nuclear membrane and cytoplasmic organelles, had been identified as a downstream partner of ifitm to restrict the entry of enveloped rna and dna viruses (fu et al., ) . in addition to the protective function of ifitm proteins to reduce viral infection of host cells, ifitm proteins, particularly ifitm and , can lead to the production of virions that package ifitms and display reduced entry into target cells (compton et al., ; tartour et al., tartour et al., , . one study found that ifitm proteins interact with hiv envelope protein in viral producer cells to disrupt envelope protein processing and virion incorporation, which impairs virion infectivity (yu et al., ) ; however, another found that ifitm expression in producing cells did not affect the amount of envelope protein incorporation into progeny hiv- virions (appourchaux et al., ) . both studies reported that the level of ifitm protein incorporation into progeny virions does not correlate with the extent of infectivity reduction. moreover, recent studies have also shown that ifitms can modulate viral infection and pathogenesis via mechanisms that are not directly related to the restriction of virus entry. for example, given the resistance of human papillomaviruses (hpv) to ifitms, hpv infection of keratinocytes inhibits the expression of ifitm and ripk to escape from ifnγ and tnf-α-mediated antiproliferation and necroptosis, which is essential for establishing a persistent infection (ma et al., ) . in addition, it was demonstrated in ifitm knockout mice that ifitm limited murine cmv (mcmv) pathogenesis without directly preventing virus replication (stacey et al., ) . instead, ifitm contributed to the antiviral cellular immunity by abrogating inflammatory cytokine-driven lymphopenia including apoptosis-independent nk cell death and t cells depletion (stacey et al., ) . not surprisingly, in the arms race between pathogen and host, many viruses have evolved strategies to evade the antiviral function of host restriction proteins. for instance, transmitted founder hiv- strains establishing de novo infection are generally capable of evading ifitm restriction (foster et al., ; wang et al., ) . mutations allow the virus to escape adaptive immune responses, and/or switches in the hiv- co-receptor tropism from ccr to cxcr increases viral sensitivity to inhibition by ifitm and ifitm in endosomal compartments (foster et al., ) . moreover, iav facilitates its infection by activating p or degrading eukaryotic translation initiation factor b (eif b) to inhibit the expression of ifitm proteins (wang s. et al., ; wang et al., ) . in contrast, human coronavirus oc (hcov-oc ) and human cytomegalovirus (hcmv) hijack ifitm to promote its infectious entry and progeny virions assembly, respectively (zhao et al., ; xie et al., ) . the human ifitm locus, approximately kb long, is located on chromosome and comprises five genes: ifitm , ifitm , ifitm , ifitm , and ifitm (figure ) . as an ifn-stimulated gene (isg), ifitm , ifitm , ifitm genes each has an interferon stimulated response element (isre) in its promoter region besides the additional gamma-activated sequence (gas) in the promoter region of ifitm gene (siegrist et al., ) . however, the protein expression of ifitm and ifitm are not induced by ifns (zhang et al., ) . though ifitm - proteins are ubiquitously expressed in human tissues in the absence of ifn induction, they can be robustly up-regulated by all three types of ifns (zhao et al., ) . the ifitm promoter has binding sites for dozens of transcription factors, including polr a, myc, elf , phf , chd , taf , rest, sin ak , sin a, irf , stat , tbp, stat , stat , zbtb a, and ctcf, some of which may affect ifitm expression . as illustrated in figure , all ifitm genes contain two coding exons interspersed by one intron. both ifitm and ifitm were predicated to encode a wild typed full-length form and a truncated isoform with / amino acid residues deletion from n-terminus. most vertebrate animals have two or more ifitm genes. in many primate species, gene duplication, and divergence of ifitm has been identified (zhang et al., ; compton et al., ) . ifitm locus in many modern primate species contains multiple copies of ifitm -like genes due to gene duplication (compton et al., ) . for instance, marmoset, macaque, and africa green monkey (agm) each has five, six and eight copies of ifitm genes, respectively (compton et al., ) . comparative genomics studies indicate that ifitm is the most ancient member in the ifitm family and ifitm emerges as a rather recent genetic event in human, chimpanzee, and gorilla (compton et al., ) . the multiplicity and diversity of ifitm / indicates that there is a positive selection in ifitm evolution, which is consistent with their role in restricting pathogen invasion (compton et al., ) . ifitm proteins have several topologies that may affect their function. as shown in figure a , although ifitm may adopt three different membrane topologies (bailey et al., ) , it exists predominantly as a type ii transmembrane protein with the n-terminus in cytosol and the short c-terminus exposed to cellular exterior or in the lumen of endolysosome (bailey et al., ) . although ifitm was also reported to predominately adopt a type ii transmembrane topology, other membrane topologies may exist ( figure b ; li et al., ) . as depicted in figure , ifitms consist of intramembrane (imd) and transmembrane (tmd) domains separated by an intracellular loop (cil) and variable n and c terminal domains (ntd and ctd), respectively (bailey et al., (bailey et al., , chesarino et al., ) . while the ntd and ctd are highly variable in length and sequence among ifitm orthologs and paralogs, the canonical cd domain spanning imd to cil domains is evolutionally more conserved (bailey et al., ) . critical structure motifs and amino acid residues undergoing post-translational modifications required for ifitm oligomerization as well as their biological and antiviral functions are discussed below. compared to ifitm , ifitm and ifitm have n-terminal -aa or -aa extension which was previously regarded to figure | ifitm gene structure. human ifitm genes located in chromosome p . are indicated, and the exons of immunity-related ifitm genes (ifitm , ifitm , and ifitm ) are indicated in blue color. the putative transcripts of ifitm and ifitm are also shown below. the transcription factor binding regions derived from ucsc genome browser are shown, which is verified by chip-seq from the proteins tested through encode; grch /hg . two red vertical lines across transcription factor binding sites represent the influenza associated snps of rs and rs with allele frequencies, respectively. n-terminally truncated ifitm isoform ( ifitm ) that is presumably generated by rs is indicated. be absent in rs -c encoding ifitm isoform. although deletion of this n-terminal -aa region significantly impaired its ability to inhibit the infection by iav, vsv, and denv, the deletion apparently did not affect its ability to enhance hcov-oc pp infection john et al., ) . interestingly, -ifitm enhanced inhibition of hiv- fusion (compton et al., ) . moreover, -ifitm , a n-terminal -aa truncated isoform of ifitm , which is derived from an alternatively initiated rna transcript (figure ) , demonstrated a more potent suppression on hiv- infection than that by fulllength wild-type ifitm (wu et al., ) . in fact, a recent study argues that the -ifitm , rather than the full-length ifitm and ifitm , is the effective restriction factor of hiv- with cxcr -tropism (wu et al., ) . the yxx motif of ifitm is an endocytic signal essential for endocytosis and localization of ifitm to endocytic vesicles and lysosomes (jia et al., ) . artificial mutations of yeml motif (y d, y a, or l a) result in an accumulation of ifitm in the plasma membrane and reduced inhibition of viruses that enter the cytoplasm at endocytic vesicles/lysosomes, such as iav, human coronaviruses nl and - e (chesarino et al., a; jia et al., ; williams et al., ; zhao et al., ) , but enhanced inhibition of viruses that directly enter cells at the plasma membranes, such as hpiv- and hiv- (jia et al., ; compton et al., ; rabbani et al., ) . intriguingly, replacement of ifitm tyrosine (y) with either alanine (a) or aspartic acid (d) to mimic unphosphorylated or phosphorylated ifitm converted the antiviral protein to enhance the entry of sars-cov and mers-cov . these studies suggest that the ntd, particularly yxx motif, plays important roles in ifitm / subcellular localization and antiviral activity. ifitm are phosphorylated by the protein-tyrosine kinase fyn on tyrosine (y ), which results in its plasma membrane accumulation and decreased antiviral activity against influenza viruses (jia et al., (jia et al., , chesarino et al., a) . this result is consistent with the mutagenesis studies of yxx motif, where y is part of an endocytosis signal that can be blocked by phosphorylation (chesarino et al., a) . additionally, phosphorylation of ifitm by fyn and mutagenesis of y also lead to decreased frontiers in microbiology | www.frontiersin.org ifitm ubiquitination, suggesting modification of y as an important mechanism to control ifitm trafficking and degradation (chesarino et al., a) . compared to ifitm and ifitm , human ifitm has a relatively long c-terminal region of amino acid residues. the krxx motif serves as a sorting signal for ifitm . substitution of two basic residues kr with alanine (kr/aa) in ifitm reduced its distribution in lamp -positive lysosomes but enriched its localization in cd -positive multivesicular bodies . the kr/aa mutant ifitm executed increased activity to inhibit the infection by jaagsiekte sheep retrovirus (jsrv) and a amphotropic murine leukemia virus (mlv) . interestingly, although sars-cov and hcov-nl share the ace receptor for infection of host cells, deletion of the c-terminal , , or amino acids did not apparently affect the activity of ifitm to inhibit sars-cov entry but enhanced the activity of ifitm to inhibit the entry of hcov-nl . however, further deletion of the c-terminal , , or amino acids significantly attenuated or even abolished the ability of ifitm to inhibit the entry of sars-cov, but did not apparently affect the entry of nl . more strikingly, deletion of c-terminal -aa or -aa converted ifitm to a potent enhancer of mers-cov and hcov-oc entry (zhao et al., . a recent mutagenesis study indicated that amino acid residues - in the ctd domain of ifitm differentially modulates its activities in target cell protection and negative imprinting of progeny hiv- infectivity (appourchaux et al., ) . these studies clearly indicate that the ctd of ifitms contains multiple structure motifs that regulate the subcellular localization and antiviral functions. the canonical cd domain comprises imd to cil domains and is evolutionally conserved (bailey et al., ) . imd domain (residues - ) is a hydrophobic region and possesses an amphipathic alpha helix spanning residues - (chesarino et al., ) . it was demonstrated recently that either deletion of this alpha helices or mutations altering amphipathicity largely impaired or even abolished ifitm antiviral activity, suggesting figure | schematic diagram of ifitm / topology and structural determinants essential for their modulation of viral entry. five structural domains, including the n-terminal domain (ntd), amphipathic helix domain (ahd), intracellular loop (cil), transmembrane domain (tmd), and c-terminal domain (ctd), are illustrated. two membrane-associated domains (ahd and tmd) are embodied in light orange. in ifitm , three hydrophilic residues (s , n , and t ) in amphipathic helix are labeled in violet. in ntd domain, yeml motif required for ifitm endocytosis are depicted in blue with red circle and ppny motif recruiting nedd e ligase are shown in green. in cil domain, svks motif required for ifitm to inhibit iav, denv, and llov infection is indicated with purple circle. the residues with post-translational modification, including phosphorylation (y and y labeled with red circle), palmitoylation (c , c , and c marked with light blue), and ubiquitination (highlighted in orange), are indicated. in addition, two residues (f and f ) essential for oligomerization are marked with yellow. in ifitm , c-terminal -aa residues critical for modulating human coronaviruses entry is indicated in blue, and kr dibasic motif constituted as ifitm sorting signal is depicted with red circle. a critical role in ifitm antiviral function, presumably through affecting membrane physical properties (chesarino et al., ) . in addition, the svks motif residing in cil domain is essential for ifitm to inhibit iav and denv infection, but replacement of this motif with four residues of alanine promoted cellular entry driven by lloviu virus (llov) glycoprotein (wrensch et al., ) . ifitm proteins can be palmytoylated at three cystines in cd domain by multiple zinc finger dhhc domain-containing palmitoyltransferases (zdhhcs) . while cys localizes close to the amphipathic alpha helix in the imd domain, cys and cys are adjacent to the tmd domain. mutagenesis studies indicated that substitution of those three cysteine residues with alanine alters the ifitm distribution from punctate clusters in the cellular membrane to a more diffused pattern and the resulting palmitoylation-deficient mutant showed impaired or even abolished antiviral activity (yount et al., ) . interestingly, when other lipid modification sites, such as myristoylation and prenylation, were introduced in ifitm ntd or ctd domains, the antiviral function of palmitoylation-deficient ifitm mutant could be restored. these findings imply that anchoring ifitm protein to membranes, but not structure alteration by s-palmitoylation, is important for ifitm restriction of virus entry (yount et al., ; chesarino et al., b) . human ifitm proteins possess four conserved lysine residues (figure ) that can be ubiquitinated by e ubiquitin ligase such as nedd (yount et al., ; chesarino et al., ) . previous studies demonstrated that each lysine residue can be modified with mono-and poly-ubiquitination through lys- and lys- linkages (yount et al., ) . by replacing all four residues of lysine with alanine, the mutant ifitm demonstrated endolysosomal distribution and execute hyperactivity to restrict iav infection than wild type (yount et al., ) . however, our studies demonstrated that ubi-deficient ifitm lost antiviral activity against all the viruses tested, including iav (zhao et al., . the discrepancy of those studies is not clear. importantly, ubiquitination and endocytosis of ifitm is required for mtor inhibitor-induced degradation of ifitm (shi et al., ) . in addition, ifitm k can be monomethylated by lysine methyltransferase set and demethylated by histone demethylase lsd (shan et al., (shan et al., , . while vesicular stomatitis virus (vsv) and iav infection increased ifitm -k me levels by promoting the interaction between ifitm and set and disassociation from lsd to attenuate ifitm antiviral activity, ifn-α reduced ifitm -k me levels and increased its antiviral activity (shan et al., (shan et al., , . ifitm proteins function as homo-or hetero-oligomers. two phenylalanine residues (f and f ) are essential for ifitm homo-and hetero-oligomerization (john et al., ) . ifitm bearing f a and f a mutations (ifitm / fa) showed reduced ability to inhibit the entry of hcov-nl , but lost ability to inhibit or enhance the entry of all other tested viruses (zhao et al., . the antiviral activity of many naturally existing human ifitm variants has been tested in cell cultures. as shown in table and mentioned in previous sections, although snp rs is predicted to encode a splice variant specifying a n-terminally truncated isoform ifitm (everitt et al., ) , the putative -ifitm protein has not been detected in the tissues or cells of affected subjects (wu et al., ; makvandi-nejad et al., ) . however, subcellular localization and antiviral activity of genetically engineered -ifitm have been extensively investigated in cultured cells (john et al., ; compton et al., ) . interestingly, ifitm was reported recently to have a similar n-terminally truncated isoform ( -ifitm ) expressed in human innate immune cells and cd + t cells (wu et al., ) . compared with the full-length wildtype ifitm , -ifitm demonstrated an increased plasma membrane accumulation and enhanced antiviral activity against hiv- , particularly, hiv- with cxcr tropism (compton et al., ; wu et al., ) . in addition, expression and antiviral activity of some human nonsynonymous ifitm variants have also been investigated in cell cultures (john et al., ) . while many of the snps results in undetectable levels of ifitm in the transfected cells, presumably due to the reduced stability of mutant proteins, and thus loss of antiviral activity against iav, a few snps did not apparently alter the amount of ifitm proteins but impaired the antiviral activity against iav. it will be interesting to test all the ifitm variants against a group of viruses and identify the snps that potentially impact the control and pathogenesis of specific viral infections in humans. as a potent viral restriction factor, ifitms play a pivotal role in limiting the infection by multiple viruses and any genetic variation affecting the ifitm expression or function might contribute to viral pathogenesis. thus, natural variation in ifitm genes and its association with illness severity has been extensively investigated. currently, a dozen of snp in ifitm have been reported, some of which may modulate ifitm expression, affect rna splicing, or result in nonsynonymous or synonymous variants ( table ) . several snps affecting function of ifitms have been investigated for their association with morbidity, severity and prognosis of microorganism infection (everitt et al., ; shen et al., ; zhang et al., zhang et al., , allen et al., ) . the most studied snp associated with severe outcomes of iav infection is snp rs , which is a nonsynonymous variation in the first exon of ifitm (everitt et al., ) . the substitution of the major allele of t with alternative allele of c was predicted to alter ifitm mrna splicing and generate a n-terminally truncated variant of ifitm with amino acid residues deletion ( -ifitm ) (everitt et al., ) . the snp rs has a higher prevalence in the population of east asia than europe ( . vs. . ) (everitt et al., ) . moreover, the homozygous cc genotype and heterozygous tc genotype have significantly higher frequency in east asia than that in europe ( . vs. . and . vs. . , respectively) (everitt et al., ) . rs located at the ifitm promoter is associated with severe influenza in three human cohorts . snp rs , wherein the majority g allele is replaced with a minor a allele, controls ifitm promoter activity and determines the expression level. compared to the g allele, a allele has activities of decreased irf binding and increased ctcf binding, thus resulting in lower ifitm expression. snp rs has diverse allele and genotypic frequencies in different human populations ( table ) . the allele frequency of rs -g is higher in the population of east asia and africa than that in europe ( . and . vs. . , respectively) . also, the homozygous gg genotype has several groups previously reported that ifitm snp of rs is associated with the susceptibility and severity of patients with seasonal influenza and pandemic h n and h n iav infection (everitt et al., ; zhang et al., ; pan et al., ) . as summarized in table , everitt et al. ( ) first discovered that a higher allelic frequency of rs -c exists in caucasian hospitalized influenza patients than healthy control. moreover, they observed a remarkably higher frequency of the homologous cc genotype in hospitalized influenza patients compared to the normal european population ( . % vs. . %) (everitt et al., ) . importantly, another group reported that the minor c allele of rs in the caucasian population is much more prevalent in han chinese and japanese and is associated with disease severity in patients with ph n / iav infection (zhang et al., ) . in line with this observation, results obtained from study of h n iav infection revealed that h n infected patients with rs -cc genotype exhibited accelerated disease progression and increased mortality rate than patients with the tc and tt genotype . these studies collectively suggest that rs -c is a risk allele associated with severe iav infection. however, several recent studies from european or africa-american ethnic groups did not support the association of rs with severe influenza infection (mills et al., ; lopez-rodriguez et al., ; randolph et al., ) . the rare frequency of rs -c in european population may account for this discrepancy. through meta-analysis of studies, rs t > c was associated with risk to severe influenza infection with odds ratio of . ( % ci . , . ) in both european and east asian populations, but for the mild infection, the results remained uncertain (prabhu et al., ) . although the association of rs with the susceptibility to influenza infection and disease severity was observed by many studies, the mechanism for this association is largely unknown. as mentioned above, although rs -c is speculated to encode a n-terminal truncated ifitm with attenuated antiviral activity to impede iav entry, the truncated variant has not been detected so far (everitt et al., ; makvandi-nejad et al., ) . moreover, the homozygous cc genotype was demonstrated to only express full-length ifitm at a similar level to tt genotype (wu et al., ) . thus rs -c genetic variant does not appear to affect the biochemical nature and expression of ifitm . obviously, further investigation to determine whether rs -c influences ifitm gene splicing or protein levels in a cell type-specific manner and whether this snp co-segregates with a different causative allele is warranted. a recent study found that another ifitm snp rs has a strong association with disease severity in three influenza cohorts . rs is located in the promoter region of ifitm (figure ) . the substitution of the majority allele of g with minority allele of c reduces ifitm expression level and consequently results in the reduced number of antiviral cd + t cells in lung tissue upon iav infection . in cohort flu , a cohort of naturally acquired influenza infection, a higher frequency of homozygosity of risk a allele was observed in patients with severe illness than the mild cases ( . % vs. . %) ( table ; allen et al., ) . also, an increased frequency p-value refers to the differences of allele frequencies between mild influenza patients and severe influenza patients. frontiers in microbiology | www.frontiersin.org of the a allele was found in patients who suffered with severe influenza in other two cohorts (table ; allen et al., ) . thus, rs -a is a risk allele associated with severe iav infection and its association with other viral infection disease deserves to be investigated in future. in addition to influenza, snp of rs was also observed to have association with development of aids and hantaan virus associated hemorrhagic fever with renal syndrome (hfrs) (zhang et al., ; xu-yang et al., ) . zhang et al. ( ) reported that rs is associated with rapid progression of aids, but not the susceptibility to hiv infection. patients of aids rapid progressors had a higher frequency of rs cc and ct genotype than non-progressors (zhang et al., ) . compared with tt genotype, more patients with cc/ct genotypes were characterized with higher viremia level and more significantly reduced cd t + cells (zhang et al., ) . in addition, the association of rs -c and homozygous cc genotype with disease severity in hfrs patients infected by hantaan virus was reported recently (xu-yang et al., ) . a higher allelic frequency of rs -c was observed in severe hfrs patients hospitalized than healthy han chinese controls ( . % vs. . %). also, severe hfrs patients had a higher frequency of rs cc than patients with mild hfrs and healthy han chinese. along these lines, this group also reported that the viral titer detected in the plasma of hfrs patients with rs -cc genotype was more significantly alleviated than those with the tc and tt genotype (xu-yang et al., ) . thus, rs c is a risk allele associated with severity of aids and hfrs, indicating that snp rs may have a profound impact on the pathogenesis of multiple viral diseases. ifitms are a group of small fusogenic proteins that restrict the infection of broad spectrum of viruses by inhibiting the fusion of viral and cellular membranes. however, whether ifitms inhibit hemifusion, the process whereby the outer, but not the inner, leaflet of the viral and cellular membranes merge, or the transition from hemifusion to pore formation remains to be rigorously determined. it is also important to note that ifitms do not always inhibit virus entry, but may promote membrane fusion under selected conditions, such as in the case of hcov-oc infection (zhao et al., ) . moreover, our recent studies indicated that specific mutations can flip the biological activity of ifitms, from inhibiting to promoting the infection of selected human coronaviruses . those later findings strongly suggest that the fusogenic activity of ifitms might be bidirectional and could be regulated by viral and host factors. the elucidation of molecular mechanisms underlying ifitm-mediated innate immunity via modulation of viral entry into host cells as well as the negative imprinting of progeny virions (virions incorporating ifitms display decreased infectivity) will open new avenues for future research. we have only just begun to appreciate the role of ifitm proteins in innate antiviral defenses. genetic association studies of rare and common variants may explain population-specific and individual variance in susceptibility to common viral pathogens and identify specific ifitm -virus interactions. expansion of genetic studies incorporating gene knock-down or knockoff screens, single cell transcriptomics, epigenetic modification, and targeted sequencing for different viral infections will provide needed insights into cellular mechanisms and pathways involved in ifitm-mediated host response to viral exposure and infection. all the authors participated in the draft and revision of this review article. the project was supported by grants from the u.s. national institutes of health (ai ), the national natural science foundation of china ( and ), the commonwealth of pennsylvania through the hepatitis b foundation as well as federal funds from the national cancer institute, national institutes of health, under contract hhsn e. this project was supported in part by the intramural research program of the nih, national cancer institute, center for cancer research. the content of this publication does not necessarily reflect the views or policies of the department of health and human services, nor do mention of trade names, commercial products, or organizations imply endorsement by the u.s. government. snp-mediated disruption of ctcf binding at the ifitm promoter is associated with risk of severe influenza in humans the antiviral effector ifitm disrupts intracellular cholesterol homeostasis to block viral entry interferon-inducible transmembrane protein (ifitm ) restricts reovirus cell entry functional mapping of regions involved in the negative imprinting of virion particles infectivity and in target cell protection by the interferon ifitm limits the severity of acute influenza in mice interferoninduced transmembrane protein is a type ii transmembrane protein ifitm-family proteins: the cell's first line of antiviral defense the ifitm proteins mediate cellular resistance to influenza a h n virus, west nile virus, and dengue virus ifitm requires an amphipathic helix for antiviral activity phosphorylation of the antiviral protein interferon-inducible transmembrane protein (ifitm ) dually regulates its endocytosis and ubiquitination regulation of the trafficking and antiviral activity of ifitm by post-translational modifications e ubiquitin ligase nedd promotes influenza virus infection by decreasing levels of the antiviral protein ifitm ifitm proteins incorporated into hiv- virions impair viral fusion and spread natural mutations in ifitm modulate post-translational regulation and toggle antiviral specificity ifitm restricts influenza a virus entry by blocking the formation of fusion pores following virus-endosome hemifusion defining the range of pathogens susceptible to ifitm restriction using a knockout mouse model ifitm restricts the morbidity and mortality associated with influenza resistance of transmitted founder hiv- to ifitm-mediated restriction zmpste defends against influenza and other pathogenic viruses ph optimum of hemagglutinin-mediated membrane fusion determines sensitivity of influenza a viruses to the interferon-induced antiviral state and ifitms the interferonstimulated gene ifitm restricts west nile virus infection and pathogenesis palmitoylation on conserved and nonconserved cysteines of murine ifitm regulates its stability and anti-influenza a virus activity distinct patterns of ifitm-mediated restriction of filoviruses, sars coronavirus, and influenza a virus respiratory dc use ifitm to avoid direct viral infection and safeguard virus-specific cd + t cell priming the n-terminal region of ifitm modulates its antiviral activity by regulating ifitm cellular localization identification of an endocytic signal essential for the antiviral action of ifitm identification of five interferon-induced cellular proteins that inhibit west nile virus and dengue virus infections the cd domain of ifitm is required for both ifitm protein association and inhibition of influenza a virus and dengue virus replication the host restriction factor interferon-inducible transmembrane protein inhibits vaccinia virus infection a sorting signal suppresses ifitm restriction of viral entry ifitm proteins restrict viral membrane hemifusion amphotericin b increases influenza a virus infection by preventing ifitm -mediated restriction ifitm and severe influenza virus infection. no evidence of genetic association the ifitm proteins inhibit hiv- infection human papillomavirus downregulates the expression of ifitm and ripk to escape from ifngamma-and tnfalpha-mediated antiproliferative effects and necroptosis lack of truncated ifitm transcripts in cells homozygous for the rs -c variant that is associated with severe influenza infection the palmitoyltransferase zdhhc enhances interferoninduced transmembrane protein (ifitm ) palmitoylation and antiviral activity ifitm and susceptibility to respiratory viral infections in the community ifitm- and ifitm- but not ifitm- restrict rift valley fever virus ifitm rs -c variant increases potential risk for severe influenza virus infection in chinese population ifitms restrict the replication of multiple pathogenic viruses the interferon-stimulated gene ifitm restricts infection and pathogenesis of arthritogenic and encephalitic alphaviruses association between ifitm rs polymorphism and influenza susceptibility and severity: a meta-analysis identification of interferon-stimulated gene proteins that inhibit human parainfluenza virus type evaluation of ifitm rs association with severe pediatric influenza infection the ifitms inhibit zika virus replication histone demethylase lsd restricts influenza a virus infection by erasing ifitm -k monomethylation negative regulation of interferon-induced transmembrane protein by set -mediated lysine monomethylation a functional promoter polymorphism of ifitm is associated with susceptibility to pediatric tuberculosis in han chinese population mtor inhibitors lower an intrinsic barrier to virus infection mediated by ifitm the small interferon-induced transmembrane genes and proteins the antiviral restriction factor ifn-induced transmembrane protein prevents cytokine-driven cmv pathogenesis ifitm proteins are incorporated onto hiv- virion particles and negatively imprint their infectivity interference with the production of infectious viral particles and bimodal inhibition of replication are broadly conserved antiviral properties of ifitms enhanced survival of lung tissue-resident memory cd (+) t cells during infection with influenza virus due to selective expression of ifitm influenza a virus facilitates its infectivity by activating p to inhibit the expression of interferon-induced transmembrane proteins influenza a virus-induced degradation of eukaryotic translation initiation factor b contributes to viral replication by suppressing ifitm protein expression the v loop of hiv- env determines viral susceptibility to ifitm impairment of viral infectivity early hypercytokinemia is associated with interferon-induced transmembrane protein- dysfunction and predictive of fatal h n infection interferon-inducible transmembrane proteins of the innate immune response act as membrane organizers by influencing clathrin and v-atpase localization and function interferon-induced cell membrane proteins, ifitm and tetherin, inhibit vesicular stomatitis virus infection via distinct mechanisms ifitm is a tight junction protein that inhibits hepatitis c virus entry ifitm polymorphism rs -c restricts influenza a viruses interferon-induced transmembrane protein-mediated inhibition of host cell entry of ebolaviruses ifitm proteins inhibit entry driven by the mers-coronavirus spike protein: evidence for cholesterol-independent mechanisms delta ifitm differentially restricts x and r hiv- human cytomegalovirus exploits interferon-induced transmembrane proteins to facilitate morphogenesis of the virion assembly compartment swine interferoninduced transmembrane protein, sifitm , inhibits foot-and-mouth disease virus infection in vitro and in vivo interferon-induced transmembrane protein inhibits hantaan virus infection, and its single nucleotide polymorphism rs influences the severity of hemorrhagic fever with renal syndrome s-palmitoylation and ubiquitination differentially regulate interferon-induced transmembrane protein (ifitm )-mediated resistance to influenza virus palmitoylome profiling reveals s-palmitoylation-dependent antiviral activity of ifitm ifitm proteins restrict hiv- infection by antagonizing the envelope glycoprotein interferon-induced transmembrane protein- rs -c is associated with rapid progression of acute hiv- infection in chinese msm cohort interferon-induced transmembrane protein- genetic variant rs -c is associated with severe influenza in chinese individuals evolutionary dynamics of the interferon-induced transmembrane gene family in vertebrates interferon induction of ifitm proteins promotes infection by human coronavirus oc identification of residues controlling restriction versus enhancing activities of ifitm proteins on entry of human coronaviruses we want to thank the critical comments and suggestions by dr. jinhong chang and julia ma on this manuscript. key: cord- - teb jrn authors: filippini, antonio; d'amore, antonella; palombi, fioretta; carpaneto, armando title: could the inhibition of endo-lysosomal two-pore channels (tpcs) by the natural flavonoid naringenin represent an option to fight sars-cov- infection? date: - - journal: front microbiol doi: . /fmicb. . sha: doc_id: cord_uid: teb jrn nan in the present opinion article we highlight evidence from different laboratories to drive the attention of the scientific community on the role played by endo-lysosomal two-pore channels (tpcs) in viral infection. in particular, cross linking our recent data and existing literature, we focus on evidence indicating that virus intracellular pathway could be targeted by a novel occurring tpcs inhibitor, the flavonoid naringenin. a conceptual framework is presented for considering such a strategy as a promising approach to limit the infection mediated by the novel coronavirus sars-cov- . our hypothesis offers a perspective on a novel molecular target, tpcs, which could be exploited for a pharmacological blockade of sars-cov- infectivity. the coronaviruses are emerging viruses that are able to cross the species barrier and cause severe diseases in humans. two such recent events are the highly pathogenic serious acute respiratory syndrome-related cov (sars-cov) that became apparent in southern china in and middle east respiratory syndrome-related cov (mers-cov), which emerged in . in the present dramatic outbreak of coronavirus disease- (covid- ) that is caused by sars-cov- [recently reviewed in (lai et al., ) ], while science and medicine are striving to develop efficient treatments, we urge researchers to take into serious consideration a novel pharmacological strategy, highly promising for efficient and safe prophylaxis and therapy. what we recommend is to focus on the role played by the endo-lysosomal two-pore channel family (tpcs) in viral infection and on the feasibility of blocking the intracellular pathway of the virus by inhibiting these channels. cross-analysis of data published over different times, experimental models and approaches gives direct and indirect evidence in support of this proposal. first of all, sakurai et al. ( ) demonstrated that tpc is required for release of the ebola viral genome into the host cell during ebola virus entry pathway and, interestingly, tpc inhibitors such as tetrandrine have proven capable of blocking virus trafficking and prevented infection in vitro and in mice in vivo. intriguingly, our recent evidence has shown that the activity of human tpc channels can be inhibited by a natural flavonoid compound, in fact present in citruses and tomatoes, naringenin (pafumi et al., ) . in our opinion this evidence gives priority to naringenin (nar) for testing as a safe potential weapon against the present infection. the rationale for a defense line based on inhibiting lysosomal pro-viral activity through tpc inhibition is further supported by the following direct and indirect data. it has been shown (gunaratne et al., ) that knockdown and pharmacological inhibitors of both tpc , mainly expressed in late endosomes/lysosomes, and tpc , which mainly localizes to early endosomes, attenuate intracellular trafficking of coronavirus mers-cov through the endolysosomal system, even though the data were obtained using an artificial virion. besides tpc , nar is also an inhibitor of tpc activity with an ic of about µm therefore larger than for tpc (about µm) (pafumi et al., ) . relevant and very recent in vitro evidence has shed light to the efficacy of chloroquine to fight sars-cov- infection through lysosomal alkalinization (touret and de lamballerie, ; wang et al., ) . as a matter of fact, chloroquine acts as a weak base and accumulates in the lysosomes quenching their acidic ph, thereby halting autophagic degradative flux (homewood et al., ) . in line with this evidence, interestingly, it has been found that loss of tpc leads to an increase in melanosome/lysosome ph (cang et al., ; ambrosio et al., ; bellono et al., ) . in fact, tpc was shown to be involved in the control of human melanosome luminal ph: actually in tpc -ko human melanotic mnt- cells, and in primary melanocytes subjected to tpc knockout by the crispr/cas gene editing system, the lumen of melanosomes is more alkaline than in control cells (ambrosio et al., ) . bellono et al. ( ) also hypothesized that tpc can regulate melanosome ph producing a cation counterflux to enhance v-atpase h + transport into the melanosome lumen, consistent with the requirement for an inward cation current in lysosomal acidification (steinberg et al., ) . in addition, cang et al. ( ) demonstrated a shift toward alkalinization in tpc −/− macrophage lysosomes after starvation. since viral replication takes place in specific cellular compartments induced by viral proteins which modify cell organelles to create sites for replication, hidden from innate immunity, membrane fusion mechanisms are crucial events in the infection process. to this purpose, the virus s protein consists of two subunits, s and s , with s providing the receptor binding function through the entry receptor ace and s providing fusion activity. interestingly, the subunits are cleaved from the complete s by host cell proteases (cysteine proteases cathepsin b and l, furin proteases and cellular serine protease tmprss ) and, following receptor binding by s , the fusion mechanism of s acts to bring the viral and cellular vesicles membranes into such close proximity that fusion occurs (reviewed in alsaadi and jones, ) . in this context, it should be noted that the opening of tpcs induces a strong sodiumdriven depolarization in the endo-lysosomal membrane (wang et al., ; boccaccio et al., ; cang et al., ; lagostena et al., ) , which is supposed to enhance membrane fusion mechanisms (wang et al., ) . in line with this hypothesis, cos- cells transfected with human tpc have larger lysosomes than cells transfected with a non-functional form of the channel. moreover, it was recently shown (freeman et al., ) that tpcs are directly involved in sodium efflux, which, in parallel with chloride movement, regulates osmolyte release in endocytic vacuoles, with significant modification of vacuolar surface-tovolume ratio. therefore, inhibition of tpcs should both impair the fusogenic potential of the endo-lysosomal system and alter the normal trafficking, which, in turn, could be a limit for viral replication (alsaadi and jones, ) . very recently, unique features of tpc in the response to different agonists have been published (gerndt et al., ) expanding the characterization of this channel, hence the range of potential approaches to pharmacologically control the intracellular pathway of the virus. the use of nar, one of the main flavonoids present in the human diet, as a specific inhibitor of tpcs (benkerrou et al., ) has several advantages. nar is a hydrophobic molecule able to cross biological membranes and to reach the intracellular compartments (endosomes and lysosomes) where tpcs are localized. the toxicity of nar is low: concentrations greater than mm do not affect human hepatocytes viability (nahmias et al., ) and, in mice, doses up to , mg/kg given by intraperitoneal injection did not induce marked elevation of liver enzymes or cause animal death (nahmias et al., ) . interestingly, in the same study (nahmias et al., ) , nar was shown to be effective to reduce hepatitis c virus secretion by % when added at µm in infected huh . . human hepatoma cell line. moreover, that nar treatment could be a promising strategy to inhibit virus replication and infection is further confirmed by interesting studies on the influenza a virus, dengue virus and zika virus (dong et al., ; frabasile et al., ; cataneo et al., ) . antiviral effect of some flavonoids and nar through blocking viral proteases activity in different experimental models has been also reported (de sousa et al., ; lulu et al., ; lim et al., ; jo et al., ) . of note, nar has been shown to ameliorate acute inflammation (jin et al., ) as well as lung fibrosis , which could represent a therapeutic advantage. in particular, zeng et al. demonstrated that nar suppresses inflammatory cytokine production through both transcriptional and posttranscriptional mechanisms (by regulating lysosome function) resulting in the inhibition of tnf-α and il- secretion by macrophages and t cells (jin et al., ; zeng et al., ) . clinical trials analyzing the therapeutic potential of nar have been recently reviewed (salehi et al., ) and an important clinical trial on the pharmacokinetics and metabolism of nar has just been reported, indicating the strong interest around this compound (bai et al., ) . while this manuscript was under review, an article by ou et al. ( ) demonstrated that tpc is a key player for sars-cov- entry in /hace cells, consistent with our findings and further supporting our hypothesis. in conclusion, these considerations offer a perspective on specific molecular targets, tpcs, and underpin a role for naringenin as pharmacological blockade of sars-cov- infectivity providing further support for exploration of tpcs inhibition as novel antiviral therapy. the raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualified researcher. af and fp conceived the hypothesis and analyzed the data. af and ac shared the study. af, ad'a, fp, and ac designed the conceptual framing and wrote the manuscript. this work was supported by progetti di ricerca di ateneo, la sapienza university of rome (italy) to af. membrane binding proteins of coronaviruses tpc controls pigmentation by regulating melanosome ph and size pharmacokinetics and metabolism of naringin and active metabolite naringenin in rats, dogs, humans, and the differences between species corrigendum: a melanosomal two-pore sodium channel regulates pigmentation a perspective on the modulation of plant and animal two pore channels (tpcs) by the flavonoid naringenin the phosphoinositide pi( , )p mediates activation of mammalian but not plant tpc proteins: functional expression of endolysosomal channels in yeast and plant cells the voltage-gated sodium channel tpc confers endolysosomal excitability mtor regulates lysosomal atpsensitive two-pore na(+) channels to adapt to metabolic state the citrus flavonoid naringenin impairs the in vitro infection of human cells by zika virus flavonoids as noncompetitive inhibitors of dengue virus ns b-ns protease: inhibition kinetics and docking studies a dual character of flavonoids in influenza a virus replication and spread through modulating cell-autonomous immunity by mapk signaling pathways the citrus flavanone naringenin impairs dengue virus replication in human cells lipid-gated monovalent ion fluxes regulate endocytic traffic and support immune surveillance agonist-mediated switching of ion selectivity in tpc differentially promotes lysosomal function naadp-dependent ca + signaling regulates middle east respiratory syndrome-coronavirus pseudovirus translocation through the endolysosomal system ph and the anti-malarial action of chloroquine naringenin ameliorates acute inflammation by regulating intracellular cytokine degradation inhibition of sars-cov cl protease by flavonoids the human twopore channel is modulated by cytosolic and luminal calcium severe acute respiratory syndrome coronavirus (sars-cov- ) and coronavirus disease- (covid- ): the epidemic and the challenges inhibitory effect of flavonoids against ns b-ns protease of zika virus and their structure activity relationship naringenin and quercetin-potential anti-hcv agents for ns protease targets apolipoprotein b-dependent hepatitis c virus secretion is inhibited by the grapefruit flavonoid naringenin characterization of spike glycoprotein of sars-cov- on virus entry and its immune crossreactivity with sars-cov naringenin impairs two-pore channel activity and inhibits vegf-induced angiogenesis ebola virus. two-pore channels control ebola virus host cell entry and are drug targets for disease treatment the therapeutic potential of naringenin: a review of clinical trials a cation counterflux supports lysosomal acidification of chloroquine and covid- remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus ( -ncov) in vitro tpc proteins are phosphoinositide-activated sodium-selective ion channels in endosomes and lysosomes naringenin as a potential im-munomodulator in therapeutics naringenin ameliorates radiation-induced lung injury by lowering il- β level the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © filippini, d'amore, palombi and carpaneto. this is an openaccess article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- - czobqy authors: byun, hyewon; gou, yongqiang; zook, adam; lozano, mary m.; dudley, jaquelin p. title: erad and how viruses exploit it date: - - journal: front microbiol doi: . /fmicb. . sha: doc_id: cord_uid: czobqy endoplasmic reticulum (er)-associated degradation (erad) is a universally important process among eukaryotic cells. erad is necessary to preserve cell integrity since the accumulation of defective proteins results in diseases associated with neurological dysfunction, cancer, and infections. this process involves recognition of misfolded or misassembled proteins that have been translated in association with er membranes. recognition of erad substrates leads to their extraction through the er membrane (retrotranslocation or dislocation), ubiquitination, and destruction by cytosolic proteasomes. this review focuses on erad and its components as well as how viruses use this process to promote their replication and to avoid the immune response. although endoplasmic reticulum (er)-associated degradation (erad) has been most thoroughly defined in yeast, recent studies in higher organisms have revealed the conservation of this process and its components. multiple diseases, including parkinson's, alzheimer's, cancer, and infectious processes, result from failure of erad, confirming its significance for correct cell function. predictably, viruses have exploited various aspects of this key cellular machinery to further their propagation. nonetheless, the complexity of erad and the number of players involved necessitates a review of its features prior to a description of how viruses have manipulated erad to their advantage. in understanding how viruses exploit erad, we learn more about the cellular process, but also how we might alter the outcome of viral diseases. a majority of newly synthesized proteins in mammalian cells are either misfolded or misassembled (hoseki et al., ) . approximately % of new proteins are synthesized in association with the er (brodsky and wojcikiewicz, ) . the er quality control system both senses and disposes of terminally misfolded proteins by erad, a process that is conserved in eukaryotes (vembar and brodsky, ; merulla et al., ) . this process detects misfolded proteins in the er lumen, and then extracts them through membrane channels in an energy-dependent manner for delivery to cytosolic proteasomes (olzmann et al., ) . protein extraction through er membrane channels is known as dislocation or retrotranslocation (hampton and sommer, ) . because protein folding depends on multiple cellular components (merulla et al., ) , protein overexpression or the presence of mutant proteins may sequester limiting components, leading to accumulation of misfolded proteins in the er lumen. a more general failure of the erad process may occur if proteins are unable to fold within a reasonable time, resulting in inefficient retrotranslocation and proteasomal degradation. levels of erad-associated factors also may be affected by the intraluminal concentration of misfolded proteins. inability of the erad system to destroy misfolded proteins is associated with more than diseases, including neurological illnesses (alzheimer's and parkinson's), cystic fibrosis, infectious diseases, diabetes, and cancer (guerriero and brodsky, ) . particularly relevant to the subject of this review, viruses can produce large quantities of glycoproteins in a short period of time, which may overwhelm erad, leading to the accumulation of misfolded proteins, cell death, and associated pathology (franz et al., ) . although erad is vital to the maintenance of healthy cells, many parts of this process are not well characterized. multiple aspects of erad have been described in yeast (thibault and ng, ) , including the nature of the er channel and the components needed to identify misfolded proteins during and after translation. protein translocation across the er membrane is the prerequisite for erad. translation of many transmembrane proteins involves recognition of a hydrophobic signal peptide (sp) emerging from the ribosome by signal recognition particle (srp), which is associated with the trimeric sec complex. many of the sps are cleaved by signal peptidase, which is associated with the luminal side of the translocon (auclair et al., ) . the sec complex provides the aqueous channel for co-translational transfer of proteins across the er membrane (loibl et al., ) . recent evidence indicates that translocation across the er membrane can occur through an srp-independent process (denic, ; johnson et al., ) . based on recent experiments in yeast, more than % of signal-containing proteins fail to use srp, including tail-anchored (ta) proteins and short secretory proteins (johnson et al., ; ast et al., ) . instead, these proteins are targeted by the get pathway to the sec translocon that is associated with the sec / complex rather than through docking to the srp receptor (rapoport, ; ast et al., ) . one large class of srp-independent proteins includes the glycosylphosphatidylinositol (gpi)-anchored proteins, which contain both an n-terminal signal sequence and a c-terminal gpi anchor (ast et al., ) . this n-terminal signal is less hydrophobic than typical srp targets. furthermore, the sec translocon has been implicated as the channel for retrotranslocation (kiser et al., ) , and it has been proposed that protein transfer can be either forward or reverse with respect to the er lumen (johnson and haigh, ) . therefore, sec appears to complex with a number of different proteins, leading to a highly flexible and dynamic structure, where association with different proteins/protein complexes leads to transit in or out of the er (figure ). reports in yeast indicate that proteins can be o-mannosylated prior to n-glycosylation (ecker et al., ) , and both types of glycosylation are believed to occur co-translationally (loibl et al., ) . these glycosylases also have been shown to be associated with the translocon (chavan and lennarz, ) , and experiments indicate competition for different glycosylation sites (loibl et al., ) . the protein o-mannosyl transferases (pmts) and the oligosaccharyltransferases (osts) are transmembrane proteins, but the latter catalyzes addition of oligosaccharides to nascent polypeptides on asparagine residues (breitling and aebi, ) . the osts prefer nxt/s sequences in an unfolded or flexible protein domain, and the unfolded state may be facilitated by the ost complex associated with the translocon (breitling and aebi, ) . glycosylation near the c-terminal end of the protein is less efficient, perhaps due to competition between osts and protein folding (ben-dor et al., ; breitling and aebi, ) . pmts also are essential for erad in yeast. a pmt mutant showed increased degradation of a typical erad substrate (arroyo et al., ) . moreover, addition of oligosaccharides can be prevented by nearby cysteines and disulfide bond formation (allen et al., ) . thus, glycosylation is one determinant of the correct folding of a protein in the er lumen (breitling and aebi, ; figure a ). the oligosaccharides on er luminal proteins are critical for their correct folding or selection for erad. the nascent n-glycosylated protein has a three-branch structure with glucose mannose -n-acetylglucosamine -asparagine (aebi et al., ; merulla et al., ) . trimming of the first two glucose residues on one branch then allows interactions with two er-resident chaperone/lectin proteins, calnexin and calreticulin, which may lead to protein folding (brodsky, ) . removal of the third glucose causes release from these lectins and exit from the er (smith et al., ; olzmann et al., ) , but re-addition of this glucose by udp-glucose:glycoprotein glucosyltransferase allows reassociation (shenkman et al., ) . proteins retry folding until removal of three or four mannose residues triggers erad (lederkremer and glickman, ; shenkman et al., ) . correctly folded proteins leave the er after one or two mannose residues have been cleaved (shenkman et al., ) . mannose removal is achieved using er mannosidase i (ermani), the er degradation-enhancing α-mannosidase-like proteins (edems) and/or the golgi-resident protein man c (gonzalez et al., ; hirao et al., ; olivari et al., ; hosokawa et al., ) . several lectins, os- and xtp -b, then interact via their mrh domains with the mannose-trimmed proteins, allowing their association with the retrotranslocon (bernasconi et al., ; christianson et al., ; hosokawa et al., ) . os- and xtp -b also associate with different proteases, lonp and carboxypeptidase vitellogenic-like protein (cpvl), respectively, suggesting that some substrates may be partially degraded prior to dislocation (christianson et al., ; olzmann et al., ) . nonetheless, multiple attempts are made to refold proteins before their triage through erad. the role of chaperones includes recognition of inappropriate glycosylation as well as refolding efforts, but proteins delivered to the retrotranslocon may require unfolding and partial proteolysis to allow their transit through the narrow membrane channel (gogala et al., ) . non-glycosylated proteins can be subjected to erad, but detection of misfolding of these proteins does not involve calnexin and calreticulin (brodsky, ) . notably, the non-lectin chaperone bip is involved in erad targeting of both types of proteins (ushioda et al., ) , yet also serves to prevent leakage of calcium out of the er lumen (schäuble et al., ) . in addition, targeting of unglycosylated proteins to the proteasomes involves edem (shenkman et al., ) , which, like bip, recognizes misfolded glycoproteins, as well as the transmembrane herp protein (usa p in yeast; okuda-shimizu and hendershot, ) . both glycosylated and their non-glycosylated derivatives are recruited to the erderived quality control compartment (erqc) near the nucleus in the presence of a proteasomal inhibitor (shenkman et al., ) . thus, these studies suggest that targeting of misfolded proteins for erad is similar for glycoproteins and non-glycosylated proteins (shenkman et al., ) . interaction of lectin-type and other chaperones with erad substrates allows association with members of the protein disulfide isomerase (pdi) family, which generally are characterized by one or more thioredoxin-like motifs (cxxc; brodsky and skach, ) . interestingly, these proteins can form, break, or rearrange disulfide bonds as well as act as chaperones (benham, ) . the yeast pdi family is composed of five members (pdi , mpd , mpd , eug , and eps ), although only pdi is essential (farquhar et al., ) . in mammalian cells, pdi is one of the best characterized family members, but there are at least such enzymes (benham, ; grubb et al., ) . pdi family proteins are generally confined by a kdel retention sequence (benham, ) to the er, which has an oxidizing environment (costantini et al., ) . the oxidoreductase erp , which is localized near the er-golgi intermediate compartment (ergic), may provide some protection for proteins that might be routed for erad by calnexin (frenkel et al., ) . in addition, some pdi members can escape the secretory system and appear at the cell surface (benham, ) . for example, a disintegrin and metalloproteinase (adam ; also known as tumor necrosis factor alpha-converting enzyme or tace) has been shown to be regulated by an extracellular activity of pdi (bass and edwards, ; willems et al., ; düsterhöft et al., ) . pdi members also have a role in erad, with different requirements for different substrates (grubb et al., ) . in hepatic cells, pdi promotes the folding of apolipoprotein b (apob) figure | the erad process. (a) substrate recognition. many nascent polypeptides (curved line) have one or more high-mannose carbohydrates (shown as a branched structure), which must be recognized and processed in a timely manner to allow exit from the er. binding of these er-luminal proteins to substrates is affected by folding to their native conformations. folding involves formation and breakage of disulfide bonds by members of the pdi family, such as erp and erp , and is facilitated by chaperone proteins, such as bip. specific carbohydrates are bound by different chaperones/lectins in the er lumen. these proteins include ermani, edem, os- , xtp -b, calreticulin, and calnexin. recognition of erad substrates probably results in assembly of the retrotranslocon (shown here as herp and the translocon/bip complex). herp is thought to facilitate oligomerization of the hrd e ligase. bip binds to a number of glycosylated and non-glycosylated erad substrates and provides a barrier on the er luminal side of the translocon. (b) retrotranslocation. recognition of misfolded or misassembled proteins triggers the assembly of the retrotranslocon. current evidence indicates that multiple types of retrotranslocons are possible (see text). a typical retrotranslocon/dislocon is shown containing derlin, the e ligase hrd and its partner sel l, which then recruits the cytosolic atpase p . derlin has transmembrane domains with both the n-terminus and c-terminus in the cytosol. presumably some or all of the recognition components, such as pdi and ermani, disengage as the substrate passes through the translocon. all retrotranslocation events appear to involve p . the retrotranslocon is shown with bip opening the sec channel for substrate passage into the cytosol. (c) ubiquitination of erad substrates. retrotranslocation exposes erad substrates to cytosolic e (unknown), e (shown here as ube g ), and e enzymes (e.g., hrd ). a polyubiquitin chain is produced as the substrate is engaged by the e and e proteins. multiple e s may be responsible for the polyubiquitin chains that then bind to the p partner proteins, ufd and npl . the substrate is shown moving through the translocon into the center of the p hexamer. (d) proteasomal degradation. once the substrate has been retrotranslocated, the bip protein seals the luminal side of the translocon. the retrotranslocon may then be disassembled prior to engagement of a new substrate. the retrotranslocated proteins must be modified by removal of carbohydrate and ubiquitin chains for insertion into the narrow channel of the proteasome. it is possible that p substitutes for the s lid, which provides access to the proteasome channel and the energy for unfolding of substrates. degraded polypeptides are shown emerging from the s lid. this model suggests that there are retrotranslocon-specific proteasomes. www.frontiersin.org through its chaperone activity, whereas erp or erp expression leads to erad (grubb et al., ) . further, various cell types express different pdi proteins, allowing differential regulation of substrates (benham, ; pescatore et al., ) and, presumably, their erad targeting. protein folding involves both formation of disulfide bonds and cis/trans isomerization of peptide bonds preceding proline residues (hebert and molinari, ) . certain erad substrates appear to be dependent on proline isomerization (bernasconi et al., b) , and such refolding events may be necessary for transit through the retranslocon by elimination of turns in substrate secondary structure (määttänen et al., ) . erad requirements for peptidyl-prolyl cis/trans isomerases (ppis) depend on whether the substrate is strictly in the er lumen or is tethered to the er membrane (bernasconi et al., b) . the ppi protein cyclophilin b was needed for erad of a luminal target, but not the same target with a transmembrane domain (bernasconi et al., b) . requirement for ppis during erad may depend on proline residues in the cis configuration (bernasconi et al., b) , potentially by conversion into trans peptidyl-prolyl bonds, thus eliminating secondary structures that hinder retrotranslocation (määttänen et al., ) . mammalian cells have erad factors that are not present in yeast. as observed for other pathways (tsai and weissman, ) , erad components identified in yeast have multiple family members in higher eukaryotes; e.g., instead of a single derlin in yeast (der p), mammalian cells have three proteins (derlin- , - , and - ; oda et al., ) . derlins are multiple membranespanning domain proteins that have been proposed to be part of the retrotranslocon channel and/or regulatory factors for retrotranslocation (brodsky, ; figure b ). in addition, derlin- has a cell-type specific distribution (oda et al., ) , suggesting that recognition of certain substrates may be involved in its function. derlins are related to rhomboid proteases, such as rhbdl , which is an er-resident transmembrane protein that cleaves unstable single-membrane-spanning or polytopic membrane proteins (fleig et al., ) . rhbdl also is upregulated by er stress and binds to the cytosolic aaa atpase p (see below; fleig et al., ) . in contrast to the rhomboid proteases, the derlins lack proteolytic activity, suggesting that these proteins bind to erad substrates and target them to e ligases for ubiquitination and to p for membrane extraction (brodsky, ) . cleavage of erad substrates by rhbdl (fleig et al., ) , sp peptidase (spp; loureiro et al., ) , or proteases associated with os- and xtp -b (olzmann et al., ) may occur prior to retrotranslocation of some substrates (tsai and weissman, ) . on the other hand, it has been proposed that derlins form a six-transmembrane structure with a gate that allows association and unfolding of substrates or access to other retrotranslocon components, such as p (see below; olzmann et al., ) . the p atpase (cdc in yeast) is bound to derlin- and derlin- through their shp domains (greenblatt et al., ) . suppressor/enhancer of lin -like (sel l) appears to link luminal factors that recognize misfolding and inappropriate glycosylation, such as os- , xtp -b, edems, erdj , and the pdi protein erp , to components of the retrotranslocon (olzmann et al., ; williams et al., ) . the transmembrane sel l protein (hrd p in yeast) also participates in regulation of erad by sequestering edem and os- into er-derived vesicles known as edemosomes (bernasconi et al., a) . inducible knockout of sel l in mice leads to death of adult mice from acute pancreatic atrophy (sun et al., ) . sel l expression is required for stability of the e ligase hydroxymethylglutaryl reductase degradation protein (hrd ), and its loss leads to er stress and attenuates translation, leading to cell death. other proteins have been described, such as erlins and and tmub , which may act as adapters between polytopic membrane substrates and e ligases (olzmann et al., ) . the ubiquitin ligases (e s) have been proposed to be a structural part of the retrotranslocon channel (brodsky, ) , but their role is considerably more complex ( figure c ). several e ligases associated with erad are multiple membranespanning proteins with cytosolic ring domains (smith et al., ; ruggiano et al., ) . in yeast, where erad has been studied most extensively, a prototypical transmembrane e , such as hrd p (also called syvn ; nadav et al., ; kikkert et al., ) , can promote erad of a luminal substrate (erad-l). the erad process also involves hrd p (sel l in metazoans) as well as usa p and der p (carvalho et al., ) . herp may assist with hrd oligomerization (carvalho et al., ) , nevertheless, the other components appear to be dispensable if hrd p is overexpressed, consistent with a role for hrd p in erad substrate transfer across the membrane (carvalho et al., ) , although such overexpression may be toxic due to inappropriate protein degradation (denic et al., ) . thus, protein adapters appear to be necessary to achieve substrate specificity (smith et al., ) . hrd p-mediated erad requires oligomerization and transmembrane domains as well as ubiquitin ligase activity (carvalho et al., ) . overexpression of a dominant-negative ring mutant of the hrd ligase prevented erad of a non-glycosylated substrate, but a dominant-negative fbs mutant (a component of scf e ligases) did not (shenkman et al., ) . dependence on hrd also is affected by tethering of the substrate to the er membrane. splice variants of the human beta-site amyloid precursor cleaving enzyme (bace) with the same deletion mutation in the ectodomain are degraded through hrd if they are luminal (erad-l s substrates), but disposal occurs in a hrd independent manner if the variant has a transmembrane domain (erad-l m substrates; bernasconi et al., a) . therefore, hrd recognizes substrates for ubiquitination and, perhaps, modifies the translocon in the er membrane. multiple e ligases participate in erad. these ligases include the transmembrane proteins gp /amfr (fairbank et al., ), trc (stagg et al., ) , rma /rnf (el khouri et al., ) , march /teb (doa in yeast; kreft and hochstrasser, ; olzmann et al., ) , and chip (matsumura et al., ). an additional − membrane-spanning e s may be involved in erad (stagg et al., ) . other e ligases associated with erad frontiers in microbiology | virology are localized to the cytosol, where they recognize misfolded glycoproteins that already have been retrotranslocated (yoshida et al., ; shenkman et al., ) . these ubiquitin ligases are members of the cytosolic scf (s-phase kinase-associated protein (skp )-cullin (cul )-f-box) family, where the f-box components of the scf complex recognize the n-glycans of the retrotranslocated substrate, e.g., fbs and fbs (yoshida, ) . furthermore, e s may work together to direct substrates for degradation (olzmann et al., ) . the p protein (cdc in yeast) is a member of the aaa atpase family (erzberger and berger, ) that functions during erad in a complex with several cofactors that have a ubiquitin-x (ubx) or ubx-like domain (schuberth and buchberger, ; figure ). these cofactors include the heterodimer nuclear protein localization homolog (npl )-ubiquitin fusion degradation (ufd ; meyer et al., ; wolf and stolz, ) , p , ubxd , ubxd , ufd /plaa, vcip , and ataxin- (meyer et al., ) . the ufd l and npl proteins are believed to form a heterodimer, where npl is needed to stabilize ufd l (nowis et al., ) . the heterodimer acts as a substrate adapter to the p atpase associated with the retrotranslocon (bays and hampton, ) . ufd l and npl bind to k -linked and k -linked polyubiquitin chains, respectively, which have been added by e ligases associated with the retrotranslocon (ye et al., ; komander et al., ) . in yeast, the cdc atpase binds to the hrd e ligase in a ring-dependent manner (hampton and sommer, ) , and the transmembrane ubx (sel ) protein acts as an adapter using a uba domain (neuber et al., ; schuberth and buchberger, ) . several other ubiquitin ligases bind p directly or through cofactors (alexandru et al., ) . the p cofactors act as ubiquitin-binding proteins, although p also has ubiquitin-binding activity (ye et al., ; meyer et al., ) . the adapter-p complexes may recognize different substrates and perform independent functions, such as membrane protein segregation and trafficking, as well as directing substrates to the proteasome (ritz et al., ) . alternatively, other models suggest that derlins are involved in unfolding of substrates as well as providing contacts with p and its associated factors (greenblatt et al., ) . the p atpase binds ubiquitin chain editors that can extend shorter chains as well as deubiquitinating enzymes (dubs; jentsch and rumpf, ; sowa et al., ) . two atpase domains (d and d ; meyer et al., ) within p form two stacked hexameric rings that provide the energy for protein remodeling and substrate extraction from the membrane or through the retrotranslocon (hampton and sommer, ) . mutations in the d domain result in dominant-negative proteins that bind, but fail to release, substrates (pye et al., ) . mutant proteins have been widely used to study p function in erad and its myriad other activities (meyer et al., ) . cytosolic chaperones, such as hsp , also may provide energy for extraction of membrane proteins with misfolded cytoplasmic domains (erad-c substrates; taxis et al., ; hrizo et al., ) . once extraction from the er membrane has occurred, p recruits peptide n-glycanase (pngase) to cleave n-linked glycans from glycosylated substrates (hirsch et al., ; li et al., ; figure d ). in addition, p binds to a deubiquitinating enzyme yod , presumably so that polyubiquitin chains will not interfere with insertion into the proteasome (ernst et al., ) . the proteasome is a highly complex structure with a s lid that has an atpase activity very similar to that of p (lipson et al., ; matouschek and finley, ) . these enzymes may function synergistically to deliver substrates to the s core (hampton and sommer, ) . alternatively, p may deliver certain substrates directly to the proteasome core (matouschek and finley, ) . the proteasome core is composed of subunits arranged into four rings, each composed of seven subunits (bhattacharyya et al., ) . proteolytic activity is sequestered in the center of a narrow chamber formed by the rings and, therefore, only unfolded proteins can enter the chamber (groll et al., ) . the s lid, p , or other activators provide docking for substrates and substrate modifying proteins as well as regulated opening of the chamber to allow access of unfolded proteins for degradation in the s core (bhattacharyya et al., ) . many questions remain about erad components and how they identify and interact with different substrates. similar to our analysis of other cellular and molecular biological processes through virology, studies of viruses that use erad are likely to prove insightful. the ability of viruses to cause persistent infections is a consequence of downregulation or subversion of the immune response. the herpesviruses are known to cause persistent infections. one well-studied example of herpesvirus manipulation of the immune response is reduced cell expression of major histocompatibility complex class (mhc-i) molecules by the viral proteins us and us (wiertz et al., ) . both proteins are transmembrane glycoproteins and bind to newly made mhc-i to initiate retrotranslocation. despite their similar function, us and us use different pathways for mhc-i degradation (figure ) . us mediated degradation of mhc-i is independent of derlin- and involves spp (loureiro et al., ) , which cleaves many sps following their removal from nascent er-bound pre-proteins (voss et al., ) . using an sirna screen, trc was identified as the e ligase involved in mhc-i degradation by us , but knockdown of this transmembrane ring-type e had no effect on us mediated destruction of mhc-i (stagg et al., ). the us cytosolic tail interacts with spp and the p atpase (chevalier and johnson, ; loureiro et al., ) , whereas trc and us bind through their transmembrane domains (stagg et al., ; figure a) . unlike the derlin-independent mechanism proposed for us , studies of the us protein facilitated identification of derlin- and sel l as erad components (figure ; lilley and ploegh, ; ye et al., ; mueller et al., ) . us does not require spp for mhc-i degradation (loureiro et al., ), but appears to interact with the e ligase marchvii/axotrophin (flierman et al., ) . the cytosolic domain of mhc-i is required for us mediated erad targeting (story et al., ; barel et al., ) , and deletion of the c-terminal valine of mhc-i reduced interaction with derlin- (cho et al., a) . the er luminal domain www.frontiersin.org also affects degradation (barel et al., ) . in addition, mhc-i substituted with the transmembrane domain of us caused interaction with derlin- and proteasomal degradation (cho et al., b) . the p atpase does not appear to interact directly with mhc-i, but requires the interaction of mhc-i cytosolic domain with the c-terminal domain of derlin- (cho et al., a) . cho et al. speculated that us recognizes mhc-i through its cytosolic domain and transfers it to derlin- , which then interacts with the p atpase for membrane dislocation (cho et al., a ; figure b ). therefore, studies of the herpesvirus us and us proteins revealed that the same substrate does not always use the same erad pathway, and presumably these viral proteins act as adapters that recognize different parts of mhc-i for targeting to the dislocon. herpesviruses use another mechanism to decrease levels of mhc-i. the mouse gammaherpesvirus (mhv ) encodes an e ligase (mk ) that ubiquitinates newly made mhc-i heavy chains for proteasomal degradation (boname and stevenson, ) . the mk ligase also is associated with the transporterassociated with antigen processing (tap) as well as p and derlin- (wang et al., ) . polyubiquitination of mhc-i did not require lysines , but could occur on serine and threonine residues in the heavy chain c-terminal tail via the recruitment of the ube j e enzyme (see figure ; wang et al., wang et al., , herr et al., ) . these data indicate that multiple erad mechanisms can be used by viruses to diminish the adaptive immune response. like the herpesviruses, retroviruses also manipulate the immune system through erad. early studies indicated that human immunodeficiency virus type (hiv- )-infected cells had decreased levels of both cd mrna and protein (hoxie et al., ) . cd acts as the receptor for binding the viral envelope (env) protein (mcclure et al., ) . furthermore, cd participates in t-cell activation by binding to both the t-cell receptor and mhc class ii molecules on antigen-presenting cells. cd + t cells secrete cytokines that control antibody production, phagocytic cell function, and cytotoxic t-cell responses, making them crucial for adaptive immune responses (tubo and jenkins, ) . hiv- encodes a number of accessory proteins, including vpu, which are not required for virus replication in tissue culture, but contribute to viral pathogenesis (strebel, ) . expression of vpu and cd by transient transfection showed dramatic decreases in cd levels, and cd depletion was dependent on serines and in vpu (magadán et al., ) . vpu-induced cd degradation has been shown to involve the erad system. knockdown of both β-trcp and β-trcp largely prevented vpu-mediated cd loss (magadán et al., ) . β-trcp and β-trcp (also known as fbw a, fbxw , fbxw a, or fwd ) are f-box proteins containing wd domains, which are associated with the scf family of ubiquitin ligases (figure ) . these protein complexes are linked to regulation of multiple pathways involving cell cycle checkpoints, nfκb, and wnt (skaar et al., ) . in addition, knockdown of p , ufd l (also called ufd ) or npl (see figure c ) blocked depletion of cd (magadán et al., ) . mutations that prevented atp binding or hydrolysis by p failed to affect cd levels (magadán et al., ) . these experiments indicated that vpu uses erad to degrade cd , but also prevents cell surface expression by retaining cd in the er, probably through transmembrane domain interactions (magadán et al., ) . moreover, vpu used an atypical e ligase to induce erad (margottin et al., ) , and this process involved scf β−trcp ubiquitination of the cd cytosolic tail on lysine, serine, and threonine residues (magadán et al., ) . thus, vpu may act as an adapter between cd , retrotranslocon components, and a cytosolic e ligase. cd degradation promotes hiv- infection by preventing re-infection, facilitating virus release by avoiding env-cd interactions during their trafficking to the cell surface, and minimizing adaptive immune responses (lanzavecchia et al., ; willey et al., ; argañaraz et al., ) . hiv- vpu also targets another cellular protein, tetherin/bst- , for erad (neil et al., ; mangeat et al., ) . tetherin is an unusual type ii membrane protein with an n-terminal frontiers in microbiology | virology knockdown of both β-trcp and β-trcp (shown to be contacting vpu) can prevent cd degradation, suggesting that either f-box protein can provide a functional scf complex for ubiquitination (magadán et al., ) . another e ligase (e ?) also may be involved. the p atpase with the adapters ufd and npl are required for cd degradation, but the ufd l protein recognizes polyubiquitinated cd . lysine and serine/threonine residues in the cd cytosolic tail are needed for ubiquitination (magadán et al., ) . transmembrane segment and a c-terminal gpi anchor (kupzig et al., ; sauter, ) . moreover, two tetherin monomers are bound together by disulfide bonds (ishikawa et al., ; kupzig et al., ) . using a unique method that only allows biotinylation of retrotranslocated molecules by cytosolic bira protein, recent experiments indicate that both cd and tetherin remain glycosylated and retain disulfide bonds during retrotranslocation (petris et al., ) . these data suggest that the typical sec channel used for translocation is insufficiently wide to accommodate retrotranslocation substrates modified with these structures (petris et al., ) , but an alternative model involving lipid droplet formation has not been confirmed . given the large number of proteins that have been implicated, a single mechanism for retrotranslocation is unlikely. despite common delivery of substrates to the proteasome via the p atpase, each of the previous examples of viral erad targeting involves different e ligases. recent evidence suggests that erad can target the retrovirus hiv- env (zhou et al., ) , a glycosylated transmembrane protein. studies of a human cd + t-cell line cem.nkr indicated that hiv- replication is restricted in these cells, which also are resistant to natural killer cell-mediated lysis (howell et al., ) . surprisingly, these cells overexpressed a mitochondrial translocator protein, tspo (braestrup and squires, ; papadopoulos et al., ) , and knockdown or knockout of this protein rescued env and hiv- production (zhou et al., ) . further experiments indicated that drugs inducing erad led to recovery of env levels and viral titers. these results suggested that the er and mitochondria communicate through juxtaposition of their membranes, so that conditions in the mitochondria influence protein folding and erad. in support of this conclusion, gp is an erad-associated e ligase (fang et al., ) localized to mitochondria-er membrane contacts (fu et al., ) . thus, mitochondria proteins may influence erad and modulate hiv- env presentation to the immune system. triggering of an innate immune response to viruses is affected by the erad process. some anti-viral signaling is controlled through mitochondria, which also cooperates with the er for lipid synthesis and calcium-controlled processes at the mitochondrialassociated membrane (mam; jacobs et al., ) . mitochondrial antiviral signaling protein (mavs; also called ips- , visa, or cardif) binds to different retinoic acid-inducible gene-i (rig-i)-like receptor (rlr) proteins, which sense cytosolic viral rnas (kawai et al., ; meylan et al., ; seth et al., ; xu et al., ) . the mavs protein is present in the mitochondrial and peroxisomal membranes, and viral rna triggers both interferondependent or independent responses, respectively, (jacobs and coyne, ; jacobs et al., ) . the levels of mavs are affected by gp , an e ubiquitin ligase that is localized to the ermitochondrial interface (mam; jacobs et al., ) . the gp ligase was detected by a high throughput rnai screen to identify genes that restricted enterovirus replication (coyne et al., ) . downregulation of gp was shown to decrease yields of vesicular stomatitis virus (vsv) and to increase type i interferon responses. some viruses, such as those inducing hepatitis b (hbv) or c (hcv), use erad to reduce the amounts of glycoproteins and particles produced. interestingly, both viruses partially induce the unfolded protein response (upr; li et al., li et al., , saeed et al., ) , which then increases the levels of certain erad components. hbv, a member of the hepadnaviridae, triggers upregulation of the glycoside hydrolase family enzymes, edem and . increased edem levels appear to bypass normal er folding of hbv glycoproteins to result in erad (lazar et al., ) . hcv, a member of the flaviviridae, induces primarily edem through the upr and splicing of x-box binding protein . further experiments suggested that elevated levels of edem and increase binding to sel l, an adapter to the retrotranslocon (figure ) . inhibition of edem binding to sel l interfered with ubiquitination of hcv env protein, e (saeed et al., ) . interestingly, infections by another member of the flaviviridae, japanese encephalitis virus, did not result in edem binding to the env proteins, indicating that not all viral family members control env proteins by this mechanism. overall, manipulation of edem levels appears to be a common mechanism to reduce viral glycoprotein levels. lowered amounts of env proteins and virus particles then contribute to avoidance of innate and adaptive immunity, leading to chronic infections (saeed et al., ; lazar et al., ) . a number of pathogens harness the erad process to facilitate various replication strategies. the best known examples are the bacterial ab toxins, particularly cholera toxin, which is thought to hijack the erad machinery for delivery to the cytosol (hazes and read, ) . cholera toxin has a catalytic a chain divided into two subunits (cta and cta ) inside a pore composed of five receptor-binding b subunits (spangler, ) . the holotoxin www.frontiersin.org binds to the ganglioside gm on the surface of gut epithelial cells, which then triggers toxin internalization and trafficking through the golgi to the er (fujinaga et al., ) . the a subunits are bound to the b subunits by disulfide bonds, and the toxin complex interacts with the er-resident enzyme pdi (figure ) . pdi is a redox-dependent chaperone that unfolds the toxin, which is then released in the oxidized state (tsai et al., ) . this unfolding event appears to be required for the ability of cta to retrotranslocate to the cytosol, where it induces the adp-ribosylation of the gαs protein and, ultimately, opening of chloride channels leading to massive diarrhea (muanprasat and chatsudthipong, ) . as noted above, retrotranslocation of erad substrates is preceded by a recognition step. the chaperone bip, which is known to be involved in identification of non-glycosylated erad substrates, and an er-resident atpase (torsin a) promote cta retrotranslocation (tsai et al., ; winkeler et al., ; forster et al., ; moore et al., ) . sel l and erdj , a co-chaperone of bip, also facilitate cta retrotranslocation, where the j domain of erdj is required (williams et al., ) . erdj also binds to sel l, likely providing interaction with the hrd e ligase (see figure ) . torsin a may provide the link to the membrane-resident derlin- protein (nery et al., ) . cta retrotranslocation appears to involve derlin- (bernardi et al., ) and the transmembrane ubiquitin ligases, hrd and gp . thus, multiple low affinity interactions are likely involved in the identification of cta as a substrate and its delivery to the retrotranslocon. similar to other retrotranslocated substrates, the cytosolic p atpase participates in cta extraction from the er membrane (abujarour et al., ; kothe et al., ) . nevertheless, cta subverts the normal erad process by avoiding polyubiquitination (rodighiero et al., ) . the hypothesis that cta avoids ubiquitination through the absence of lysines targeted for polyubiquitination was not substantiated by mutational analysis (rodighiero et al., ) . these results indicate that cta employs many of the typical components used for erad targeting, including the e ligase, but it is unclear how polyubiquitination and degradation of the substrate are avoided. therefore, retrotranslocon targeting and substrate extraction from the er membrane is not necessarily coupled to ubiquitination, although ubiquitination may be required for proteasomal degradation. viral pathogens also use erad. mouse mammary tumor virus (mmtv) is a betaretrovirus that subverts the erad process to complete its viral replication cycle. all retroviruses synthesize an unspliced viral rna that requires export from the nucleus to the cytosol for translation or packaging into virus particles (cullen, ) . the unspliced rnas of simple retroviruses have a highly structured cis-acting sequence, such as the constitutive transport element (cte) of mason-pfizer monkey virus (mpmv; bray et al., ) . the cte facilitates rna export through the typical tap/nxf -mediated pathway used by cellular mrnas (grüter et al., ) . in contrast, the complex retroviruses encode an adapter protein, such as the rev protein of hiv- (hanly et al., ) , which binds to a structured rna element near the end of the genome (daly et al., ; zapp and green, ) . mmtv also produces a rev-like protein, rem, for export of unspliced rna (mertz et al., ) , but rem binding to viral rna has additional translation-associated functions (mertz et al., b) . unlike other complex retroviruses, rem is made from an internally deleted form of the env protein, and the export function resides in a long sp of amino acids (indik et al., ; mertz et al., ) . interestingly, rem is a precursor protein that is directed to the er membrane for translation, where it appears to be cleaved by signal peptidase into the rev-like rem-sp and a c-terminal glycosylated product (rem-ct) of unknown activity (byun et al., ) . recent evidence indicates that rem-sp uses retrotranslocation for extraction from the er membrane, but, like cholera toxin, avoids proteasomal degradation (byun et al., (byun et al., , . dultz et al. ( ) first reported that rem is directed to the er membrane for translation and cleavage by signal peptidase. they also suggested that the rem precursor (the uncleaved protein) could be detected in the nucleus by fluorescence microscopy (dultz et al., ) . byun et al. ( ) showed that mutation of the predicted signal peptidase cleavage site prevented the appearance of rem-sp as detected by both western blotting and a highly sensitive reporter assay for rev-like function (mertz et al., ; byun et al., ) . this assay requires binding to a specific rna element in viral rna (müllner et al., ; mertz et al., a) . fluorescence experiments indicated that only the cleaved rem-sp enters the nucleus, whereas the uncleaved form was highly unstable and localized to the cytosol (byun et al., ) . furthermore, rem-sp activity was inhibited by expression of a dominant-negative form of the p atpase required for retrotranslocation (byun et al., ) . rem-sp function also was reduced by the expression of a dominant-negative derlin- , but not derlin- protein (byun et al., in preparation) . these results strongly suggest that rem must be cleaved by signal peptidase prior to sp retrotranslocation to the cytosol and import into the nucleus for rna binding (figure ) . experiments indicate that an altered conformation of either the n-terminal rem-sp in the cytosol or the er-luminal portion of rem affect folding and accessibility to signal peptidase, which is associated with translocons (falk and gilula, ) . first, rem tagging at the c-terminus with green fluorescent protein (rem-gfp) resulted in a stable protein that was inefficiently cleaved and had little fluorescence (mertz et al., ; byun et al., ) . rem-gfp also had very low functional activity in reporter assays (mertz et al., ) . in contrast, rem tagged at the n-terminus with gfp was cleaved normally, and gfp-rem-sp localized to the nucleoli, a result typical of other rev-like proteins (cullen, ; mertz et al., ) . second, deletion mutations of the rem c-terminus greatly affected stability of the protein (byun et al., ) . removal of the c-terminal amino acids had little effect on the cleavage or stability of the protein, but deletion of or amino acids produced a highly unstable precursor that could be rescued by the proteasomal inhibitor mg- (byun et al., ) . reduced cleavage of the precursor also was observed. surprisingly, further deletion to give only the sp (rem-sp) again yielded a stable protein (byun et al., ) . third, substitution of the leucine at position in the sp gave a stable precursor protein that was poorly cleaved by signal peptidase (mertz et al., a; byun et al., ). an independent report indicated that residues frontiers in microbiology | virology rem is a precursor protein that has an n-terminal signal peptide (rem-sp) that directs translation to the er membrane. the rem-ct enters the er lumen, where it is modified by n-glycosylation on two different sites. rem recognition for retrotranslocation is not understood, but appears to involve derlin- and, potentially, an e ligase, although ubiquitinated rem has not been observed. full-length rem is cleaved by signal peptidase, and rem-ct is released into the er lumen. similar to other retrotranslocation substrates, rem-sp is extracted from the er membrane using the p atpase. despite its dislocation into the cytosol, rem-sp escapes the proteasome and translocates into the nucleus for binding of mmtv rna. this figure is adapted from byun et al. ( ) . through act as the hydrophobic membrane anchor sequence, suggesting that position is localized in the cytosol (dultz et al., ) . recognition of rem c-terminal sequences in the er lumen, presumably by their interaction or lack of interaction with specific chaperone proteins, prevent degradation by erad. the er-luminal chaperone bip has repeatedly been detected after purification and proteomic analysis of rem-binding proteins (gou et al., manuscript in preparation) . our preliminary data indicate that rem-sp is not ubiquitinated, and it is possible that this feature protects rem-sp from proteasomal degradation. since the rem precursor and c-terminal deletion mutants are subject to erad, cleavage and association with specific cellular proteins appear to be critical for avoidance of the degradative process. the idea that viral proteins manipulate e enzymes to form alternative complexes (olzmann et al., ) would be consistent with rem-sp escape from erad. the polyomaviruses have a unique entry method that uses retrotranslocation, while avoiding erad. the bk polyomavirus (bkv) first binds to the ganglioside receptors gt b and gd b and enters through caveolae (neu et al., ) , which are composed of membrane microdomains/lipid rafts that are enriched for sphingolipids and signaling molecules (head et al., ; figure ) . particle delivery to the cytosol occurs through a phdependent step involving endosomal trafficking via microtubules to the er (eash and atwood, ; moriyama and sorokin, ; jiang et al., ) . other members of the polyomaviridae use caveolae-independent entry for er delivery (neu et al., ) . er localization of these viruses is necessary to access specific retrotranslocation components. the vp capsid proteins of polyomaviruses form pentamers during assembly that are held together by disulfide bonding (li et al., ) . each pentamer is associated with one molecule of either the minor capsid protein vp or vp (barouch and harrison, ) , which become accessible to antibodies after exposure to the unique environment of the er (norkin et al., ) . particle delivery into the er allows reduction and isomerization of disulfide bonds using erp (mouse polyomavirus; magnuson et al., ) or erp and pdi (sv ; schelhaas et al., ) to allow partial uncoating (jiang et al., ; tsai and qian, ) . the partially uncoated virion then engages the retrotranslocation machinery to allow cytosolic entry similar to cholera toxin (neu et al., ) . interestingly, different polyomaviruses use distinct derlin family members for retrotranslocation. sv uses derlin- and sel l (schelhaas et al., ) , whereas mouse polyoma virus uses derlin- ; figure ) . additional experiments indicate that exposure of vp hydrophobic sequences tethers virus particles to the er membrane, and that both bip and bap are needed for dislocation of sv to the cytosol (geiger et al., ) . bap may serve as a shuttle to the erqc that has been associated with enriched erad components (kamhi-nesher et al., ; wakana et al., ) . furthermore, use of epoxomicin or eeyarestatin , inhibitors of the proteasome or p atpase, respectively, blocked early events of bkv infection (bennett et al., ) . epoxomicin treatment of cells allowed accumulation of bkv in the calnexin-rich, bip-deficient erqc (bennett et al., ) . these results are consistent with erad extraction of polyomaviruses from the er to the cytosol, although it is has been suggested that www.frontiersin.org figure | use of erad for polyomavirus uncoating. many polyomaviruses enter through caveosomes that are enriched for viral entry receptors, triggering particle uptake through endosomes. using the microtubule network, vesicles traffic the virus to the er, where the unique environment allows structural changes to the icosahedral capsids. studies of jcv, bkv, and sv indicate that viral particles interact with pdi and erp in the er lumen to rearrange capsid proteins. in contrast, the related mouse polyomavirus (pyv) uses the pdi family member, erp , presumably for a similar function. the altered particles then appear to engage different retrotranslocons (dependent on either derlin- or derlin- ) to induce retrotranslocation to the cytosol, where the reduced calcium environment produces further capsid rearrangements. these particles then bind to the nuclear pore where uncoating occurs to allow passage of viral dna into the nucleus. this figure is adapted from neu et al. ( ). there are cell-type and virus-specific differences and that direct er to nuclear transport may occur (bennett et al., ) . low levels of calcium in the cytosol lead to further capsid destabilization and exposure of the nuclear localization signals on capsid proteins. the partially uncoated capsid then transits through the nuclear pores for initiation of viral dna replication (neu et al., ) . the preceding experiments indicate that erad is used by viruses to allow trafficking events that promote replication. mmtv rem trafficking through the er allows access to signal peptidase and cleavage of rem precursor into functional n-and c-terminal proteins. in contrast, the polyomaviruses use erad to partially uncoat virions on their path to the nucleus. importantly, both types of viruses avoid proteasomal degradation during erad, although the mechanisms remain unclear. erad may be regulated or "tuned" through the rapid turnover of specific components through the proteasomes or autophagosomes/vesicular trafficking to lysosomes (merulla et al., ) . normal secretory vesicles released from the er are - nm in diameter and have coatamer proteins, such as copii, whereas the er-derived tuning vesicles (edemosomes) lack coatamers and are - nm in diameter (bernasconi et al., b) . tuning vesicles contain sel l, edem , and os- , which are transmembrane or luminal proteins involved in erad (figure ; olzmann et al., ) . edemosomes are believed to reduce erad by disposal in acidic organelles (bernasconi et al., b) , favoring the correct folding of polypeptides (calì et al., ) . the coronaviruses are known to take advantage of erad tuning (reggiori et al., ) . many plus-stranded rna-containing viruses manipulate cellular membranes to further rna replication (paul and bartenschlager, ) . these membrane structures have been divided into invaginated vesicle/spherule type and double-membrane vesicle (dmv) type (two lipid bilayers). such vesicles allow viruses to concentrate their replication components, to separate distinct viral processes (e.g., translation, transcription, and replication), and to avoid immune detection (paul and bartenschlager, ) . severe acute respiratory syndrome coronavirus (sars-cov) and mouse hepatitis virus (mhv) induce dmvs for targeting their replication and transcription (reggiori et al., ) . the dmvs originate from er membranes and contain the non-structural transmembrane proteins nsp and nsp and viral double-stranded rna (stertz et al., ; reggiori et al., ) . nevertheless, dmvs lack markers typical of the ergic or the golgi (oostra et al., ) . recent experiments indicate that dmvs are coated with microtubule-associated protein light chain [lc ; atg in yeast (reggiori et al., ) ], which is a ubiquitin-like modifier (van der veen and ploegh, ). lc can exist in a lipidated form (covalent linkage to phosphatidylethanolamine; also known as lc -ii) or a predominantly cytosolic non-lipidated form (lc -i). lc -ii is believed to be involved in fusion of autophagosomes to lysosomes (van der veen and ploegh, ), but coronavirus dmvs display the non-lipidated lc -i (reggiori et al., ) . these ubiquitinlike modifiers recognize specific receptors that target associated vesicles to particular cellular locations (van der veen and ploegh, ). the coronaviruses appear to be redirecting vesicles destined for autophagosomes to sequestered locations in the cytosol where replication will occur. the autophagy machinery is not required for coronavirus replication, and no colocalization of viral non-structural proteins was observed with lc -ii-coated autophagosomes (reggiori et al., ) . coronavirus-induced dmvs and edemosomes both are coated with the non-lipidated lc -i protein (calì et al., ; reggiori et al., ) , which is not covalently attached to membranes like lc -ii (kabeya et al., ) . induction of autophagy in coronavirus-infected cells with rapamycin decreased the levels of edem and coronavirus (reggiori et al., ) . the viruscontaining dmvs had both edem and os- , but not other erad-associated chaperones, and virus infection interfered with erad tuning by hijacking the edemosomes. nevertheless, lc -i, but not edem and os- , is necessary for coronavirus infection, and the hijacked edemosome cargo is not degraded by proteases in the endosomes/lysosomes (reggiori et al., ) . further, the erad transmembrane adapter protein, sel l, is needed for dmv formation, capturing the er-resident edem and os- proteins (and possibly xtp -b and edem ), while using its proline-rich cytosolic domain to bind to lc -i. as expected, sel l knockdown impairs coronavirus replication (bernasconi et al., a) . the organizationally similar arterioviruses (classified with coronaviruses, toroviruses, and roniviruses into the order nidovirales; gorbalenya et al., ) subvert edemosome trafficking for their replication, although the size of the vesicles is smaller (monastyrska et al., ) . the mechanism for altering edem containing vesicular trafficking is unclear, but likely involves expression of viral non-structural proteins that span the erderived membranes (monastyrska et al., ) , perhaps through their interaction with sel l. these experiments indicate that viruses hijack edemosomes to sequester their double-stranded rna from cytosolic sensors that will trigger interferon production and innate immunity (zinzula and tramontano, ) . other components of the erad system, particularly chaperone proteins, also participate in the replication and transmission of both plant and mammalian viruses (verchot, ) . the erad system is a complex and highly regulated process controlling the disposal of misfolded or misassembled proteins that are directed to the er for translation. deregulation of this process results in pathogenic conditions, including infectious diseases. viruses exploit erad to decrease overall viral levels and allow establishment of chronic infections by minimizing antigen presentation to the immune system. trafficking of specific viral proteins or entire virion particles may involve erad for refolding or processing in the unique er environment. alternatively, viruses can use erad-associated components to form isolated lipid vesicles for replication and shelter from immune detection. virus-mediated subversion of erad can lead to degradation of molecules that are involved in innate or adaptive immunity. continued studies of viruses are certain to provide additional insights into both the erad process and the components that regulate it. further experiments may identify targets for viral therapeutics. p is in a complex with cholera toxin and influences the transport of cholera toxin and related toxins to the cytoplasm n-glycan structures: recognition and processing in the er ubxd binds multiple ubiquitin ligases and implicates p in hif alpha turnover intracellular folding of tissue-type plasminogen activator. effects of disulfide bond formation on n-linked glycosylation and secretion enhanced cd down-modulation by late stage hiv- nef alleles is associated with increased env incorporation and viral replication functional and genomic analyses of blocked protein o-mannosylation in baker's yeast a network of cytosolic factors targets srp-independent proteins to the endoplasmic reticulum signal peptidase i: cleaving the way to mature proteins amino acid composition of alpha /alpha domains and cytoplasmic tail of mhc class i molecules determine their susceptibility to human cytomegalovirus us -mediated down-regulation interactions among the major and minor coat proteins of polyomavirus adams and protein disulfide isomerase: the key to regulated cell-surface protein ectodomain shedding? biases and complex patterns in the residues flanking protein n-glycosylation sites the protein disulfide isomerase family: key players in health and disease role of cell-type-specific endoplasmic reticulum-associated degradation in polyomavirus trafficking derlin- facilitates the retro-translocation of cholera toxin the e ubiquitin ligases hrd and gp bind to and promote cholera toxin retro-translocation stringent requirement for hrd , sel l, and os- /xtp -b for disposal of erad-ls substrates cyclosporine a-sensitive, cyclophilin b-dependent endoplasmic reticulumassociated degradation role of the sel l:lc -i complex as an erad tuning receptor in the mammalian er unconventional roles of nonlipidated lc in erad tuning and coronavirus infection a dual task for the xbp -responsive os- variants in the mammalian endoplasmic reticulum: inhibiting secretion of misfolded protein conformers and enhancing their disposal regulated protein turnover: snapshots of the proteasome in action mhc class i ubiquitination by a viral phd/lap finger protein specific benzodiazepine receptors in rat brain characterized by high-affinity ( h)diazepam binding a small element from the mason-pfizer monkey virus genome makes human immunodeficiency virus type expression and replication rev-independent n-linked protein glycosylation in the endoplasmic reticulum cleaning up: er-associated degradation to the rescue protein folding and quality control in the endoplasmic reticulum: recent lessons from yeast and mammalian cell systems substrate-specific mediators of er associated degradation (erad) requirements for mouse mammary tumor virus rem signal peptide processing and function retroviral rem protein requires processing by signal peptidase and retrotranslocation for nuclear function segregation and rapid turnover of edem by an autophagy-like mechanism modulates standard erad and folding activities retrotranslocation of a misfolded luminal er protein by the ubiquitin-ligase hrd p the molecular basis of coupling of translocation and n-glycosylation human cytomegalovirus us chimeras containing us cytosolic residues acquire major histocompatibility class i and ii protein degradation properties the c-terminal amino acid of the mhc-i heavy chain is critical for binding to derlin- in human cytomegalovirus us -induced mhc-i degradation forced interaction of cell surface proteins with derlin- in the endoplasmic reticulum is sufficient to induce their dislocation into the cytosol for degradation defining human erad networks through an integrative mapping strategy os- and grp deliver mutant alpha -antitrypsin to the hrd -sel l ubiquitin ligase complex for erad cysteineless non-glycosylated monomeric blue fluorescent protein, secbfp , for studies in the eukaryotic secretory pathway comparative rnai screening reveals host factors involved in enterovirus infection of polarized endothelial monolayers nuclear mrna export: insights from virology specific binding of hiv- recombinant rev protein to the rev-responsive element in vitro a portrait of the get pathway as a surprisingly complicated young man a luminal surveillance complex that selects misfolded glycoproteins for er-associated degradation the signal peptide of the mouse mammary tumor virus rem protein is released from the endoplasmic reticulum membrane and accumulates in nucleoli membrane-proximal domain of a disintegrin and metalloprotease- represents the putative molecular switch of its shedding activity operated by protein-disulfide isomerase involvement of cytoskeletal components in bk virus infectious entry o-mannosylation precedes and potentially controls the n-glycosylation of a yeast cell wall glycoprotein rnf is a novel e ligase of endoplasmic reticulum-associated degradation (erad) that targets cystic fibrosis transmembrane conductance regulator (cftr) the otubain yod is a deubiquitinating enzyme that associates with p to facilitate protein dislocation from the er evolutionary relationships and structural mechanisms of aaa+ proteins the complex biology of autocrine motility factor/phosphoglucose isomerase (amf/pgi) and its receptor, the gp /amfr e ubiquitin ligase connexin membrane protein biosynthesis is influenced by polypeptide positioning within the translocon and signal peptidase access the tumor autocrine motility factor receptor, gp , is a ubiquitin protein ligase implicated in degradation from the endoplasmic reticulum protein disulfide isomerase is essential for viability in saccharomyces cerevisiae ubiquitin-dependent intramembrane rhomboid protease promotes erad of membrane proteins e - k mediates us -triggered retro-translocation of mhc class i heavy chains in a permeabilized cell system protein disulfide isomerase-like proteins play opposing roles during retrotranslocation create and preserve: proteostasis in development and aging is governed by cdc /p /vcp separate roles and different routing of calnexin and erp in endoplasmic reticulum quality control revealed by interactions with asialoglycoprotein receptor chains regulation of mitophagy by the gp e ubiquitin ligase gangliosides that associate with lipid rafts mediate transport of cholera and related toxins from the plasma membrane to endoplasmic reticulm bap and bip are essential for dislocation of sv from the endoplasmic reticulum to the cytosol structures of the sec complex engaged in nascent peptide translocation or membrane insertion identification, expression, and characterization of a cdna encoding human endoplasmic reticulum mannosidase i, the enzyme that catalyzes the first mannose trimming step in mammalian asn-linked oligosaccharide biosynthesis nidovirales: evolving the largest rna virus genome derlin- is a rhomboid pseudoprotease required for the dislocation of mutant α- antitrypsin from the endoplasmic reticulum a gated channel into the proteasome core particle protein disulfide isomerases contribute differentially to the endoplasmic reticulum-associated degradation of apolipoprotein b and other substrates tap, the human homolog of mex p, mediates cte-dependent rna export from the nucleus the delicate balance between secreted protein folding and endoplasmic reticulum-associated degradation in human physiology finding the will and the way of erad substrate retrotranslocation comparative analysis of the htlv-i rex and hiv- rev trans-regulatory proteins and their rna response elements accumulating evidence suggests that several ab-toxins subvert the endoplasmic reticulum-associated protein degradation pathway to enter target cells interaction of membrane/lipid rafts with the cytoskeleton: impact on signaling and function: membrane/lipid rafts, mediators of cytoskeletal arrangement and cell signaling in and out of the er: protein folding, quality control, degradation, and related human diseases role of the ring-ch domain of viral ligase mk in ubiquitination of non-lysine and lysine mhc i residues edem , a soluble edem homolog, enhances glycoprotein endoplasmic reticulum-associated degradation and mannose trimming a role for n-glycanase in the cytosolic turnover of glycoproteins mechanism and components of endoplasmic reticulum-associated degradation human xtp -b forms an endoplasmic reticulum quality control scaffold with the hrd -sel l ubiquitin ligase complex and bip stimulation of erad of misfolded null hong kong alpha -antitrypsin by golgi alpha , -mannosidases natural killing target antigens as inducers of interferon: studies with an immunoselected, natural killing-resistant human t lymphoblastoid cell line alterations in t (cd ) protein and mrna synthesis in cells infected with hiv the hsp molecular chaperone stabilizes apolipoprotein b from endoplasmic reticulum-associated degradation (erad) a novel, mouse mammary tumor virus encoded protein with rev-like properties molecular cloning and chromosomal mapping of a bone marrow stromal cell surface gene, bst , that may be involved in pre-b-cell growth mechanisms of mavs regulation at the mitochondrial membrane regulation of mitochondrial antiviral signaling (mavs) expression and signaling by the mitochondria-associated endoplasmic reticulum membrane (mam) protein gp cdc (p ): a "molecular gearbox" in the ubiquitin pathway? early events during bk virus entry and disassembly the er translocon and retrotranslocation: is the shift into reverse manual or automatic? post-translational translocation into the endoplasmic reticulum trc can deliver short secretory proteins to the sec translocon lc , a mammalian homologue of yeast apg p, is localized in autophagosome membranes after processing a novel quality control compartment derived from the endoplasmic reticulum ips- , an adaptor triggering rig-i-and mda -mediated type i interferon induction human hrd is an e ubiquitin ligase involved in degradation of proteins from the endoplasmic reticulum expression and degradation of the cystic fibrosis transmembrane conductance regulator in saccharomyces cerevisiae molecular discrimination of structurally equivalent lys -linked and linear polyubiquitin chains role of p aaa-atpase in the retrotranslocation of the cholera toxin a chain, a non-ubiquitinated substrate an unusual transmembrane helix in the endoplasmic reticulum ubiquitin ligase doa modulates degradation of its cognate e enzyme bst- /hm . is a raft-associated apical membrane protein with an unusual topology t cells can present antigens such as hiv gp targeted to their own surface molecules activation of erad pathway by human hbv modulates viral and subviral particle production a window of opportunity: timing protein degradation by trimming of sugars and ubiquitins hepatitis b virus x protein (hbx) activates atf and ire -xbp pathways of unfolded protein response the aaa atpase p links peptide n-glycanase to the endoplasmic reticulum-associated e ligase autocrine motility factor receptor subversion of cellular autophagy machinery by hepatitis b virus for viral envelopment characterization of self-assembled virus-like particles of human polyomavirus bk generated by recombinant baculoviruses murine polyomavirus requires the endoplasmic reticulum protein derlin- to initiate infection a membrane protein required for dislocation of misfolded proteins from the er a proteasomal atpase contributes to dislocation of endoplasmic reticulum-associated degradation (erad) substrates protein o-mannosyltransferases associate with the translocon to modify translocating polypeptide chains signal peptide peptidase is required for dislocation from the endoplasmic reticulum protein quality control in the er: the recognition of misfolded proteins multilayered mechanism of cd downregulation by hiv- vpu involving distinct er retention and erad targeting steps erp triggers a conformational change in polyomavirus to stimulate membrane binding hiv- vpu neutralizes the antiviral factor tetherin/bst- by binding it and directing its beta-trcp -dependent degradation a novel human wd protein, h-beta trcp, that interacts with hiv- vpu connects cd to the er degradation pathway through an f-box motif cell biology. an ancient portal to proteolysis endoplasmic reticulum protein quality control is determined by cooperative interactions between hsp/c protein and the chip e ligase hiv infection of primate lymphocytes and conservation of the cd receptor mapping of the functional boundaries and secondary structure of the mouse mammary tumor virus rem-responsive element rev and rex proteins of human complex retroviruses function with the mmtv rem-responsive element mouse mammary tumor virus encodes a self-regulatory rna export protein and is a complex retrovirus specificity and regulation of the endoplasmic reticulum-associated degradation machinery emerging functions of the vcp/p aaa-atpase in the ubiquitin system cardif is an adaptor protein in the rig-i antiviral pathway and is targeted by hepatitis c virus an autophagy-independent role for lc in equine arteritis virus replication the ero alpha-pdi redox cycle regulates retro-translocation of cholera toxin intracellular trafficking pathway of bk virus in human renal proximal tubular epithelial cells cholera: pathophysiology and emerging therapeutic targets sel l, the homologue of yeast hrd p, is involved in protein dislocation from the mammalian er identification of the rem-responsive element of mouse mammary tumor virus a novel mammalian endoplasmic reticulum ubiquitin ligase homologous to the yeast hrd tetherin inhibits retrovirus release and is antagonized by hiv- vpu torsina participates in endoplasmic reticulum-associated degradation the polyomaviridae: contributions of virus structure to our understanding of virus receptors and infectious entry ubx links the cdc complex to er-associated protein degradation caveolar endocytosis of simian virus is followed by brefeldin a-sensitive transport to the endoplasmic reticulum, where the virus disassembles destabilization of the vcp-ufd -npl complex is associated with decreased levels of erad substrates derlin- and derlin- are regulated by the mammalian unfolded protein response and are required for er-associated degradation characterization of an erad pathway for nonglycosylated bip substrates, which require herp edem regulates er-associated degradation by accelerating de-mannosylation of folding-defective polypeptides and by inhibiting their covalent aggregation lipid droplet formation is dispensable for endoplasmic reticulum-associated degradation the mammalian endoplasmic reticulum-associated degradation system localization and membrane topology of coronavirus nonstructural protein : involvement of the early secretory pathway in replication translocator protein ( kda): new nomenclature for the peripheral-type benzodiazepine receptor based on its structure and molecular function architecture and biogenesis of plus-strand rna virus replication factories protein disulfide isomerase is required for platelet-derived growth factor-induced vascular smooth muscle cell migration, nox nadph oxidase expression, and rhogtpase activation cd and bst- /tetherin proteins retro-translocate from endoplasmic reticulum to cytosol as partially folded and multimeric molecules going through the motions: the atpase cycle of p protein translocation across the eukaryotic endoplasmic reticulum and bacterial plasma membranes coronaviruses hijack the lc -i-positive edemosomes, er-derived vesicles exporting short-lived erad regulators, for replication endolysosomal sorting of ubiquitylated caveolin- is regulated by vcp and ubxd and impaired by vcp disease mutations role of ubiquitination in retro-translocation of cholera toxin and escape of cytosolic degradation quality control: er-associated degradation: protein quality control and beyond role of the endoplasmic reticulum-associated degradation (erad) pathway in degradation of hepatitis c virus envelope proteins and production of virus particles counteraction of the multifunctional restriction factor tetherin bipmediated closing of the sec channel limits ca + leakage from the er simian virus depends on er protein folding and quality control factors for entry into host cells membrane-bound ubx recruits cdc to ubiquitin ligases and their substrates to ensure efficient er-associated protein degradation ubx domain proteins: major regulators of the aaa atpase cdc /p identification and characterization of mavs, a mitochondrial antiviral signaling protein that activates nf-kappab and irf a shared endoplasmic reticulum-associated degradation pathway involving the edem protein for glycosylated and nonglycosylated proteins mechanisms and function of substrate recruitment by f-box proteins road to ruin: targeting proteins for degradation in the endoplasmic reticulum defining the human deubiquitinating enzyme interaction landscape structure and function of cholera toxin and the related escherichia coli heat-labile enterotoxin the trc e ligase ubiquitinates mhc class i molecules before dislocation from the er the intracellular sites of early replication and budding of sarscoronavirus the cytosolic tail of class i mhc heavy chain is required for its dislocation by the human cytomegalovirus us and us gene products hiv accessory proteins versus host restriction factors sel l is indispensable for mammalian endoplasmic reticulum-associated degradation, endoplasmic reticulum homeostasis, and survival use of modular substrates demonstrates mechanistic diversity and reveals differences in chaperone requirement of erad the endoplasmic reticulum-associated degradation pathways of budding yeast cellular entry of polyomaviruses protein disulfide isomerase acts as a redox-dependent chaperone to unfold cholera toxin a ubiquitin-binding rhomboid protease aimed at eradication cd + t cells: guardians of the phagosome glycosylation-independent erad pathway serves as a backup system under er stress ubiquitin-like proteins one step at a time: endoplasmic reticulumassociated degradation the er quality control and er associated degradation machineries are vital for viral pathogenesis mechanism, specificity, and physiology of signal peptide peptidase (spp) and spp-like proteases bap is an itinerant protein that moves between the peripheral endoplasmic reticulum (er) and a juxtanuclear compartment related to erassociated degradation requirements for the selective degradation of endoplasmic reticulum-resident major histocompatibility complex class i proteins by the viral immune evasion molecule mk ubiquitination of serine, threonine, or lysine residues on the cytoplasmic tail can induce erad of mhc-i by viral e ligase mk ube j ubiquitinates hydroxylated amino acids on erassociated degradation substrates the viral e ubiquitin ligase mk uses the derlin/p endoplasmic reticulum-associated degradation pathway to mediate down-regulation of major histocompatibility complex class i proteins sec -mediated transfer of a membrane protein from the endoplasmic reticulum to the proteasome for destruction thiol isomerases negatively regulate the cellular shedding activity of adam human immunodeficiency virus type vpu protein regulates the formation of intracellular gp -cd complexes the erdj -sel l complex facilitates cholera toxin retrotranslocation bip-dependent export of cholera toxin from endoplasmic reticulum-derived microsomes the cdc machine in endoplasmic reticulum associated protein degradation visa is an adapter protein required for virus-triggered ifn-beta signaling function of the p -ufd -npl complex in retrotranslocation from the er to the cytosol: dual recognition of nonubiquitinated polypeptide segments and polyubiquitin chains recruitment of the p atpase and ubiquitin ligases to the site of retrotranslocation at the endoplasmic reticulum membrane a membrane protein complex mediates retro-translocation from the er lumen into the cytosol f-box proteins that contain sugar-binding domains glycoproteinspecific ubiquitin ligases recognize n-glycans in unfolded substrates sequence-specific rna binding by the hiv- rev protein the mitochondrial translocator protein, tspo, inhibits hiv- envelope glycoprotein biosynthesis via the endoplasmic reticulum-associated protein degradation pathway strategies of highly pathogenic rna viruses to block dsrna detection by rig-i-like receptors: hide, mask, hit we thank dr. jon huibregtse for helpful comments and suggestions on the manuscript and marianna grenadier for the figures. this work was supported by nih grants r ca and r ai . the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. key: cord- -ovnzlpa authors: chen, mengmeng; liu, xing; hu, bo; fan, zhiyu; song, yanhua; wei, houjun; qiu, rulong; xu, weizhong; zhu, weifeng; wang, fang title: rabbit hemorrhagic disease virus non-structural protein induces apoptosis in rabbit kidney cells date: - - journal: front microbiol doi: . /fmicb. . sha: doc_id: cord_uid: ovnzlpa rabbit hemorrhagic disease (rhd) is a highly contagious disease caused by rabbit hemorrhagic disease virus (rhdv). previous research has shown that rhdv induces apoptosis in numerous cell types, although the molecular mechanisms underlying the apoptosis induced by rhdv are not well understood. one possible factor is non-structural protein (nsp ), a c-like protease that plays an important role in processing viral polyprotein precursors into mature non-structural proteins. to fully establish a role for nsp , the present study examined the effects of ectopic expression of the protein in rabbit (rk ) and human (hela and hepg ) cells. we found that nsp suppressed cell viability and promoted apoptosis in all three cell types in a dose-dependent manner. we also identified increased caspase- , - , and - activities in rk cell, and an increased bax to bcl mrna ratio. mechanistically, the ability of nsp to induce apoptosis was impaired by mutation of the catalytic his residue. our study has shown that rhdv nsp can induce apoptosis in host cells and is likely an important contributor to rhdv-induced apoptosis and pathogenesis. rabbit hemorrhagic disease (rhd) is a highly contagious disease characterized by acute liver damage and disseminated intravascular coagulation (dic) (alonso et al., ; jung et al., ) . the first outbreak of rhd was reported in in the jiangsu province of china (zhu et al., ) and has quickly spread to most parts of the world (calvete et al., ; dalton et al., ) . this has led to the deaths of millions of rabbits and hares, representing a serious threat to their populations. the etiologic agent that causes rhd is rabbit hemorrhagic disease virus (rhdv) (abrantes et al., ) , a calicivirus of the lagovirus genus. rhdv contains both genomic rna (grna) and additional subgenomic rna sequences (sgrna). the genomic rna consists of a positive-sense single-stranded rna molecule that is nucleotides in length and includes two slightly overlapping open reading frames (orfs), orf and orf . orf encodes a large polyprotein that is cleaved into mature non-structural proteins - (nsp - ) and the major structural protein (vp ) (meyers et al., (meyers et al., , . of the non-structural proteins, nsp is known to be a trypsin-like cysteine protease (allaire et al., ; boniotti et al., ; wirblich et al., ) that is released from larger precursors molecules via proteolytic cleavage at the n and c termini. mutagenesis analysis has suggested that three amino acids in particular (his , asp , and cys ) play an important role in nsp function as a catalytic triad (wirblich et al., ) . although the first outbreak of rhd was more than years ago, the mechanisms underlying its pathogenesis are still not fully understood. the liver is believed to be the main site of rhdv reproduction, with viral replication leading to liver cell apoptosis and necrosis (jung et al., ) . it is also known that systemic hemorrhagic diathesis and dic can lead to rabbit death. these are most likely consequences of liver cell loss through rhdv-induced apoptosis (alonso et al., ; trzeciak-ryczek et al., ) . hepatocytes are indeed the first choice cells in this study, but so far there is no stable rabbit liver cell line, which brings some difficulties to the research. studies have shown that rhdv infection can not only cause liver cell apoptosis, but also cause the apoptosis of multiple cells in various organs such as heart, spleen, lung, kidney, etc., (alonso et al., ) . therefore, a stable kidney cell line from rabbit (rk cell) was selected as the object to study cell apoptosis in this research. studies have also shown that macrophages and endothelial cells also display the morphological hallmarks of apoptosis (gelmetti et al., ) . further research has found that granulocyte and lymphocyte apoptosis also occurs in rabbits infected with various rhdv strains (marques et al., ; niedzwiedzka-rystwej and deptula, ; teixeira et al., ; niedzwiedzka-rystwej et al., ) . more recently, studies have indicated that n-acetyl cysteine (san-miguel et al., ) , cardiotrophin (tunon et al., b) , and melatonin (tunon et al., a) , can attenuate liver damage and prolong survival in rhdv-infected rabbits. this is likely due to the induction of various anti-apoptotic factors and their inherent anti-apoptotic effects. altogether, these findings suggest that apoptosis plays an important role in rhd-mediated symptoms and may be a key determinant of disease pathogenesis. however, the exact viral components that contribute to the apoptosis-inducing effects of rhdv are unknown, and research is hampered by the fact that rhdv cannot be propagated in cell culture. to determine which viral components may be involved in rhd pathogenesis, our study employed ectopic expression of nsp in rabbit and human cells to identify any effects on apoptosis and cell viability. we first amplified nsp from the full-length cdna clone of rhdv strain nj- through pcr and cloned the product into a pcdna . - his vector for expression and subsequent analysis. this showed that nsp expression could induce apoptosis in rk , hela, and hepg cells, likely via modification of caspase expression and altered bax and bcl expression ratios. this effect was ameliorated by mutation of the catalytic his residue of nsp , suggesting involvement of this residue in nsp apoptosis induction. altogether, our findings indicate that nsp contributes to rhdv-induced apoptosis of host cells and is therefore central to rhdv pathogenesis in rabbits. oryctolagus cuniculus (rabbit) kidney cells (rk ), human liver cancer cells (hepg ) and human cervical cancer cells (hela) were purchased from the american tissue culture collection (atcc; manassas, va, united states). they were cultured in dulbecco's modified eagle's medium (dmem; gibco; thermo fisher scientific, waltham, ma, united states) supplemented with % fetal bovine serum (fbs; gibco) in a humidified atmosphere of % air and % co . genes encoding wild-type nsp were amplified using pcr from the full-length cdna clone of rhdv strain nj- . single-point mutant plasmids (targeting the catalytic triad of his , asp , and cys ) were constructed using a mut express ii fast mutagenesis kit v (vazyme biotech, nanjing, china). the primer pairs used are shown in table . the genes were then cloned into the multiple cloning site of a pcdna . - his vector (genscript, nanjing, china). the resultant plasmids were designated pcdna . -nsp , h n, d g, and c g. the plasmids were used to transfect the rk cells with lipofectamine reagent (invitrogen; thermo fisher scientific), according to manufacturer's instructions. untransfected cells and vector pcdna . - his transfected rk cells were used as mock and vector controls, respectively. rk cells were transfected with pcdna . -nsp , h n, d g, c g, or pcdna . - his vector for h in -well culture plates and then rinsed with phosphate-buffered saline (pbs). the cells were then fixed with chilled % ethanol for min at c and washed three times with pbs plus tween- (pbst). cells were incubated with an anti-his monoclonal antibody ( : in pbst; genscript) for h at • c, washed, and then a secondary dye-coupled antibody (fitc-conjugated goat anti-mouse igg; southern biotech, birmingham, al, united states) was added at a dilution of : and incubated at • c for h. after rinsing five times with pbs, the plates were analyzed via fluorescence microscopy (zeiss, oberkochen, germany). an mtt cell proliferation and cytotoxicity assay kit (beyotime, shanghai, china) was used to detect any potential effects of nsp on cell proliferation according to manufacturer's instructions. assays were performed and h post transfection (hpt) using the previously described lipofectamine protocol. following the final incubation step with formazan, optical density was determined using a microplate reader (bio-rad laboratories, hercules, ca, united states). the relative cell number of transfected rk cells was compared with that of pcdna . - his vector or pcdna . -nsp . each experiment was performed in triplicate. trypan blue dye exclusion assay: rk cells were transfected with pcdna . -nsp and cell viability was determined by . % trypan blue solution staining using automated cell counter (jimbio, china) at h, h and hpt. empty vector-transfected rk cells are shown as controls. a tunel assay kit (roche, basel, switzerland) was used to detect dna fragmentation in response to apoptotic stimuli according to the manufacturer's instructions. the cells were then analyzed by fluorescence microscopy for tunel staining (zeiss). for apoptosis analysis, × cells (including rk , hela, and hepg cells) were seeded onto plates and transfected with recombinant plasmids (including pcdna . -nsp and mutants) or empty vector for h. the cells were then washed with pbs and resuspended in µl binding buffer supplemented with µl annexin v ( µg/ml) and µl propidium iodide (pi) ( µg/mg) for min in the dark. next, µl binding buffer was added, and the samples were immediately screened using flow cytometry to assess annexin v staining and permeability. caspase- - , and - activities were measured using colorimetric assay kits (beyotime) according to manufacturer's instructions. rk cells transfected with pcdna . -nsp and pcdna . - his vector for h were harvested by centrifugation and incubated in lysis buffer on ice for min. the lysate was then centrifuged at , rpm and c for min and the final protein content was determined using a pierce bca protein assay kit (thermo fisher scientific). next, µg of protein was incubated for h at c with devd-pna colorimetric substrate, ietd-pna, and lehd-pna for the caspase- , , and assays, respectively. activity was estimated by measuring absorption at nm and subtracting the background values obtained from wells without colorimetric substrate. rk cells were transfected with nsp for , , or h. total rna was then extracted using the total rna kit i (omega bio-tek, norcross, ga). isolated rna ( µg) was then used for subsequent cdna synthesis using a primescript rt reagent kit (takara bio, kusatsu, japan). specific primers for rabbit bcl and bax were used for real-time pcr analyses with sybr master mix (takara bio) and an abi real-time pcr system (applied biosystems, foster city, ca, united states). bcl and bax mrna levels were normalized to glyceraldehyde- -phosphate dehydrogenase (gapdh) expression, and data are presented as fold change relative to the medium-only control. primers used for real-time pcr are listed in table . all experiments were performed independently in triplicate. significant differences between groups were determined with one-or two-way analysis of variance (anova) using graphpad prism . (graphpad software, inc., san diego, ca, united states). a threshold p-value of . was considered statistically significant. the expression of nsp in transfected rabbit kidney cells (rk ) was determined using a his-nsp fusion protein and immunofluorescent staining (figure ) . the fluorescent signal was observed in the cytoplasm of pcdna . -nsp -transfected cells at hpt and was found primarily aggregated in the cytoplasm. no fluorescent signaling was detected in the control. to assess the effect of nsp on rk cell growth, rk cells were assayed using mtt and trypan blue dye. in mtt assay, rhdv-nsp reduced the relative cell number of rk cells to and % of control cell at and hpt (figure a) , respectively. in trypan blue dye exclusion test, the viable cell rate of rk cells transfected with nsp reduced to , and % at , and hpt ( figure b) , so there is a slight discrepancy between the results of mtt assay and trypan blue dye exclusion test. the reason for the difference in results may be the different experimental principles. trypan blue dye is used to examine cell viability by detecting cell membrane integrity, while in mtt assay the activity of mitochondrial reductase enzymes is used as an indicator of cell viability. above all, both of the results indicate nsp expression may affect cell metabolism reducing growth of the cells. to examine if the decrease in cell viability induced by nsp was due to increased apoptosis in host cells, we transfected rk cells with either empty pcdna . - his or pcdna . -nsp plasmids and analyzed apoptosis via annexin v/pi staining with flow cytometry. as showed in figure c , h after transfection with pcdna . -nsp , the percentage of apoptotic cells was markedly increased to % (including % early apoptotic rate and % late apoptotic rate). this result presented a difference comparing dates presented in figures a,b . the main reason for this difference is that the results from both mtt and trypan blue dye exclusion test reflect the level of living cells, but cells under way in apoptosis may still counted as living cells in those method. however, in annexin v/pi assay, early apoptotic cells and late apoptotic cells were detected. in agreement with these observations, a tunel analysis further confirmed the presence of an apoptotic cell population in pcdna . -nsp transfected rk cells. as shown in figure d , compared to vector control, tunel-positive cells (green) were observed in pcdna . -nsp -transfected rk cells. however, only adherent cells were detected in tunel assay as a large number of apoptotic cells in the supernatant were discarded with the culture medium during the experiment. therefore, the apoptotic rate indicated by tunel assays was only %. in addition, we found that percentage of apoptotic rk cells induced by nsp was increased in a dose-dependent manner ( figure a) . moreover, similar results were also obtained in human hela and hepg cells (figures b,c) , indicating that the effect was not limited to rk cells and that nsp -induced apoptosis is not cell specific. nsp promotes activation of caspase- , - , and - caspases are cysteine proteases that play fundamental roles in the apoptotic responses of cells to different stimuli. to gain insight into the mechanism of nsp -induced apoptosis, we examined the activities of caspase- , caspase- , and caspase- . these proteins are key components of the caspase cascade and are central to the intrinsic and extrinsic pathways of apoptosis. we found that caspase- activity was first observed in nsp -transfected cells at hpt and upregulated . -fold at hpt ( figure a) . furthermore, the activities of caspase- and caspase- , which are representative initiator to caspases in the death receptor-mediated and mitochondrial apoptotic pathways, respectively, were also measured. as shown in figures b,c the caspase- and caspase- activities were also increased in nsp -transfected cells and maximal increases were observed at hpt ( . -fold and . -fold increases in activity, respectively, when compared with that of the pcdna . - his vector control). this is consistent with the increase in apoptotic cells observed in pcdna . -nsp -transfected cells using annexin v/pi flow cytometry and tunel. previous research has shown that n-acetyl cysteine can attenuate liver damage and prolong survival in rhdv-infected rabbits (san-miguel et al., ) . this protective effect is related to the apoptosis regulator bcl- , a family of evolutionarily related proteins. these proteins govern mitochondrial outer membrane permeabilization and can be either pro-apoptotic (bax) or anti-apoptotic (bcl ). we studied the effects of nsp on these proteins and found bax expression was also significantly higher in nsp transfected cells at and hpt when compared with the vector control ( figure a ). the expression of bcl was significantly lower in nsp -transfected cells at and hpt ( figure b ) relative to vector control. overall, there was an increased ratio of bax to bcl in nsp transfected cells compared with that of vector control (figure c ), indicating that nsp -induced cell apoptosis involves bcl- family members. nsp is a c-like serine protease that contains a canonical catalytic triad of his , asp , and cys . to assess if empty vector and nsp -transfected rk cells fixed at hpt were labeled with tunel (green) and then counterstained with dapi (blue). nsp expression was also determined by immunofluorescence using fitc-conjugated murine anti-his monoclonal antibodies (green). scale, µm. results are from one representative of three independent experiments. * p < . ; * * p < . ; * * * p < . . these residues are involved in nsp -triggered apoptosis, three single-point mutants, his asn (h n), asp gly (d g), and cys gly (c g), were generated in the wild-type nsp protein. a diagram of the protein mutant residue positions is illustrated in figure a . initially, protein expression for each mutant was confirmed using indirect immunofluorescence ( figure b) . next, the apoptosis rates induced by each mutant were analyzed by flow cytometry using annexin v/pi staining of rk cells. as shown in figure c , all mutants induced some degree of apoptosis. however, the level of apoptosis induced by the h n mutant was significantly lower than that of wild-type nsp , implying that his took part in the apoptosis induction. apoptosis plays an important role in the pathogenicity of a wide variety of viruses (garg et al., ; van den berg et al., ; okamoto et al., ; elizalde et al., ; guo et al., ) . like many other positive-strand rna viruses, rhdv-infection can also induce cell apoptosis in vivo and is believed to contribute to rhd pathogenesis (alonso et al., ) . previously, rhdv research was limited due to the lack of a stable cell system to culture the virus in vitro. so far, it has remained unclear which viral components contribute to rhdv-induced apoptosis. rhdv nsp is a c-like protease. functionally, rhdv nsp is similar to the c proteases of . h after transfection, the apoptosis rate were detected by annexin v/pi double staining combined with flow cytometry. the doses of pcdna . -nsp are , , and ng, respectively. results are from one representative of three independent experiments. * * p < . ; * * * p < . . figure | rhdv-nsp enhances the activity of caspase- , , and in rk cells. rk cells were transfected with pcdna . -nsp or empty vector ( ng). activity kinetics of (a) caspase- , (b) caspase- , and (c) caspase- was measured at the indicated time points post transfection using a caspase- / / assay kit. results are from one representative of three independent experiments. * p < . ; * * p < . . picornaviruses (wirblich et al., ) , although much more is known of the picornavirus genome. these proteases show serine protease activity and are structurally homologous to cellular serine proteases of the trypsin family. the cleavage reactions they mediate are required for releasing the different functional viral polyproteins. in addition to this protease activity, it has been reported that picornavirus c proteases, including those from coxsackievirus b , enterovirus , and poliovirus, can also induce apoptosis (barco et al., ; li et al., ; calandria et al., ; lin et al., ; zaragoza et al., ; chau et al., ) . the role of c-like protease induced apoptosis has also been characterized in severe acute respiratory syndrome-associated coronavirus (lin et al., ) . however, whether rhdv nsp with similar structure can induce apoptosis has not been reported. in this study, rhdv-nsp was cloned into pcdna . -his vector. his-nsp fusion protein was found primarily aggregated in the cytoplasm of rk cells detected by immunofluorescence assay (figure ) . this is slightly different with the results from urakova et al. ( ) . urakova team perform research about subcellular localization of recombinant non-structural rhdv proteins. their study pointed out the nsp (rhdv protease) was found to accumulate in nuclear and cytoplasmic compartments at h after transfection (urakova et al., ) . we speculated that one reason for this discrepancy may be due to the different time points we choosed. in our experiment, we found that very few nsp + positive cells was detected and a large number of cells occured apoptosis and shed into the culture medium without detection at hpt by indirect immunofluorescence assay. therefore, we changed the detection time and detected the expression of nsp protein in rk cells at hpt. another reason is the limitations of our method. we did not analyze the fluorescence signal in the nucleus at hpt. trypan blue test showed that a decrease in cell viability observed in pcdna . -nsp transfected rk cells which was further confirmed by mtt assay. both of the tests showed that nsp protein is cytotoxic to the cells, and the cytotoxicity was time dependent (figures a,b) . to confirm the decrease in cell viability was due to the induction of apoptosis, the quantification of cellular apoptosis was performed by flow cytometry and tunel assay. we found a marked increase in tunel positive cells in pcdna . -nsp transfected rk cells ( figure d ) and the increased number of annexin v+ cells (lower right quadrant, figure c ) and annexinv+/pi+ cells (upper right quadrant, figure c ) confirmed induction of apoptosis by pcdna . -nsp in rk cells. in addition, nsp induced apoptosis in a dose-dependent manner in rk cells ( figure a) . this was replicated in human hela and hepg cells (figures b,c) , indicating that the effect is not limited to rk cells. based on the above results, we speculated and nsp is highly likely to be responsible for the apoptosis observed during rhdv infection. caspase- is a major effector caspase in both the extrinsic and intrinsic apoptotic pathways acting on enzymes that are indispensable for chromatin condensation and dna fragmentation (duprez et al., ; lan et al., ) . a previous studies has shown a marked increase in caspase- activity at h ( . -fold) and h post-inoculation ( . -fold) in rhdv-infected animals, indicating apoptosis activation (garcia-lastra et al., ) . consistent with these results, our current study found that caspase- activity were activated in nsp -transfected cells, indicating that nsp induces rk cells apoptosis through activation of the effector caspase, caspase- . furthermore, we found that the caspase- (representative initiator caspase in the death receptor-mediated apoptotic pathway) and caspase- (representative initiator caspase in mitochondrial apoptotic pathway) activities were also increased in nsp -transfected cells. these results suggest that rhdv nsp induced caspase-dependent apoptosis in rk cells via both the death receptor-mediated and mitochondrial apoptotic pathways. however, the activation of the initiator caspase- can also be related to the mitochondrial apoptotic pathway via cleavage of bid (a member of the pro-apoptotic bcl- family) and translocation of the truncated bid to mitochondria (wang et al., ; lan et al., ) . therefore, to clarify the detailed mechanisms of nsp -induced apoptosis, further investigations that focus on the kinetics of signal transduction factors are necessary. conversely, bcl , an anti-apoptotic protein that promotes cell survival (polcic et al., ) , acts to regulate the intrinsic and extrinsic pathways through broad molecular interactions with other critical cellular proteins. meanwhile, bax, a protein that also features a bcl- like conserved domain, plays a vital role in promoting programmed cell death. the balance in bax and bcl expression can affect mitochondrial cytochrome c release, promoting apoptosis during increased bax to bcl ratios (pepper et al., ; li et al., ) . a study also suggested that apoptosis induced by rhdv-infection is related to the modulation of bcl and bax genes (san-miguel et al., ) . this is in agreement with our results where the relative expression levels of bcl and bax mrna changed significantly in nsp expressing cells. more importantly, we found that the ratios of bax to bcl were higher in nsp expressing cells, further promoting apoptosis. the amino acids his , asp , and cys play an important role in nsp function as a catalytic triad (wirblich et al., ) . asn and gly were chosen as residue substitutions, as they do not have similar structural functions to the wild-type residues, cannot form disulfide bridges, and are different in size and chemistry and could thus damage proteolytic activity (boniotti et al., ) . so three single-point mutants were constructed and the apoptosis induction of each mutant were confirmed by flow cytometry. our data showed that mutation of the clike protease active site (specifically the his residue) impaired nsp -induced apoptosis indicate that the mechanism of rhdv nsp promoted apoptosis is similar to the c proteases of picornaviruses. moreover, our result is consistent with previous research showing that mutating sites involved in protease activity impairs apoptosis induced by an enterovirus c protease in human neural cells (li et al., ) . in conclusion, our findings demonstrate that rhdv nsp significantly contributes to cell apoptosis and suggest that the protein alone is sufficient to induce apoptosis in rk , hela, and hepg cells. this appears to be linked to the activation of caspase- , - and - , and increase in bax to bcl ratios, which promotes apoptosis. in addition, we have shown that a his residue in the catalytic domain of the protein is involved in the apoptotic effect. thus, nsp is important in the apoptosis induced by rhdv and is a central mediator of rhd pathogenesis. mc designed and performed the experiments and analyzed the data. mc and bh wrote the manuscript. xl, zf, ys, hw, wz, rq, and wx assisted in performing some experiments. fw contributed essential ideas and discussion. all authors read and approved the final manuscript. we thank editage (www.editage.cn) for english language editing. rabbit haemorrhagic disease (rhd) and rabbit haemorrhagic disease virus (rhdv): a review picornaviral c cysteine proteinases have a fold similar to chymotrypsin-like serine proteinases programmed cell death in the pathogenesis of rabbit hemorrhagic disease poliovirus protease c(pro) kills cells by apoptosis identification and characterization of a c-like protease from rabbit hemorrhagic disease virus, a calicivirus individual expression of poliovirus apro and cpro induces activation of caspase- and parp cleavage in hela cells rabbit haemorrhagic disease: cross-protection and comparative pathogenicity of gi. /rhdv /b and gi. b/rhdv lagoviruses in a challenge trial coxsackievirus b proteases a and c induce apoptotic cell death through mitochondrial injury and cleavage of eif gi but not dap /p /nat conventional and real-time rt-pcr 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( ) . viral protease cleavage of inhibitor of kappabalpha triggers host cell apoptosis. proc. natl. acad. sci. u.s. a. , - . doi: . /pnas. zhu, j., miao, q., tan, y., guo, h., li, c., chen, z., et al. ( ) . the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © chen, liu, hu, fan, song, wei, qiu, xu, zhu and wang. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- -rqc kvsl authors: crémet, lise; gaborit, benjamin; bouras, marwan; drumel, thomas; guillotin, florian; poulain, cécile; persyn, elise; lakhal, karim; rozec, bertrand; vibet, marie-anne; roquilly, antoine; gibaud, sophie title: evaluation of the filmarray(®) pneumonia plus panel for rapid diagnosis of hospital-acquired pneumonia in intensive care unit patients date: - - journal: front microbiol doi: . /fmicb. . sha: doc_id: cord_uid: rqc kvsl the filmarray(®) pneumonia plus panel (fapp) is a new multiplex molecular test for hospital-acquired pneumonia (hap), which can rapidly detect bacteria, viruses, and resistance genes. we aimed to compare the diagnosis performance of fapp with conventional testing in intensive care unit (icu) patients who required mechanical ventilation, with clinically suspected hap. a total of samples [ bronchoalveolar lavages (bal(ds)) and endotracheal aspirates (eta(ds)) obtained at hap diagnosis, and eta obtained during follow-up (eta(tt))], were analyzed independently by routine microbiology testing and fapp. patients had paired bal(ds) and eta(ds). the positivity thresholds of semi-quantified bacteria were ( )– ( ) cfus/ml or ( ) copies/ml for bal, and ( ) cfus/ml or copies/ml for eta. respiratory commensals (h. influenzae, s. aureus, e. coli, s. pneumoniae) were the most common pathogens. discordant results for bacterial identification were observed in / ( . %) bal(ds) and / ( . %) eta(ds), and in most cases, fapp identified one supplemental bacteria ( / bal(ds) and / eta(ds)). an absence of growth, or polybacterial cultures, explained almost equally the majority of the non-detections in culture. no linear relationship was observed between bin and cfus/ml variables. concordant results between paired bal(ds) and eta(ds) were obtained in / ( . %) patients with fapp. one of the resistance genes detected with fapp (meca/c and mrej) was not confirmed by conventional testing. overall, fapp enhanced the positivity rate of diagnostic testing, with increased recognition of coinfections. implementing this strategy may allow clinicians to make more timely and informed decisions. hospital-acquired pneumonia (hap) is the most frequent cause of nosocomial infection in intensive care unit (icu) patients, with dramatic effects on patients' outcomes. international experts have developed guidelines to prevent and improve the management of hap (kalil et al., ; torres et al., ) . among strategies proposed, optimization of empiric antimicrobial therapy is of major importance. this entails administrating early appropriate antimicrobial therapy, while limiting overuse of broad-spectrum antibiotics. hence, european guidelines suggest using narrow-spectrum empiric therapy (amoxicillin-clavulanate, cefotaxime, ceftriaxone, and fluoroquinolones) in patients without risk factors for multidrugresistant (mdr) pathogens in case of early-onset hap (first days of hospitalization). however, making such choice is not so obvious in icu patients, and adherence to guidelines is associated with a high rate of unnecessary broad-spectrum antibiotics (roquilly et al., ; ekren et al., ) . microbiological confirmation of hap is a crucial step for tailoring antibiotic therapy. nevertheless, current culture methods take - h to obtain antimicrobial susceptibility results. moreover, traditional techniques fail to recover pathogens in up to % of clinically-diagnosed hap (roquilly et al., ) . recently, syndromic multiplex molecular tests have emerged as powerful tools for rapid diagnostics (meningitis/encephalitis, gastroenteritis, bacteraemia, pneumonia) (couturier and bard, ; poole and clark, ) . initially based on qualitative dna detection, those approaches were not suitable for diagnosing pneumonia caused by common colonizers of the upper airways (e.g., streptococcus pneumoniae, haemophilus influenzae). the filmarray r pneumonia plus panel (fapp) is a new panel for hap, which offers potential advantage to detect and quantify in a single test, respiratory pathogens ( bacteria, viruses) and antibiotic resistance genes. the aim of this study was to assess the performances of this new molecular test on bronchoscopy specimens [bronchoalveolar lavages (bal) and/or endotracheal aspirates (eta)] from icu patients with hap requiring mechanical ventilation. the study protocol was approved by our local ethical committee (gneds, nantes, france). patients and relatives were informed of the trial. consent was waived according to french law. the study was conducted at the nantes university hospital (france), in icus located on two sites spaced km apart. we recruited critically ill adult patients receiving mechanical ventilation with clinically suspected hap, between october and january ( table ) . pneumonia was suspected based on european guidelines, if there were the following criteria: a new or persistent radiological pulmonary infiltrate without another obvious cause combined with two clinical signs among fever, purulent endotracheal secretions, hyperleukocytosis or the respiratory specimens were analyzed in parallel by routine microbiology testing and fapp, as soon as they arrived at the microbiology laboratory. the turnaround times from samples to validated results were recorded. results of routine microbiology testing were analyzed independently of fapp. the biofire r filmarray r pneumonia plus panel (biomérieux) was performed according to the manufacturer's instructions, with a handling time of ∼ min. briefly, the respiratory sample collected with a flocked swab (∼ µl) and then mixed with a sample buffer, was injected along with an hydration solution in the reagent pouch "pneumonia plus panel, " which was then inserted into the filmarray r instrument. the test consisted of automated nucleic acid extraction, purification, amplification, detection, and analysis with each target reported as "detected" or "not detected." a semi-quantitative measurement reported into bins (i.e., , , , and ≥ bacterial dna copies/ml) was provided for bacteria, if detected. the panel included bacteria, atypical bacteria, viruses, and antimicrobial resistance genes ( table ) . each resistance marker was reported only if the potential microorganism harboring the gene was concomitantly detected in the sample. clinicians were left blinded to the fapp results. bal were considered as positive with fapp when at least one microbial target was detected (at ≥ copies/ml for semiquantified bacteria). for eta, in order to match the culture threshold that differentiate commensalism from pathogenicity (≥ cfus/ml), we set up a bin threshold of ≥ copies/ml to consider the semi-quantitative bacterial targets as positive. the agreement between fapp and culture was measured for each bacterial pathogen in the form of negative percent agreement (npa), positive percent agreement (ppa) and overall percent agreement (opa), and their two-sided percent confidence intervals. in order to explain discrepant results, cultures were reread after routine final reports in light of results obtained with fapp. concordance was calculated based on the original culture reading. at the time of hap diagnosis, fapp yielded positive results with significant levels (i.e., ≥ bin in bal and ≥ bin in eta for semi-quantified bacteria) in / patients. thus, as shown in figure a , . % ( / ) bal ds , and . % ( / ) eta ds were positive for at least one target. of these, more than half were positive for at least two pathogens ( / ( . %) for bal ds , and / ( . %) for eta ds ), leading to the diagnosis of coinfection in / patients (figure ) . multiple detections per positive specimen were not higher in eta ds than in bal ds , since bacteria with bin results of were considered as negative in eta (it represented bacteria in eta ds ). of note, if the cutoff had been used for eta, . % ( / ) eta ds would have been positive, and multiple targets would have been detected in . % ( / ) of these specimens (figure ) . a maximum of pathogens ( bacteria and one human rhinovirus/enterovirus) was detected in one patient (bal ds and eta ds ). the most common pathogens detected at diagnosis were h. influenzae, s. aureus, e. coli, s. pneumoniae, and k. pneumoniae, which were found in ( %), posiƟve ( figure ). the panel identified viruses at diagnosis [human rhinovirus/enterovirus ( patients), coronavirus ( patients), influenza a ( patients), adenovirus ( patients), parainfluenza viruses ( patients), and rsv ( patient)] in / patients ( . % ( / ) bal ds , and . % ( / ) eta ds ). in most cases, it corresponded to viral-bacterial co-infections ( patients, including one with multiple viruses (adenovirus and influenza a) and s. pneumoniae) (supplementary table s ). an atypical bacteria (m. pneumoniae) was detected with other bacteria in one patient. the positivity rate of eta tt obtained during follow-up was . % ( / ), and bacteria were below the cutoff in eta tt (figures a, ). four types of resistance genes were detected in patients: meca/c and mrej (one patient), and the ctx-m esbl ( patients), either alone ( patients) or combined with a carbapenemase (bla ndm in one patient, and bla oxa− −like in one another). the median turnaround time (from sample collection to results) was h min (bal ds or eta ds ). at hap diagnosis, culture identified one or more bacteria in / patients ( patients who benefited from a fast track multiplex pcr routinely ordered by clinicians, yielding an overall positive detection in / patients. two or more bacterial pathogens were identified and reported in / patients, in a higher proportion of bal ds ( / , . %) than eta ds ( / , . %), certainly because bal are more distal than eta and are normally not contaminated. indeed, this property might have encouraged microbiologists to identify and report any bacteria found in these distal specimens rather than concluding to "polymicrobial flora." thus, only . % ( / ) of culture-negative bal ds had results reported as "mixed bacterial flora" vs. . % ( / ) of culture-negative eta ds (supplementary table s ). the most frequent bacteria detected by culture were h. influenzae, s. aureus, e. coli, s. pneumoniae, and k. pneumoniae in ( %), ( %), ( %), ( %), and ( %) patients, respectively (figure ). culture showed a lower positivity rate of . % ( / ) for eta tt collected during follow-up, with a high proportion of culture-negative results reported as "no growth" ( / , . %) (supplementary table s ). regarding ast, enterobacteriaceae resistant to thirdgeneration cephalosporins were found on average days after specimens collection, in / patients. in cases, it was esblproducing strains (k. pneumoniae or e. coli), while in the others cases, high-level cephalosporinases were confirmed with additional tests (mastdiscs tm d c), in strains of e. cloacae complex ( patients), s. marcescens ( patient), and e. coli ( patient). two esbl-producing k. pneumoniae that were resistant to ertapenem ± imipenem, were also confirmed to be carbapenemase (ndm or oxa- like) producers, by means of an immuno-chromatographic test (coris bioconcept resist- o.k.n.) performed days after specimen collection. all strains of p. aeruginosa detected in patients were susceptible to ceftazidime. the mean turnaround time from sample collection to results validation was h for bal ds , and h for eta ds . in total, at hap diagnosis, just over half of the specimens were concordant for the bacterial identification ( / ( . %) bal ds and / ( . %) eta ds ) (figure and table ). in most of the discordant specimens ( / ( . %) bal ds and / ( . %) eta ds ), fapp identified one supplemental bacterial pathogen, which was most often confirmed by fapp in the paired respiratory sample and/or in the eta tt collected - days later (figure ) . by rereading the plates in light of fapp results after final report, we showed that an absence of significant growth, or polybacterial cultures impeding the accurate visualization of non-predominant pathogens, explained almost equally the majority of the non-detections in culture (figure ) . in the rest of the cases, the corresponding bacteria had not been searched on the plates (s. pyogenes or s. agalactiae in mixed flora, or because of an impossibility due to proteus invasion) (figure ) . furthermore, in patients [ / ( . %) bal ds and / ( . %) eta ds ], culture yielded bacteria that were not targeted by fapp (citrobacter koseri, hafnia alvei, morganella morganii, raoultella planticola, stenotrophomonas maltophilia, and streptococcus pseudopneumoniae), and two fapp false-negative results were observed: k. oxytoca (one bal ds with a pure culture at cfus/ml), and h. influenzae (one polymicrobial eta ds with h. influenzae at > cfus/ml) ( figure e and table ). the atypical bacteria m. pneumoniae found in one patient with fapp, had not been searched with conventional methods at the time of hap diagnosis, but was subsequently confirmed with an in-house real-time pcr. the performance data for each fapp bacterial target are provided in table s ). not surprisingly, most discrepancies ( / , . %) were explained by no growth of bacteria identified with fapp ( figure a) . the vast majority of the fapp-positive bacterial targets that were not reported by routine culture, had already been detected by fapp at diagnosis, either above positive threshold values ( / , . %), or not (bin result of in eta ds ) in a few cases ( / , . %) (figure b) . regarding fapp semi-quantitative results, most bacteria with bin results of in eta (i.e., below our positivity threshold) were not reported in culture ( / ( %) in eta ds , and / ( . %) in eta tt ). on the other hand, for patients with eta at diagnosis and - days later, . % ( / ) of the detections with a bin value of in eta ds were positive again in eta tt with a higher bin value (≥ copies/ml). no linear relationship was observed between the bin and cfus/ml variables (supplementary table s ). however, semi-quantitative culture results were not stratified into log ranges above positive thresholds ( cfus/ml for eta and cfus/ml for bal). eighteen resistance markers were detected with fapp in samples ( meca/c and mrej, bla ctx−m , bla ndm , and bla oxa− −like ) (supplementary table s ). all esbl and carbapenemases were confirmed by standard laboratory protocols (ast and additional tests performed in routine). among both methicillin-resistant s. aureus (mrsa) detected with fapp, one found at bin in eta tt did not grow in culture. the other corresponded to a false-positive meca/c and mrej result since a methicillin-susceptible s. aureus (mssa) was found in culture. this result was repeatable after retesting with fapp, but none of the comparator methods (bdmax tm staphsr performed on the same bal ds , or alere tm pbp a testing and cefoxitin susceptibility testing performed on several colonies) found a mrsa. no additional cases of methicillin-resistance, esbl, or carbapenemase production were found with routine microbiology testing. lastly, based on fapp results, an initial antibiotic therapy by amoxicillin-clavulanate could have been proposed in / patients, whose results ruled out pathogens with chromosomallyencoded cephalosporinase (i.e., p. aeruginosa, a. baumannii, e. cloacae complex, k. aerogenes, and s. marcescens) and/or resistance markers of the panel. however, this antibiotic would have not been optimal in / ( . %) patients. in fact, in those cases, culture brought to light bacterial strains with acquired resistance to amoxicillin-clavulanate ( h. influenzae and e. coli, in patients), or pathogens not targeted by fapp and naturally resistant to amoxicillin-clavulanate ( h. alvei, m. morganii, and s. maltophilia, in patients). a medicoeconomic evaluation is ongoing to determine what impacts fapp results would have had on care and antibiotics prescribing (guillotin et al., in preparation) . among the patients with paired bal ds and eta ds , ( . %) had the same pathogen(s) (or no pathogen) identified in both samples with fapp. of the discrepancies observed, were due to detection of one more pathogen in eta ds ( viruses, and bacteria at bin), to detection of one additional bacteria in bal ds ( of which were also detected in eta ds , but considered as negative since at bin in eta ds ). in the latter cases, the difference relied on two pathogens. if bacteria with a bin had been considered as positive in eta ds , the agreement between both types of specimens would have been less satisfactory, with / ( . %) concordant results (figures c,d) . regarding culture, concordant results were obtained in / ( . %) paired specimens. in most of the discordant cases ( / ), there was at least one additional pathogen detected in bal ds . at last, only two of all discordant pairs (n = with fapp and/or culture) were confirmed with both methods (similar results between fapp and culture) (supplementary table s ). at first developed for the detection of widely circulating respiratory viruses and selected atypical bacteria, syndromic molecular tests for respiratory tract infections continuously expand their breadth of coverage to improve diagnostic accuracy. fapp and the curetis r unyvero hospitalized pneumonia panel, are the first two, fda approved and ce marked, commercially available platforms which target a large number of lower respiratory tract pathogens and resistance genes from aspirates or bal fluids (collins et al., ; murphy et al., ) . there are no published prospective studies comparing the performances of both plateforms, but regarding their technical characteristics, fapp offers a shorter turnaround time ( min vs. - h), a smaller footprint, and the possibility to detect viral pathogens and to semi-quantify bacteria (poole and clark, ) . in this study, this test was compared to routine microbiological methods using prospectively collected bal and eta specimens obtained from icu patients at the time of suspected hap and, if possible, at a later timepoint during follow-up. as expected, implementation of fapp shortened the delay in getting results ( h min on average, one icu setting being located km away from the laboratory vs. - h with culture). in accordance with recent evaluations (lee et al., ; buchan et al., ; murphy et al., ; yoo et al., ) , fapp increased the positivity rate of diagnostic testing ( . % for bal ds , and . % for eta ds ), enabling identification of additional bacteria in . % bal ds and . % eta ds . the most common pathogens detected were consistently the same across both methods (i.e., in order of prevalence, h. influenzae, s. aureus, e. coli, s. pneumoniae, and k. pneumoniae). this pathogen distribution, which mostly corresponded to bacterial species that are part of the normal throat flora, was not really different from that described in community-acquired pneumonia. according to the latest european surveillance report on healthcare-associated infections acquired in icu in , p. aeruginosa was the most common microorganism associated with pneumonia ( . %), followed by s. aureus ( . %), klebsiella spp. ( . %), and e. coli ( . %). in the majority of cases, pneumonia was associated with intubation, and hap episodes occurred after an average length of icu stay of . - . days, depending on the country (european centre for disease prevention and control [ecdc], ). in our study, whatever the method used, p. aeruginosa was identified in only / patients, including three who did not present classic risk factors for mdr pathogens (no previous antimicrobial therapy or hospitalization in the preceding days, and length of icu stay of - days) (torres et al., ; european centre for disease prevention and control [ecdc], ). the most common pathogen of our study, h. influenzae, was detected with fapp in / patients at diagnosis, after a median length of icu stay of days, but was less frequently found in culture ( / patients). in line with our data, the majority of discrepancies previously reported between fapp and culture, concerned the same fastidious bacteria, and were explained by the higher sensitivity of the molecular test and/or antibiotics consumption before sampling (lee et al., ; yoo et al., ) . here, in just over half of the discrepant cases, h. influenzae grew on the enriched medium used for culture, but was overgrown by other pathogens or commensal bacteria, and was therefore not detected and/or not reported. thus, whether detection of h. influenzae represents true infection or colonization will be an important area for future research. it is less a question for s. aureus, which is a member of the normal nasal flora in about % of the population, but can also be regarded as an aggressive and life-threatening bacterial pathogen (laux et al., ) . however, in the same manner as for h. influenzae, discrepant results obtained for s. aureus in patients (fapp-positive but culture-negative), were not always explained by no bacterial growth. as noted previously, these findings pointed the limits of bacterial cultures, which are subject to interpretation and based on selection of dominant species assigned to play a pathogenic role, the minority species being not considered (buchan et al., ; murphy et al., ) . these results confirmed the need to inoculate selective agars for enhancing detection of specific bacteria in lower airways (chapin and doern, ; doern and brogden-torres, ) . moreover, a significant part of discrepancies was linked to a lack of growth in culture [ / ( . %) for bal ds , / ( . %) for eta ds , and / ( . %) for eta tt ]. a quater ( / ) of the patients enrolled in the study had received antibiotics before sampling at the time of hap diagnosis, while eta tt were collected under antibiotic treatment. thus, in our view, these culture-negative detections most likely corresponded to pathogens present at low abundances (i.e., below the limit of detection in culture) or to remnant dna from nonviable bacteria, notably in supplemental eta tt , rather than non-specific amplifications. in fact, fapp results from eta tt and/or paired bal ds or eta ds allowed to verify a lot of fapp-positive results for bacteria that had been undetected by culture. as a result, fapp may prove useful to guide treatment in situations of diagnostic uncertainty where patients have received antibiotics before sampling, and/or have unfavorable treatment outcomes after obtaining culture, because the higher sensitivity of this method decreases the likelihood to miss out on pathogens of the panel. an important finding of this study, was that the implementation of fapp increased the number of coinfections detected compared to conventional methods. thus, the multiplex panel identified mixed infections in / patients ( . % of positive bal ds or eta ds ), compared to / patients ( . % and . % of positive bal ds and eta ds , respectively) by culture. these data corroborate other published results, and outline that the true incidence of polymicrobial hap is probably underestimated with conventional techniques (lee et al., ; buchan et al., ; murphy et al., ; yoo et al., ) . it remains to be evaluated whether detection of more pathogens will increase cure rates, and not adversely result in unnecessary consumption of broad-spectrum antimicrobials. new research avenues have emerged in recent years about the pathophysiology of hap, because their rate of clinical cure does not commonly exceed % (roquilly et al., ) . it has been demonstrated that healthy lungs harbor a diverse and dynamic microbiota, which is profoundly altered in critically ill patients, and would play a role in the development of pneumonia. future progress in this field should help understand how to appreciate lower abundance taxa of the microbiome, over other most numerous species (panzer et al., ; roquilly et al., ) . in our study, viruses were identified in / patients with fapp, but in half of them no viral testing had been ordered, including one with an influenza a. as this virus can be responsible for severe pneumonia, and can represent a potential source of intra-hospital transmission, fapp demonstrated a concrete benefit in that case (loubet et al., ; van someren gréve et al., ) . conventional testing for respiratory viruses other than influenza, has not been universally embraced as a standard of care, especially because viral carriage is not uncommon in patients with hap (loubet et al., ; torres et al., ; papazian et al., ) . furthermore, while the interaction between influenza and s. pneumoniae or s. aureus is a major contributor to influenza mortality in community-acquired pneumonia, the consequences of viral-bacterial coinfection on the prognosis of hap is still unclear (loubet et al., ; van someren gréve et al., ) . in our study, the majority ( %) of the patients with identified viruses, were coinfected with bacteria, and patients were infected with a single virus (influenza a, rsv, coronavirus, or human rhinovirus/enterovirus). furthermore, in our opinion, additional viral targets (herpes simplex virus and cytomegalovirus) might be relevant if added to the panel, because reactivation of these viruses are indeed quite frequent in icu patients, causing nosocomial viral pneumonia that can evolve into acute respiratory distress syndrome (ards) (papazian et al., ) . one special feature of fapp, is its ability to provide semiquantitative assessment of bacterial dna targets to help in interpretation. here, we showed that in bal, copies/ml corresponded to bacterial counts of ∼ - cfu/ml. in eta, bacteria with bin results of copies/ml were not found in culture in . % of the cases ( / ). however, a small proportion ( . %) of targets quantified as copies/ml in eta ds , were recovered later with higher bin values in eta tt . thus, we show that in those potentially contaminated samples, targets quantified as copies/ml by fapp, can be reported as negative to provide results concordant with those routinely reported by culture, in accordance with current guidelines (≥ cfu/ml) (buchan et al., ) . nonetheless, this raises the important question of whether low concentration culture-negative detections with fapp are adding value in the care of icu patients. this issue is discussed in the medico-economic evaluation coupled with this study (guillotin et al., in preparation) . we found no correlation between bin ≥ and culture concentrations in both types of specimens. however, the plating method used in the present study did not allow accurate determination of relative quantities beyond cfu/ml for bal, and cfu/ml for eta. an originality of this work lies on the inclusion of patients from whom both bal ds and eta ds were collected, and could be compared. latest european and american guidelines for the management of hap provide divergent recommendations on sampling techniques to prioritize for diagnosis of hap. while scientific societies from north america place a high value on non-invasive sampling with semiquantitative cultures (i.e., eta), european guidelines suggest obtaining distal quantitative samples with invasive techniques to improve the accuracy of results, and reduce overutilization of antibiotics (kalil et al., ; torres et al., ) . in fact, endotracheal aspirates may overestimate the presence of bacteria, but they can be performed more quickly and simply, with fewer complications and resources. in our study, provided that a copies/ml threshold was applied for eta, those specimens appeared equally accurate as bal for the diagnosis of hap (concordance obtained in . % of patients with fapp vs. . % patients for conventional culture). finally, this study examined if when compared to culture, informations supplied by fapp would have had positive impacts on antibiotics prescribing. regarding the adequacy of bacteria targeted by fapp, five gram-negative species including three resistant to amoxicillin-clavulanate (h. alvei, m. morganii, and s. maltophilia) in / patients, were missed by the panel. on the other hand, the molecular test led to an increased identification of respiratory pathogens, and to the rapid detection of some genotypic markers of resistance in patients. thus, in total, for covering fapp findings, the narrow-spectrum antibiotic amoxicillin-clavulanate could have been a therapeutic option in the majority of patients ( %). nonetheless, natural or acquired resistances to amoxicillin-clavulanate would have gone unnoticed in . % of them. all carbapenemase and/or esbl-producing strains were correctly detected with the multiplex panel (ast agreed with fapp). however, it should be noted that the overall prevalence of antimicrobial resistance was low in our study, and it should also be kept in mind that a lack of detection of resistance genes does not necessarily means susceptibility to antibiotics as there are resistance mechanisms that are not detected by fapp (i.e., derepressed or plasmidic cephalosporinases, or non-ctx-m esbl). regarding methicillin resistance, consistent with previous observations, we noticed the false-positive detection of meca/c and mrej genes in one specimen containing a mssa in culture (yoo et al., ) . since this respiratory sample was polymicrobial, we hypothesized that it could have contained both a methicillin-resistant coagulase-negative staphylococcus carrying meca/c, and a mssa with an empty sccmec cassette (thus positive for mrje) (murphy et al., ) . in conclusion, our study demonstrates that fapp provides results at a speed and sensitivity never possible before, and may allow clinicians to make more informed decisions about antibiotics use and isolation of patients. there is still room for improvements in terms of breadth (amoxicillin-clavulanate naturally resistant gram-negative bacilli), resistance (mrsa), and cost, but this culture-independent technique may achieve more reliable identification of causative agents than culture. there will be a learning curve for physicians to establish how best to use fapp results in the management of icu patients with hap. to achieve maximum benefit from this new molecular test, nuances in results interpretation might be applied on the basis of clinical presentation, timing of initial antimicrobial therapy (fresh vs. post-treatment samples), sampling type (bal vs. eta), and local bacterial ecology and resistance patterns. we are currently assessing the impact of this platform on antibiotic use and patients outcome in our hospital, and are evaluating if an algorithm-based treatment plan guided by fapp would be of great benefit. all datasets generated for this study are included in the article/supplementary material. the studies involving human participants were reviewed and approved by the groupe nantais d'ethique dans le domaine de la santé (gneds). written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements. lc, ar, and sg were involved in all the aspects of the study and were the guarantors for the data. bg, mb, kl, br, and ar performed the clinical procedures. lc, td, ep, and sg performed the laboratory procedures. lc, td, fg, cp, m-av, and sg analyzed the data. lc and ar wrote the manuscript. all authors contributed to the article and approved the submitted version. this work has been supported by the biomérieux biofire medical, france. the authors maintained control over all the aspects of the study and over the content of the publication. we thank the clinical research and innovation department of the nantes university hospital for their helpful contribution to this study. the supplementary material for this article can be found online at: https://www.frontiersin.org/articles/ . /fmicb. . /full#supplementary-material practical comparison of the biofire r filmarray r pneumonia panel to routine diagnostic methods and potential impact on antimicrobial stewardship in adult hospitalized patients with lower respiratory tract infections selective media for recovery of haemophilus influenzae from specimens contaminated with upper respiratory tract microbial flora evaluation of a novel multiplex pcr panel compared to quantitative bacterial culture for diagnosis of lower respiratory tract infections direct-from-specimen pathogen identification: evolution of 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ventilatorassociated pneumonia: guidelines for the management of hospital-acquired pneumonia (hap)/ventilator-associated pneumonia (vap) of the european respiratory society (ers), european society of intensive care medicine (esicm), european society of clinical microbiology and infectious diseases (escmid) and asociación latino americana del tórax (alat) respiratory viruses in invasively ventilated critically ill patients-a prospective multicenter observational study evaluation of the biofire r filmarray r pneumonia panel for rapid detection of respiratory bacterial pathogens and antibiotic resistance genes in sputum and endotracheal aspirate specimens key: cord- -ashjw xs authors: guo, lingxi; wei, dong; zhang, xinxin; wu, yurong; li, qingyun; zhou, min; qu, jieming title: clinical features predicting mortality risk in patients with viral pneumonia: the mulbsta score date: - - journal: front microbiol doi: . /fmicb. . sha: doc_id: cord_uid: ashjw xs objective: the aim of this study was to further clarify clinical characteristics and predict mortality risk among patients with viral pneumonia. methods: a total of patients with viral pneumonia at ruijin hospital in shanghai from may to may were recruited. multiplex real-time rt-pcr was used to detect respiratory viruses. demographic information, comorbidities, routine laboratory examinations, immunological indexes, etiological detections, radiological images and treatment were collected on admission. results: ( . %) patients died within days in hospital. a predictive mulbsta score was calculated on the basis of a multivariate logistic regression model in order to predict mortality with a weighted score that included multilobular infiltrates (or = . , % ci . – . , p = . ; points), lymphocyte ≤ . (∗) ( )/l (or = . , % ci . – . , p < . ; points), bacterial coinfection (or = . , % ci . – . , p < . ; points), acute-smoker (or = . , % ci . – . , p = . ; points), quit-smoker (or = . , % ci . – . , p = . ; points), hypertension (or = . , % ci . – . , p = . ; points) and age ≥ years (or = . , % ci . – . , p = . ; points). points was used as a cut-off value for mortality risk stratification. this model showed sensitivity of . , specificity of . and a better predictive ability than curb- (auroc = . vs. . , p < . ). conclusion: here, we designed an easy-to-use clinically predictive tool for assessing -day mortality risk of viral pneumonia. it can accurately stratify hospitalized patients with viral pneumonia into relevant risk categories and could provide guidance to make further clinical decisions. statement viral infections could present with severe pneumonia, acute respiratory distress syndrome or are complicated by bacterial super-infections in many patients. influenza and other respiratory viruses are common reasons of acute pneumonia which can result in significant morbidity or mortality in the setting of high-risk factors such as extremes of age, pregnancy, obesity or chronic pre-existing conditions. cytokines and chemokines, on the other hand, are regarded as possible hallmarks of severe disease and many of them reach high serum levels in the setting of severe infection. although a variety of clinical prediction rules for pneumonia such as crb- and curb- are widely used in the assessment of community acquired pneumonia, no standard rule for the calculation of viral pneumonia severity scores has been established to our knowledge. here, we designed a easy-touse clinically predictive score for assessing mortality risk of viral pneumonia. this model showed better predictive ability with a c-index of . , sensitivity of . and specificity of . . a cut-off value of points could be used for mortality risk stratification. background viral infections, in spite of their common manifestations as mild illnesses, present with severe pneumonia, acute respiratory distress syndrome (ards) or bacterial coinfections in many patients (shorr et al., ) . in recent years, the dissemination of pcr has increased the ability to detect respiratory viruses in both upper and lower-respiratory tract samples (das et al., ) . influenza and other respiratory viruses are common reasons of acute respiratory infection. patients predisposed to bacterial infections have greater morbidity and mortality levels (hanada et al., ) . during natural infection, both the adaptive and innate immune responses play important roles in controlling respiratory virus infection (nussing et al., ) . adaptive t and b cells maintain immunological memory and provide protection against subsequent virus infections. cytokines and chemokines, on the other hand, are regarded as possible hallmarks of severe disease and many of them reach high serum levels in the setting of severe infections (la gruta et al., ) . such variables also could guide clinical decision making as well as infectious disease management. although a variety of clinical prediction rules for pneumonia such as crb- and curb- are widely used in the assessment of community acquired pneumonia (cap) (viasus et al., ; uranga et al., ) , most remain not applicable in the setting of viral infection. other reported risk factors for influenza pneumonia such as po /fio , lymphocyte count, and antigen-specific t cells are likewise useful in predicting mortality and deciding on appropriate management (viasus et al., ; shi et al., ) . to our knowledge, no standard rule for the calculation of viral pneumonia severity scores has been established. here, we aimed to further elucidate the potential risk factors and attempt to predict the probability of mortality among patients infected with respiratory viruses. a retrospective single-center observational study was conducted from may to may in ruijin hospital, shanghai, china. the study was approved by ruijin hospital ethics committee and written informed consent was obtained from all patients involved before enrolment. we retrospectively studied all hospitalized patients with positive result of multiplex real-time reverse-transcription polymerase chain reaction (rt-pcr, tib respiratory kit, roche, switzerland) aiming to detect respiratory viruses. the time period of this study was selected because of the introduction of the viral test panel. patients who were diagnosed pneumonia according to the infectious diseases society of american (idsa)/american thoracic society (ats) guidelines (charles et al., ) were enrolled in this study. patients were excluded if: ) age < years; ) had a clear alternative final diagnosis as lung cancer or other non-pneumonia illness; ) long hospitalization > months before death. hospitalized patients had initial positive rt-pcr results and of them were enrolled with pneumonia. a total of cases were excluded: final diagnosis of non-pneumonia illness (n = ), children or adolescent patient (n = ). pneumonia patients with positive viral detection were finally included in this analysis (figure ). infections due to influenza a (flua), adenovirus (adv), bocavirus, human rhinovirus (hrv), influenza b (flub), parainfluenza (piv), coronavirus (cov), respiratory syncytial virus a (rsva), respiratory syncytial virus b (rsvb), enterovirus (ev) and human metapneumovirus (hmpv) were confirmed using rt-pcr via nasal wash products. data were collected on admission including demographic information, comorbidities, routine laboratory examinations, chest radiography or ct scanning, immunological and etiological detections. we used positive bacterial culture of blood and sputum samples as the criteria for bacterial growth. the use of antiviral therapy and steroids was recorded, including the drug, start date, duration and dosage. patients were evaluated as deemed clinically appropriate at any time when pneumonia was suspected. curb- score of each patient was calculated (barlow et al., ) . length of stay and outcome state of each patient were recorded. those improved patients with hospital stay < days were followed up by a phone call to determine survival status if they were not seen in the outpatient clinic. finally, the outcome of mortality was defined as overall mortality within days. viral pneumonia patients were classified into two groups: survival group and -day death group. univariate analysis was initially used to compare risk factors for mortality separately among patients with viral pneumonia. proportions or means with sd were used to characterize the patient sample. continuous variables were compared using t-tests or one-way anova while χ or fisher exact tests were used for categorical dependent data analysis, as appropriate. the percentages of missing values of variables in our cohort were lower than %. we imputed missing data of the covariates by using multiple imputations (sterne et al., ) . conclusions of univariate logistic regression analyses with or without imputed data were unchanged. continuous variables were categorized and retained for multivariate testing. cut-off points were identified following youden's index of receiver operator characteristic (roc) curve or a clinically relevant cut-off. variables with p < . were regarded as potential risk factors and included in multivariate regression analysis against overall mortality reduced by a backward elimination procedure (conditional likelihood ratio test and elimination if p ≥ . ). data of patients was partitioned randomly into two complementary subsets: the training set of ( %) was used to establish the model; the testing set of ( %) was used to validate the analysis. for pragmatic reasons, scores for each predictors were assigned as integer values relative to the regression coefficient. cut-off points were identified following youden's index of roc. survival analysis was performed using univariate approach with kaplan-meier analysis between lowrisk and high-risk group according to the cut-off value. performance of the score was assessed by measuring the area under roc curve (auroc) while sensitivity and specificity were calculated. internal validation was assessed by auroc of bootstrapped samples. the cross-validation was assessed by calculating auroc of the testing set. roc curve and net reclassification improvement (nri) (leening et al., ) analyses were used to assess the improvement in risk predicting capacity compared with curb- . statistical analysis was performed using spss version . and r . . . all tests were two-sided and a p-value < . was considered significant. baseline characteristics of complete cases and different groups are described in table . the mean age of viral pneumonia patients was . (sd . ) years and . % were male. immune examinations between survival and dead patients are described in table lower serum levels of t-lymphocyte subtypes were noted in death group (p < . ). moreover, patients from death group were found to possess lower serum levels of t-lymphocyte subtypes (p < . ) and elevated levels of the cytokines il- r (p = . ), il- (p < . ) and il- (p = . ). we further analyzed these same immunological indices in patient subgroups with or without bacterial infections. lower levels of cd + (p < . ), cd + (p = . ) and cd + (p = . ) t-lymphocyte counts were found in the group suffering bacterial infections. as for interleukins, elevated levels of the cytokines il- r (p = . ), il- (p = . ) and il- (p = . ) were also found in bacterial group. there were no statistically significant differences in cd + /cd + , il- , il- and tnf-α in both comparisons. (figure ) . we compared the curb- score, severity and prognoses among patients with or without bacterial co-infection (table ) . patients with bacterial infections revealed striking differences in curb- scores, use of either non-invasive or invasive ventilation, icu admission rate, length of hospitalization and treatment cost as compared with those who simply suffered viral infections. multiple imputation of missing data was performed for: serum interleukins ( . % missing); pao /fio ( . % missing); t lymphocyte subsets levels ( . % missing); body mass index (bmi) and lymphocyte count (all < % missing). according to the methods and analyses above, the following categorical variables were entered in a backward stepwise logistic regression analysis: male; age ≥ years; smoking history; hypertension; lymphocyte ≤ . * /l; po /fio ≤ ; il- ≥ pg/ml; il- r ≥ pg/ml; positive sputum or sanguine culture for bacteria; fungi infection; multilobular infiltration ( table ) . in order to develop a simple and useful clinical predicting tool, relative weights were assigned according to the regression coefficient of each categorical variable (β). figure shows coefficient, odd ratio (or), % ci and calculation of the multilobular infiltration, hypo-lymphocytosis, bacterial coinfection, smoking history, hyper-tension and age (mulbsta) score. auroc of the training set was . ( % ci . to . ), and auroc of the testing set was . ( % ci . - . ). for the total patients, auroc was . ( % ci . - . ). sensitivity, specificity and corresponding risk of death of mulbsta are shown in table . patients were divided in to high-risk and low-risk groups considering the cut-off value of . the kaplan-meier survival curves for high-risk and low-risk groups are shown in figure . continuous parameters presented as mean ± sd, categorical data as n (%). in comparison, the nomogram of the full regression model in original form is shown in supplemental figure . compared with the mulbsta score, there was no difference between the auroc for the original regression model ( . vs. . , p = . ). in our cohort, mulbsta was a significantly stronger predictor of overall mortality than curb- (auroc = . vs. . , p = . , n = ) (figure ) . the average auroc of bootstrapped (n = ) mulbsta model and curb- score were . and . separately. nri of mulbsta was also improved than curb- (nri . , % ci . - . , p = . ). as curb- was commonly used to predict -day mortality, we also assessed the use of mulbsta score in -day mortality which tended to be a stronger predictor than curb- (auroc = . vs. . , p < . , n = ). in patients hospitalized with viral pneumonia, a simple prognostic tool was made for overall mortality which is useful for prediction several days after admission upon obtaining culture results. this score predicts prognoses with greater accuracy than curb- . pneumonia is a global cause of death with high shortterm and long-term mortality. though short-term mortality rates are high in this acute disease, long-term mortality within days, year and years are also noteworthy in previous studies (mortensen et al., ; uranga et al., ) . nowadays, the survival time for patients with severe lung failure with the progress of radiological image, new drugs and supporting techniques like extracorporeal membrane oxygenation (ecmo) (pappalardo et al., ) . a prospective research on viral pneumonia showed a higher -day mortality rate than overall mortality as length of hospital stay was between to days (zhou et al., ) . during hospitalization, patients in our study died between and days of hospital stay, among them ( . %) lived longer than days, which makes -day mortality worthy of attention. as immune deficiency is a close relative of mortality, evaluating immune condition could be conductive to monitor patient's general condition and estimate prognosis. in our study, all t-lymphocyte subtypes were reduced in death group reflecting the deficiency of adaptive immune response. prior research on viral infection indicated that adaptive t cells provide broader and more lasting cross-reactive cellular immunity with less limitations of strain-specific restriction, especially cd + t cells (bender et al., ). besides, the higher level of proinflammatory cytokines had been documented to attribute to severe disease and lung damage (das et al., ) . accordingly, il- r and il- , which appeared to significantly correlate with illness severity by complementing cd + t cell function (nussing et al., ) , presented with significantly higher serum levels in death group. il- r and il- were also found related to mortality in univariate regression. meanwhile, il- secreted along with adoptive transfer of th cd + t cell clones, but it was associated with delayed viral clearance and failed to cause protective effect (la gruta et al., ) . although the test of interleukin is not yet widely available, we suggest that patients could be stratified by il- r and il- regarding mortality risk. bacterial coinfection in the setting of viral pneumonia is known as another major cause of mortality. acinetobacter baumannii is one of the most commonly encountered pathogens both in prior studies and in our investigation (gao et al., ) . we further compared patients with or without bacterial infection. bacterial co-infection not only manifested with worsened outcomes but also prolonged hospital stay and significantly increased the cost of hospital care. bacterial infection is an independent predictor without other driving forces. viral pneumonia further deteriorates when bacterial infection occurs spontaneously. this process is considered to be associated with the dysregulation of t-cell, antigen-specific t cell and plasma cytokine levels (li and cao, ) . levels of inflammatory cytokines, such as il- and il- , were found to be higher in patients suffering bacterial and influenza virus co-infections than in patients infected by a sole pathogen (li et al., ) . as such, the remarkably increased il- in patients co-infected with bacteria demonstrated its predictive potential once again. despite intense efforts, the development of antiviral therapy to prevent or treat respiratory virus infections is under limitation. influenza antivirals as oseltamivir or zanamivir were commonly used on the basis of international recommendations (jefferson et al., ) . however, oral oseltamivir has a relatively strict time window and several secondary effects like nausea and renal syndromes, and it's hard to use for unconscious patients (lee et al., ) . there is no effective listed antiviral or vaccine approved for the prevention or treatment of non-influenza viruses (heylen et al., ) . in our study, oseltamivir was commonly used as antiviral therapy; while acyclovir, ganciclovir or foscarnet were used for cytomegalovirus or herpes simplex virus. nevertheless, early antiviral treatment did not prevent progression to pneumonia consistent with earlier studies (elizaga et al., ; chemaly et al., ) . the confused choice of respiratory virus therapy makes it urgent to predict mortality more accurately. to date, a variety of studies concerning respiratory viruses were found to demonstrate risk factors by multivariate regression. consistent with previous report, po /fio ≤ in combination with lymphopenia (peripheral blood lymphocyte count < . * / l) were reported to be simple and reliable predictors of influenza (shi et al., ) . multilobular infection was also noted in our study, which was also a remarkable factor in prior report (jennings et al., ) . in our study, po /fio was also statistically significant mortality predictors according to univariate analysis, while the cut-off was adjusted to . moreover, younger age, chronic comorbid conditions, morbid obesity, high-dose steroid use, hematopoietic stem cell therapy, lower levels of cd + t specific cells and a lack of early antiviral therapy were also regarded as independent risk factors for severe disease, according to prior reports (viasus et al., ; chemaly et al., ; li and cao, ) . however, none of these were significant in our study. the idsa/ats guidelines had recommended curb- (confusion, urea, respiratory rate, blood pressure, age ≥ year) as one of cap severity score (charles et al., ). however, it had a low mortality rate among patients categorized as low risk (mandell et al., ) . several studies argued that increasing age had worse predicting ability due to the fact that influenza a virus had been reported to occur in younger individuals (riquelme et al., ; bjarnason et al., ) . meanwhile, the relative mortality rate of virus infectious diseases in the elderly are reported more than twice those of the young (pawelec et al., ) . early study suggested that a high cd + t cell count and low nk activity correlated significantly with survival of infectious diseases in the elderly (ogata et al., ) , suggesting that aging could lead to increasing immunity deficiency and mortality. in our population of hospitalized viral pneumonia patients, age ≥ years was statistically associated with mortality while the weight coefficient was relatively small. it is not reasonable to completely deny the importance of age, but appropriate weight adjustment may enhance the predictive capacity of the model. all parameters identified in the mulbsta score are easy to get clinically and all examinations are recommended to be done on admission of hospitalization. roc and nri analysis suggests that our new score has better predictive capacity in comparison with curb- . moreover, the mulbsta score shows promise for the risk stratification of patients hospitalized with viral pneumonia. the death rates for each grade ( table ) suggest the following risk categories: mulbsta - ('low-risk' , mortality = . %); mulbsta - ('high-risk' , mortality = . %). a higher mulbsta score might be used as a good predictor of prognosis. some limitations of this study should also be acknowledged. the retrospective single-center design leads to missing data and unavoidable biases in identifying and recruiting participants. the sample size was relatively small in order to build up a predicting score. despite these limitations, the study was designed to reflect the 'real life' clinical situation. clinical information was meticulously gathered using standard protocols by admitted medical team. this score might assist clinicians in making appropriate decisions and optimizing the use of hospital resources. we found that the 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t cells and aging predicting mortality in hospitalized patients with h n influenza pneumonia mortality prediction to hospitalized patients with influenza pneumonia: po /fio combined lymphocyte count is the answer viruses are prevalent in non-ventilated hospital-acquired pneumonia multiple imputation for missing data in epidemiological and clinical research: potential and pitfalls predicting -year mortality after hospitalization for communityacquired pneumonia biomarkers for predicting short-term mortality in community-acquired pneumonia: a systematic review and meta-analysis factors associated with severe disease in hospitalized adults with pandemic (h n ) in spain disease severity and clinical outcomes of community-acquired pneumonia caused by non-influenza respiratory viruses in adults: a multicentre prospective registry study from the cap-china network this study was approved by the coordinating ethics committee of ruijin hospital affiliated to shanghai jiao tong university school of medicine (no. - ) . written informed consents were obtained from all patients involved before enrolment. in our study, patients from to years old were included. the consent obtained from the participants was both informed and written. mz and xz conceived or designed the work. lg, dw, and yw collected the data. lg, dw, and ql analyzed and interpreted the data.lg, dw, ql, and jq drafted the manuscript. all authors critically revised the manuscript and approved the final version of the manuscript to be published. the supplementary material for this article can be found online at: https://www.frontiersin.org/articles/ . /fmicb. . /full#supplementary-material key: cord- -avjw kx authors: das, shubhagata; dunbar, sherry; tang, yi-wei title: laboratory diagnosis of respiratory tract infections in children – the state of the art date: - - journal: front microbiol doi: . /fmicb. . sha: doc_id: cord_uid: avjw kx in the pediatric population, respiratory infections are the most common cause of physician visits. although many respiratory illnesses are self-limiting viral infections that resolve with time and supportive care, it can be critical to identify the causative pathogen at an early stage of the disease in order to implement effective antimicrobial therapy and infection control. over the last few years, diagnostics for respiratory infections have evolved substantially, with the development of novel assays and the availability of updated tests for newer strains of pathogens. newer laboratory methods are rapid, highly sensitive and specific, and are gradually replacing the conventional gold standards, although the clinical utility of these assays is still under evaluation. this article reviews the current laboratory methods available for testing for respiratory pathogens and discusses the advantages and disadvantages of each approach. acute respiratory tract infections are one of the leading causes of childhood morbidity and mortality worldwide, and it has been estimated that globally, respiratory infections are responsible for about million deaths in children between and years of age (bryce et al., ; kallander et al., ) . approximately % of these respiratory infection cases are caused by viral pathogens such as influenza a and b, respiratory syncytial virus (rsv) a and b, parainfluenza virus types - , adenovirus, rhinovirus, human metapneumovirus (hmpv), and others (mahony, ) . the non-specific clinical presentation of respiratory infections poses a considerable challenge to the differential diagnosis of these pathogens. early and accurate diagnosis of the causative pathogens in respiratory infections is essential to administer appropriate antiviral or antibacterial therapy, initiate effective infection control measures, and reduce the length of hospital stay (barenfanger et al., ; byington et al., ; akers et al., ) . in the last decade, there has been a remarkable improvement in the diagnosis of respiratory pathogens with the availability of molecular and pointof-care (poc) testing. although an increasing number of laboratories are adopting rapid molecular assays, conventional testing methods, such as culture and immunodiagnostics are still used. this review focuses on current laboratory methods for testing for respiratory pathogens, and discusses the advantages and disadvantages of each approach. electron microscopy (em) is one of the oldest direct examination methods that has been implemented for both clinical viral diagnosis and study of viral ultrastructure and pathogenesis in developed countries (roingeard, ) . historically, em has played an instrumental role in identifying novel viral strains in several outbreak situations, such as the coronavirus associated with the severe acute respiratory syndrome (sars) outbreak (falsey and walsh, ; ksiazek et al., ) . however, despite several advantages, the use of em has been limited in respiratory viral diagnosis as it is expensive, laborious, time-consuming, and has a greater turnaround time (approximately - h including specimen preparation), and is often insensitive when compared to other diagnostic methods (goldsmith and miller, ; zhang et al., ) . additionally, em requires strict control of experimental conditions, a high concentration of viral particles (> l − ), and considerable technical skill and expertise for accurate analysis. detection of viruses by observing the cytopathic effect and hemadsorption in cell culture has been considered the "gold standard" for diagnosis of respiratory viral pathogens for decades. viruses such as adenovirus, influenza a/b, rsv, and human parainfluenza viruses are the most common respiratory viruses that are isolated and detected by cell culture (olsen et al., ; winn and koneman, ) . the traditional tube culture method is advantageous for growing a wide variety of viruses, including novel or unknown viruses, but takes days and often weeks to provide results. over the years, modified cell culture methods such as the centrifugation enhanced shell-vial method has reduced the turnaround time from to days to h (dilnessa and zeleke, ) . shell-vial culture using combination cell lines allows simultaneous detection of multiple respiratory viruses and, as compared to conventional culture, has similar sensitivity for parainfluenza - ( % vs. %) and influenza a/b ( % vs. %), and significantly higher sensitivity for rsv ( % vs. %) (lasala et al., ) . despite these advantages, many clinically relevant viruses are difficult to grow in culture (such as rhinovirus and coronavirus) and may produce variable results (ieven and goossens, ; hodinka, ) . additionally, multiple freezing and thawing of the samples prior to testing can reduce the viral titer, thus effecting the growth in culture. therefore, as compared to molecular tests, both traditional tube and shell-vial culture methods are laborious, exhibit higher false negative rates, and have longer turnaround times, making viral culture less clinically relevant (leland and ginocchio, ; ginocchio, ; hematian et al., ) . culture is also the gold standard for detection of atypical (bacterial) respiratory pathogens, which is followed by identification and antimicrobial susceptibility testing by various manual or automated methods (tenover, ) . bacterial culture can also often be insensitive, especially when specimen adequacy is not determined by gram stain or if specimens were collected after antibiotic exposure (ats and idsa, ; woodhead et al., ; harris et al., ) . bacterial culture is also labor intensive, requires substantial technical expertise, and has a typical turnaround time of - h if antibiotic susceptibility testing is performed, and can therefore be considered inadequate for optimal patient care and guiding effective antimicrobial therapy (tenover, ) . rapid immunoassays (rias) can deliver test results in less than min and are usually performed in the poc setting, thus allowing the test results to be incorporated into the clinical decision-making algorithm (weinberg and walker, ) . among the four primary ria formats (latex agglutination, horizontal flow devices, lateral flow devices, and optical immunoassays), the lateral-flow immunoassay (lfia) is the most versatile and popular immunochromatographic method. rias are relatively inexpensive, easy to perform, and most of them have waived status in the united states according to the clinical laboratory improvement act (clia) guidelines, thereby making them invaluable in outpatient clinics, primary care, emergency, and low resource settings (ginocchio, ) . currently, commercially available rias are mostly limited to the detection of influenza a virus, influenza b virus, and rsv. numerous studies have revealed that rias demonstrate overall poor sensitivity for influenza and rsv ( - %); however, they have a higher median specificity ( to %) as compared to cell culture (who, ; leland and ginocchio, ; ginocchio, ) . in the pediatric population, commercially available immunoassays have demonstrated high sensitivity ( %) for detection of rsv, and a systematic review of published studies has further revealed that the sensitivity of rsv rias is relatively higher for children ( %) than adults ( %) (slinger et al., ; chartrand et al., ) . the higher sensitivity can be attributed to the fact that pediatric patients often shed higher titers of respiratory viruses and for a longer time as compared to adults (englund et al., ; kawai et al., ) . despite the overall lower sensitivity, rias have been deemed a valuable diagnostic tool in the emergency department as they can significantly decrease the length of stay, additional ancillary testing, and antibiotic prescriptions for those children who do test positive for influenza (abanses et al., ; blaschke et al., ) . direct fluorescent antibody (dfa) testing of nasopharyngeal wash specimens is considered a rapid and reliable method for detecting respiratory viral infections. commercial dfa kits have demonstrated high sensitivity and specificity for multiple respiratory viruses such as hmpv ( and %), adenovirus ( and %), rsv ( and %), and parainfluenza viruses ( and . %), although the results can be subjective and require technical expertise for accurate interpretation (landry and ferguson, ; ohm-smith et al., ; rocholl et al., ; aslanzadeh et al., ; vinh et al., ) . the high specificity ( - %) of dfa indicates that the test can be used as a reliable detection method, especially during the initial days of the illness, as was shown by shafik et al. ( ) for rsv in children. pathogen-specific antibodies typically appear about weeks after the initial infection and can be detected by serological tests. serological tests can successfully identify antibodies to most respiratory pathogens such as rsv, adenovirus, influenza a and b, parainfluenza - virus, etc., and can detect mixed infections from hospitalized children suffering from acute respiratory infections, with the exception of infants for whom an antibody response is usually undetected (hall et al., ; chkhaidze et al., ) . however, it has been reported that serological assays are significantly less sensitive for the detection of parainfluenza virus and adenovirus when compared to molecular methods, such as rt-pcr (kuypers et al., ) . kuypers et al. ( ) found that rt-pcr detected % more specimens from pediatric patients that were positive for at least one respiratory virus than were detected by fluorescent antibody assay (fa). fa testing, in addition to rt-pcr, is useful for epidemiological studies as it increases the probability of identifying acute viral infections and has been used for accurate assessment of respiratory viruses other than influenza in children (sawatwong et al., ; feikin et al., ; zhang et al., ) . for bacteria, serological testing is particularly challenging, especially for identifying atypical bacterial agents such as mycoplasma pneumoniae. with varied sensitivity ( % - %) and specificity ( % - %) as compared to pcr, serological testing has limited usefulness clinically (beersma et al., ; wellinghausen et al., ) . the clinical utility of serologic tests is further limited because they require both acute and convalescent sera to monitor seroconversion or to identify a four−fold increase in antibody titer (loeffelholz and chonmaitree, ) . additionally, serological tests are not relevant for identifying frequently recurring viral infections as the serum igm levels are lower due to repeated exposure to vaccines or circulating viruses. for optimal virus specific igm testing, an acute-phase serum specimen should be obtained early in the course of illness (dunn, ) . a wide variety of newer, nucleic acid amplification tests (naats) for the detection of respiratory pathogens are commercially available. these tests are listed in table and described below according to complexity and pathogen coverage. the accuracy of respiratory virus detection by molecular tests is not only dependent on their specific assay chemistry, but is also critically affected by the type, quantity, and quality of specimens collected (dunn, ) . several types of specimens can be used for detection of respiratory viruses, including: bronchoalveolar lavage (bal), throat swab, nasopharyngeal (np) washes, np aspirates, lung aspirates, and np swabs, although the appropriate specimen type depends on the specific patient population. for example, in pediatric patients, obtaining nasal washes and nasal aspirates is more technically challenging when compared to np swabs, especially in severely ill children and in poor resource settings (hammitt et al., ) . it has been observed that for optimal test results, specimens should be collected within - days after onset of symptoms to ensure that the sample has a high concentration of virus particles, viral antigen or viral nucleic acids, should be transported to the testing laboratory in refrigerated condition ( - • c) in appropriate transport media, and testing should be performed within h (grys and smith, ). detection of respiratory pathogens by naats such as pcr, nucleic acid sequence-based amplification (nasba), transcription mediated amplification (tma), strand displacement amplification (sda), loop-mediated isothermal amplification (lamp), rolling circle amplification (rca), etc., have gained immense popularity over the past decade. large syndromic panels that can detect multiple pathogens simultaneously are beneficial for infection control, timely treatment decisions, and are substantially less expensive than detecting individual pathogens by monoplex real-time rt-pcr (reijans et al., ; schreckenberger and mcadam, ) . these multiplex respiratory panels vary in terms of their complexity (high, moderate, or waived), throughput, pathogens detected, ability to subtype, instrumentation (batched or random access), workflow (stepwise or integrated sample-to-answer), ease of use, and time to result (several hours to minutes) (caliendo, ; krunic et al., ; mahony et al., ) . studies have reported increased diagnostic yield ( % vs. %) and considerably higher sensitivity ( - %) and specificity ( - %) for these assays when compared to conventional diagnostic methods such as dfa, viral isolation, and immunoassays (reijans et al., ; gharabaghi et al., ) . in comparison to other naat-based molecular methods, high-plex syndromic panels demonstrate comparable performance, although sensitivity and specificity might vary for individual targets (sails et al., ) . for example, greater than % specificity was observed for all targets except enterovirus/rhinovirus ( %), and the overall sensitivity ranged between ( - %) when the nxtag rpp and the anyplex ii rv assays were evaluated for detection of respiratory pathogens in hospitalized children (brotons et al., ) . syndromic panels can also detect bacterial infections and viral-bacterial coinfections, which is vital for developing effective treatment plans and prevention strategies (brealey et al., ; brotons et al., ) . however, for novel pathogen strains, such as flu a(h n pdm ), panel tests often fail to identify the subtype and require confirmation by other tests (bryce et al., ) . syndromic respiratory panels with high sample throughput can usually run - specimens and are mostly classified as high complexity assays according to clia guidelines, which indicates that running these panel requires considerable knowledge, training, and experience (fda, b) . it has been noted that these high throughput panels are ideal for batch testing, especially during outbreak situations and respiratory viral seasons when sample volumes are unusually high (ginocchio et al., ; crawford et al., ; tang et al., ) . despite the advantages, implementation of high throughput panels can be challenging because of demanding sample preparation, processing, and result interpretation procedures, and the turnaround time varies from approximately - h (beckmann and hirsch, ) . moderate complexity sample-to-answer molecular test systems have gained immense popularity over the last few years because of their ease of use, faster turnaround time, and efficient workflow. currently, the united states. fda-cleared sampleto-answer test systems include both low-plex, targeted assays and high-plex panels that can detect multiple pathogens and pathogen classes. these tests usually have low to moderate sample throughput (e.g., - samples/run), but have faster turnaround times when compared to batched, high throughput, multiplex panels (popowitch et al., ) . the high-plex, sampleto-answer panels can detect up to - pathogens based on the panel size. most of these qualitative assays allow random access that ensures that tests can be performed on demand, and the reactions occur in closed cassettes or cartridges, thereby minimizing the risk of contamination (poritz et al., ; popowitch and miller, ) . comparative studies evaluating the performance of these assays has reported more than % agreement between different commercial platforms, and greater than % sensitivity and % specificity in most cases (babady et al., ) . however, some high-plex sample-to-answer tests have demonstrated lower sensitivity for certain pathogens such as adenovirus ( %), influenza a(h n pdm ) ( %), and influenza b virus ( %) (popowitch et al., ) . cross-reactivity between adenovirus subtypes has also been reported for some assays, but the differentiation of these groups is not deemed clinically important for routine diagnosis (lin et al., ; gray et al., ) . despite a short turnaround time of - h, the clinical utility of these assays has been questioned since no statistically significant reduction in antibiotic prescription rates or mortality has been observed between patients who tested positive for a non-influenza virus and those who tested negative ( % vs. % respectively) (green et al., ) . in some cases, a reduction in length of hospital stay ( days vs. days) and duration of isolation ( h vs. h) was observed (rogers et al., ; akers et al., ) . the primary concerns for the highplex sample-to-answer assays are high cost per test, and the inability to order customized, targeted panels (ramanan et al., ) . multiplex assays such as the verigene rp flex panel (luminex) offer flexible testing and payment options by allowing users to order any combination of targets for an individual sample. low-plex integrated respiratory test systems typically target - pathogens per assay. comparative studies evaluating the performance of these assays has reported more than % concordance between different commercial platforms, and greater than % sensitivity and specificity in most cases (dugas et al., ; selvaraju et al., ; mcmullen et al., ) . the total turnaround time per sample for these assays varies between ∼ - h, and the number of samples that can be tested in a standard -h work shift depends on the number of instruments available in the testing laboratory (ling et al., ) . smaller panels targeting a few pathogens that can be run independently (random access) or simultaneously (random batch) could be a viable option for controlling laboratory costs. therefore, when considering among various testing options, it is recommended that in addition to clinical performance, other assay characteristics such as ease of use, panel composition, total turnaround time, hands-on time, and cost be considered before implementing an assay in various clinical settings (juretschko et al., ) . nucleic acid amplification based poc testing is relatively new in the realm of respiratory viral diagnosis and has generated contradicting opinions regarding their implementation and clinical utility. molecular poc assays have extremely short turnaround times (< min), minimal hands-on time ( - min), and can be easily operated by non-laboratory staff members, thereby making them suitable for near patient implementation and testing (brendish et al., ; peters et al., ; ling et al., ) . currently most of the united states. fdacleared commercial poc assays are limited to the detection of influenza a/b and rsv viruses, with the exception of the filmarray r rp ez (biofire) which detects targets (fda, c) . most of the studies evaluating the clinical performance of these assays have reported high sensitivity ( - %) and specificity (> %) for detecting influenza a/b and rsv in pediatric and adult patients (bell et al., ; nie et al., ; popowitch and miller, ; gibson et al., ; ling et al., ) . however, these studies have also shown that the clinical performance varies and sensitivity is significantly low for influenza b ( . - . %); therefore, the results should be interpreted with caution and additional confirmatory testing should be considered, depending on the clinical circumstances (jokela et al., ; busson et al., ; davis et al., ) . molecular poc tests can be costly and more prone to incorrect results and contamination, as the assays are usually performed by non-laboratory personnel and poor training, failure to follow manufacturer's instructions, and failure to perform adequate quality control might affect the accuracy of results (azar and landry, ) . currently, more evidence-based studies are required to understand the overall clinical utility of molecular poc testing, since no correlation has been observed between implementation of these tests and reduction of antibiotic usage or hospital length of stay (andrews et al., ; brendish et al., ) . molecular testing has considerably improved the diagnosis of respiratory pathogens and is being considered as the new "gold standard". although these tests have gained immense popularity, factors such as the patient population (adult, pediatric, and immunocompromised), the size of the laboratory, the purpose of testing (routine or urgent care), and cost/benefit ratio should be considered before implementing a particular assay. for example, during the - influenza outbreak, two nosocomial cases were identified at memorial sloan kettering cancer center on january , . despite implementing appropriate infection control measures, five more cases were identified few days later on january , . a delay in reporting the positive influenza result was observed as the laboratory was following the routine diagnostic algorithm, and this might have resulted in the subsequent cases of nosocomial infection. following the - outbreak, the laboratory implemented the filmarray r rp for routine diagnosis and the xpert r xpress flu/rsv, a rapid molecular poc system, for seasonal use during epidemics and pandemics because the needs for routine testing and urgent care are completely different in these situations. molecular tests are highly sensitive and specific and result in higher pathogen detection, and therefore physicians and attending clinicians should evaluate test results before initiating a particular course of treatment. rapid molecular tests are best suited for routine diagnosis and outbreak situations; whereas, conventional methods, such as cell culture or em could be considered for identifying novel strains of the pathogens. with the introduction of new technologies, newer assays will be developed for respiratory and other pathogens. for example, using np swab specimens from children, graf et al. 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sloan kettering cancer center and the luminex corporation (sk - ) and by an nih/nci cancer center support grant p (ca ). conflict of interest statement: sda and sdu are employees of luminex corporation.the remaining author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © das, dunbar and tang. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- - ca cfy authors: izumida, mai; kamiyama, haruka; suematsu, takashi; honda, eri; koizumi, yosuke; yasui, kiyoshi; hayashi, hideki; ariyoshi, koya; kubo, yoshinao title: fragments of target cells are internalized into retroviral envelope protein-expressing cells during cell-cell fusion by endocytosis date: - - journal: front microbiol doi: . /fmicb. . sha: doc_id: cord_uid: ca cfy retroviruses enter into host cells by fusion between viral and host cell membranes. retroviral envelope glycoprotein (env) induces the membrane fusion, and also mediates cell-cell fusion. there are two types of cell-cell fusions induced by the env protein. fusion-from-within is induced by fusion between viral fusogenic env protein-expressing cells and susceptible cells, and virions induce fusion-from-without by fusion between adjacent cells. although entry of ecotropic murine leukemia virus (e-mlv) requires host cell endocytosis, the involvement of endocytosis in cell fusion is unclear. by fluorescent microscopic analysis of the fusion-from-within, we found that fragments of target cells are internalized into env-expressing cells. treatment of the env-expressing cells with an endocytosis inhibitor more significantly inhibited the cell fusion than that of the target cells, indicating that endocytosis in env-expressing cells is required for the cell fusion. the endocytosis inhibitor also attenuated the fusion-from-without. electron microscopic analysis suggested that the membrane fusion resulting in fusion-from-within initiates in endocytic membrane dents. this study shows that two types of the viral cell fusion both require endocytosis, and provides the cascade of fusion-from-within. cell-cell fusion occurs in various physiological and pathological conditions, such as the formations of muscle (abmayr and pavlath, ) and placenta (mi et al., ) , organ repair by stem cells (rodic et al., ) , and malignant transformation (lu and kang, ) . interestingly, syncytiotrophoblasts are formed by endogenous retroviral envelope (env) proteins called syncytins (malassiné et al., (malassiné et al., , . membrane fusion mechanism in retroviral entry has been well studied. however, cell-cell fusion mechanism by retroviral env proteins is less characterized. pathology of many placental abnormalities including eclampsia remains to be elucidated. some of these disorders may be induced by impaired syncytiotrophoblast formation. therefore, it is important to resolve cell-cell fusion mechanism induced by the env protein for identification of placental diseases caused by impaired syncytin functions and for development of new therapeutic approaches against such diseases. here, we challenged to elucidate the mechanism of cell-cell fusion by env proteins of ecotropic murine leukemia virus (e-mlv) and human immunodeficiency virus type (hiv- ). there are two types of cell-cell fusion induced by retroviruses. when fusogenic viral env protein alone is expressed, the cells fuse with neighboring susceptible cells, called fusion-from-within. on the other hand, when viral particles are inserted into interface between two host cells and simultaneously fuse with the both cells, syncytia are formed, called fusion-from-without. membrane fusion activity of the e-mlv env protein is regulated by its c-terminal -amino acid segment called r peptide. the r peptide is cleaved after virion budding. the r peptide-containing env protein does not induce fusion-fromwithin, but the r peptide-truncated env (r-env) does, showing that the r peptide cleavage after virion release activates the fusogenicity required for the viral entry (rein et al., ; kubo and amanuma, ) . in the case of hiv- , the precursor gag protein inhibits the env-induced cell fusion (murakami et al., ) . therefore, syncytium formation is efficiently induced, when the wild type hiv- env protein alone is expressed in susceptible cells. e-mlv particles bind to mouse cationic amino acid transporter (mcat ) as the infection receptor, and then are internalized into endosomes by host cell endocytosis. endosomal cathepsin proteases are activated by endosome acidification, and digest the viral env protein to potentiate its membrane fusion activity (katen et al., ; kumar et al., ) . the viruses finally enter host cells by fusion between viral envelope and host cell endosome membranes. this viral entry cascade is found not only in the e-mlv infection but also in infections by ebola virus (chandran et al., ) and sars coronavirus (belouzard et al., ) . in hiv- infection, it has been shown that hiv- uses the endocytic process as a mean of infection in some circumstances (miyauchi et al., ) . however, the mechanistic details of cell-cell fusion induced by retroviral env proteins are less clear. some studies have indicated that virus-cell membrane fusion during viral infection and cell-cell membrane fusion are different. for example, lymphocyte function-associated antigen- (lfa- ) regulates hiv- mediated-cell fusion but not viral transmission (pantaleo et al., ) , and e-mlv env mutants containing amino acid substitutions at the r peptide cleavage site do not induce infection but mediate syncytium formation in xc cells (kubo and amanuma, ) . additionally, it has been reported that cellular transformation by the h-ras oncogene activates the e-mlv virion-induced fusion-from-without but not infection (wilson et al., ) , and that actin inhibitors suppress hiv- virion-induced fusion-from-without but not viral entry in np derived cells (kondo et al., ) . using an endocytosis inhibitor and a dominant negative mutant of dynamin, we probed requirement of endocytosis for the retroviral env-induced fusion-from-within. because size of an endosome is much smaller than that of a cell, a whole cell cannot be encapsulated into an endosome. to examine how retroviral env-induced cell fusion uses endocytosis pathway, we performed fluorescence, time-lapse, and electron microscopies. small fusion pores were observed in membrane dents at the interface of cells. these results suggested that membrane fusion for the syncytium formation initiates in the membrane dents before intracellular endosome vesicles are formed. additionally, the fluorescence microscopic analysis revealed that fragments of the target cells are internalized into the env-expressing cells. this result suggested that endocytosis in the env-expressing cells is important for the cell-cell fusion, whereas that in the target cells is for the viral entry. it is thought that the fusion-from-without is induced by membrane fusion at cell surface (kondo et al., ) . interestingly, the endocytosis inhibitor attenuated the fusionfrom-without induced by e-mlv, suggesting that endocytosis is involved in the fusion-from-without. finally, we proposed a cascade of the viral env-induced fusion-from-within. human embryo kidney (hek) t, human te , human hela, mouse sc- , and african green monkey cos cells were maintained in our laboratory. te cells expressing mcat (te mcat ) were constructed as already reported . np cells expressing cd and cxcr (np /cd /x ) were kindly provided by dr. h. hoshino (soda et al., ) . these cell lines were cultured in dulbecco's modified eagle medium (dmem) with % fetal bovine serum (fbs), % penicillin-streptomycin. the cdna clone of the e-mlv infection receptor (mcat ) was kindly provided by dr. j. m. cunningham (albritton et al., ) . the cd cdna (endosome marker) was molecularly cloned from hela cells by rt-pcr with the la taq (takara) and kod-plus (toyobo) dna polymerases in our laboratory. the mlv wt env (r+env) and r peptide-truncated mlv env (r-env) expression plasmids have been shown previously (kubo and amanuma, ). an expression plasmid encoding c-terminally gfp-tagged cd was constructed in our laboratory. hiv- env (jd ) and nuclear localization signal-containing lacz (nls-lacz) expression plasmids were kindly provided by dr. u. hazan (dumonceaux et al., ) and dr. l. chang (chang et al., ) , respectively. an expression plasmid encoding the dominant negative mutant of dynamin that inhibits endocytosis was obtained from dr. s. l. schmid (damke et al., ) . fusion-from-within induced by retroviral env proteins was measured as follows. hek t cells seeded in -cm plates were transfected with pcdna . , mlv r-env, or hiv- env ( µg) together with nls-lacz ( µg) by the fugene hd transfection reagent (promega), and incubated for - h at • c. target cells (te mcat cells for the mlv r-env, and np /cd /x cells for the hiv- env) were pretreated with dynasore ( µm) or ca me ( µm) for h. the transfected and treated cells were co-cultured for h and stained with -bromo- -chloro- -indoly-d-galactopyranoside (x-gal). we counted blue nuclei by a light microscope (leica). the fusion index was calculated as follows: number of blue nuclei in syncytia/total number of blue nuclei × . the target and env-expressing cells were always co-cultured at ratio of : . to assess the effect of the dynamin dominant negative mutant on fusion-from-within, hek t cells seeded in -cm plates were transfected with the nls-lacz ( . µg) and env expression plasmids ( . µg) together with the dynamin mutant expression or pcdna . plasmid ( . µg). these cells were washed by pbs, co-cultured with te mcat , or np /cd /x cells h after the transfection, and stained with x-gal h after the co-culture. fusion-from-without induced by e-mlv particles was measured as follows. target mouse sc- cells were seeded onto wells, and pretreated with dynasore ( and µm) or ca me ( and µm) for h. undiluted culture supernatants from ecotropic friend mlv env-expressing telceb cells were inoculated to the pretreated sc- cells. the areas of syncytia were measured by the image j software h after the inoculation. hek t, te mcat , sc- , helamcat cells were treated with the inhibitors for h. the cells were collected and treated with trypan blue. numbers of unstained and stained cells were counted using a counting chamber, and ratios of unstained live cells per total cells were calculated. for observation by fluorescence microscopy and time lapse analysis, hek t cells were cultured in glass slides or glass bottom dishes, and transfected by the env expression plasmid ( . µg) together with gfp or cd -gfp expression plasmid ( . µg). target cells were stained with the cell tracker orange (takara) for h. the transfected and stained cells were cocultured, fixed, and observed under a conforcal fluorescence microscope (olympus). time lapse imaging (astec or keyence) was performed on the live cells. for observation by an electron microscope, hek t cells were transfected by the hiv- env plasmid ( . µg). the cells were co-cultured with np /cd /x cells for min. the co-cultured cells were fixed with . % glutaraldehyde overnight at • c. after fixation in osmium tetroxide and ethanol-dehydrated, samples were embedded in epoxy resin. sections ( - nm) were stained with uranyl acetate and then with lead nitrate, and observed using a transmission electron microscope (jem- , jeol). endocytosis of host cells is required for the retroviral infection. to examine whether the cell-cell fusion also requires endocytosis, we investigated syncytia between different fluorescencelabeled env-expressing and host cells by confocal fluorescence microscopy. the e-mlv r-env or hiv- env expression plasmid was transfected to hek t cells together with a gfp expression plasmid. te cells expressing the e-mlv receptor (te mcat ) were stained with the cell tracker orange, and co-cultured with the transfected cells. interestingly, many small orange dots under green background were observed in fused cells induced by the e-mlv r-env (figure a upper panel) and the hiv- env (figure a lower panel) . relatively large orange signals represent unfused cells on syncytia. when t cells were transfected with the non-fusogenic r peptide-containing env expression plasmid together with the gfp expression plasmid, and co-cultured with the cell tracker orange-stained te /mcat cells, such orange dots were not detected in the gfp-expressing cells (figure b upper panel) . similarly, when the r-env-transfected t cells were co-cultured with unstained te mcat cells and cell tracker orange-stained te cells, small orange dots were not observed in green fused syncytia ( figure b middle and lower panels). when unsusceptible orange cells were located near fused cells, the syncytia were not enlarged. these results show that the small orange dots are specifically generated in fused cells. these observation suggested three possibilities; (i) small fragments derived from the target cells are internalized into the env-expressing cells, (ii) the target cells are fragmented after enclosing the whole target cells by the env-expressing cells like phagocytosis, or (iii) after the cell fusion, the target cell cytoplasm are not mixed with the env-expressing cell cytoplasm in the fused cells, and are fragmented. because size of endosomes is much smaller than that of cells, and because t cells do not have an ability of phagocytosis, the second possibility is unlikely. because the cell tracker orange is a low molecular size compound and more rapidly diffuses than the gfp protein, the third possibility is also unlikely. thus, the first possibility is most likely, and the small orange dots might represent intracellular vesicles such as endosomes/lysosomes. to assess the hypothesis, the e-mlv r-env was transfected to hek t cells together with a cterminally gfp tagged cd (cd -gfp) expression plasmid, since cd is specifically localized to late endosomes and lysosomes (pols and klumperman, ) . target cells were stained with the cell tracker orange, and co-cultured with the env plus cd -gfp-expressing cells - days after the transfection. we found that almost all of small orange dots in fused cells were co-located with cd -gfp signals (figure a) . this result indicated that parts of the target cells were encapsulated into late endosomes/lysosomes of the env-expressing cells during the cell fusion, and strongly supported the first possibility. to analyze cell fusion dynamics, we performed time-lapse imaging analysis of cell-cell fusion induced by the e-mlv r-env protein. as the result, time course of the cell fusion was as follows. first, a cell tracker orange-stained target cell contacted to an envexpressing green cell (figure b and supplementary video ) . second, the cell color of the target cells was changed from orange to yellow, indicating that cytoplasm is mixed resulted from the membrane fusion. third, those cells were merged to form a symcytium. due to low resolution of the time-lapse fluorescence microscope, orange dots were not observed in fused cells. additionally, the time-lapse imaging showed that ruffling structures of cell membranes bound each other, and cell-fusion occurred ( figure c) . surprisingly, these cells were already fused cells, indicating that fused cells can fuse with fused cells. these results suggested that membrane fusion during the envinduced syncytium formation takes place in ruffling membranes, then cytoplasm are mixed by membrane fusion, and finally the cells were merged to form a syncitium. this result also excludes the possibility that phagocytosis is involved in the cell fusion. to confirm whether fragments of the target cells are internalized into the env-expressing cells by endocytosis, the env-expressing or target cells were treated with an inhibitor of endocytosis (dynasore), endosomal cathepsin b (ca me), or endosome acidification (concanamycin a). syncytium formation induced by the e-mlv r-env (figure a left panel) or by the hiv- env (figure a right panel) was more efficiently inhibited when the env-expressing cells were treated than when the target cells were treated, showing that the syncytium formation requires endocytosis, cathepsin b, and endosome acidification in the envexpressing cells. because the treatment with each inhibitor did not affect cell viability (figure b) , the suppression of cell fusion by the inhibitors should not result from unexpected effects of the inhibitors. additionally, when the e-mlv r-env (figure c left panel) or hiv- env (figure c right panel) expression plasmid was transfected to hek t cells together with a dominant negative mutant of dynamin, cell-cell fusion was inhibited. these results suggest that endocytosis in the env-expressing cells are required for the cell-cell fusion. unfortunately, the target cells stably expressing the dynamin dominant negative mutant were not obtained. when the target cells were transiently transfection with the dynamin mutant, syncytium formation was not affected due to the mutant expression in only several % of transfected cells. thus, the impact of dynamin mutant expressed in target cells on the cell-cell fusion could not be analyzed. the above results showed that endocytosis in the env-expressing cells is required for the retroviral env-induced cell-cell fusion. to know how endocytosis is involved in the cell fusion, electron microscopic observation at the cell-cell contact area was performed. it was observed that small parts of a cell were invaginated into another cell at the cell-cell contact area like endocytosis (figures a,b) . two types of fused membrane were observed by the electron microscopy; (i) a relatively wide area of plasma membrane at the cell-cell contact region disappeared by membrane fusion (figure c , red box), and (ii) a small membrane region at a tip of a membrane dent disappeared by membrane fusion (figure upper panel) . because in the former type of fused membrane, relatively wide membrane area always disappeared, it was thought that their fusion pores were already enlarged. in contrast, the fusion pores observed in the membrane dents were smaller and not enlarged yet, suggesting that the fusion pores in the membrane dents are nascent and newly formed. these results suggested that the membrane fusion starts at membrane dents. additionally, long and narrow membrane vesicles were detected in cytoplasm of fused cells (figure d) . these compartments should be derived from plasma membrane remaining between the membrane fusion sites when more than two membrane fusions occurred at a cell-cell contact area. thus, membrane fusions should occur at gaps of the long and narrow compartments ( figure d, red arrows) . interestingly, a round vesicle was observed at a gap of long and narrow compartments (figure lower panel) . it was thought that the vesicle is formed by membrane fusion at the root of invaginating membrane, which results in cytoplasm mixing. this result also supported the conclusion that the membrane fusion starts at the membrane dent. such intracellular vesicles were not observed at many of the nicks of the long and narrow compartments ( figure d) . the nicks without vesicles might be formed by the membrane fusion at a tip of a membrane dent mentioned above (figure upper panel). thus, it was suggested that the membrane fusion at a tip of a membrane dent more preferentially occurs than that at a root. it is thought that the fusion-from-without is induced by membrane fusion at cell surface, and does not required endocytosis. to confirm this issue, target cells were pretreated with dynasore, ca me, or concanamycin a, and were inoculated with e-mlv to form fusion-from-without. contrast to the previous theory, these inhibitors significantly suppressed the fusion-from-without (figure ) . this result showed that endocytosis, cathepsin b, and endosome acidification are required for the fusion-from-without. in this study, our fluorescence and electron microscopic observations showed that fragments of the target cells were internalized into the env-expressing cells. in addition, the treatment of the env-expressing cells with an endocytosis inhibitor reduced syncytium formation more effectively than that of target cells. these results indicated that endocytosis in the env-expressing cells triggers the cell-cell fusion. target cells treated with the inhibitor also reduced syncytium formation, because the target cells become envexpressing cells after the fusion with the env-expressing cells. the e-mlv r-env (left panel) or hiv- env (right panel) was transfected to hek t cells together with the nls-lacz expression plasmid. the transfected hek t cells were co-culture with te mcat or np /cd /x cells pretreated with indicated inhibitor (black bars). the transfected cells were treated with indicated inhibitors, and were co-cultured with te mcat or np /cd /x cells (gray bar). this experiment was repeated three times. fusion indexes in the control cells were always set to , and relative fusion indexes ± sd are shown. asterisks indicate statistically significant differences between treatments of the env-expressing or target cells. (b) hek t, te mcat , sc- , and helamcat cells were treated with indicated inhibitor. ratios (%) of live cells per total cells are indicated. this experiment was repeated three times. (c) hek t cells were transfected with the mlv r-env (left panel) or hiv- env (right panel) expression plsmid together with the nls-lacz expression plasmid. the cells were also simultaneously transfected with the dynamin dominant negative mutant expression plasmid or pcdna . . this experiment was repeated three times. fusion indexes in the control cells were always set to , and relative fusion indexes ± sd are shown. asterisks indicate statistically significant differences between transfection with pcdna . and dynamin mutant. based on our results, we propose the cascade of retroviral envinduced cell-cell fusion as follows (figure ) . first, a target cell (orange) contacts with an env-expressing cell (green) through ruffling membranes. second, parts of the target cell are inserted into the env-expressing cell at the contact region by endocytosis. third, membrane fusion occurs at the endocytic membrane dent, and cytoplasm of the target cells are mixed with that of the env-expressing cells (yellow). vesicles containing the target cell cytoplasm (orange) remain in the fused cell. fourth, cells are merged completely to form a syncytium. it was thought that a little proportion of invaginating endosomes participates for cellcell fusion, because many small vesicles containing the target cytoplasm were detected in the fused cells. we showed here two patterns of membrane fusion. one membrane fusion occurs in the tip of membrane dent, and the other occurs at the root of membrane dent (figure ) . the membrane dents might be induced by endocytosis. because dynamin is involved in pinching endosomes off and does not affect the membrane dent formation, dynasore can inhibit the membrane fusion at a root of membrane dent, but not that at a tip. the membrane fusion at a root of membrane dent might be induced by dynamin-mediated scission of both the target and env-expressing cell membranes. the membrane fusion at a tip of membrane dent might result from activation of the env fusogenicity by acidification of spaces between target and env-expressing cells. our study cannot exclude the possibility that the membrane fusion resulting in cell-cell fusion occurs other places than membrane dents. it was difficult to find the membrane dents with fusion pores, suggesting that the nascent fusion pore is unstable and easily enlarged. further study is required to elucidate the mechanism by which endocytosis promotes the cell fusion. the treatment of env-expressing cells with the cathepsin b inhibitor also suppressed the cell-cell fusion induced by the env proteins of e-mlv and hiv- . although the e-mlv infection requires cathepsin b, the inhibitor does not attenuate hiv- env-mediated infection . how is cathepsin b involved in the hiv- env-induced cell fusion? further study is needed to understand this issue. interestingly, it was observed that a fused cell fuses with a fused cell. because fused cells express the env protein, it is thought that the infection receptor proteins in the fused figure | fusion-from-without requires endocytosis. mouse sc- cells were pretreated with dynasore, ca me, or concanamycin a, and were inoculated by e-mlv. sizes of fused cell areas are measured. this experiment was repeated three times. sizes of fused cell areas in control cells are always set to , and relative values ± sd are indicated. asterisks indicate statistically significant differences between sizes of fused areas in solvent-and inhibitor-treated cells. figure | membrane fusion occurs at membrane dents. hiv env-transfected and np /cd /x cells were co-cultured. the cells were observed under an electron microscopy. cells are occupied by the env proteins, and the fused cells are not susceptible to the env-induced cell fusion. nascent syncytia might partially express the infection receptors that are not occupied by the env proteins yet, and fuse with the env-expressing cells. alternatively, when the expression level of env protein is low, the free infection receptor molecules still remain on cell surface of the fused cells, and can fuse with env-expressing cells. this study found that the fusion-from-without also requires endocytosis. when a viral particle is internalized into a target cell by endocytosis, another cell bound to the virion may be also internalized into the cell. further study is needed to understand the mechanism of fusion-from-without. in summary, this study found that the fusion-from-within induced by the e-mlv or hiv- env protein requires endocytosis in the env-expressing cells, although the viral entry requires endocytosis in target cells. the fusion pore is initiated in endocytic membrane dents. the fusion-from-without also requires endocytosis. mi, hk, eh, y. koizumi, ky, and y. kubo performed experiments. ts helps the experiment of electron microscopy. mi, hh, ka, and y. kubo analyzed the data. mi and y. kubo wrote the manuscript. myoblast fusion: lessons from flies and mice a putative murine ecotropic retrovirus receptor gene encodes a multiple membranespanning protein and confers susceptibility to virus infection activation of the sars coronavirus spike protein via sequential proteolytic cleavage at two distinct sites endosomal proteolysis of the ebola virus glycoprotein is necessary for infection efficacy and safety analyses of a recombinant human immunodeficiency virus type derived vector system induction of mutant dynamin specifically blocks endocytic coated vesicle formation spontaneous mutations in the env gene of the human immunodeficiency virus type ndk isolate are associated with a cd -independent entry phenotype infection of xc cells by mlvs and ebola virus is endosome-dependent but acidification-independent infectious entry by amphotropic as well as ecotropic murine leukemia viruses occurs through an endocytic pathway distinct requirements for hiv-cell fusion and hiv-mediated cell-cell fusion mutational analysis of the r peptide cleavage site of moloney murine leukaemia virus envelope protein host cell cathepsins potentiate moloney murine leukemia virus infection cell fusion as a hidden force in tumor progression expression of the fusogenic herv-frd env glycoprotein (syncytin ) in human placenta is restricted to villous cytotrophoblastic cells expression of herv-w env glycoprotein (syncytin) in the extravillous trophoblast of first trimester human placenta syncytin is a captive retroviral envelope protein involved in human placental morphogenesis hiv enters cells via endocytosis and dynamin-dependent fusion with endosomes regulation of human immunodeficiency virus type env-mediated membrane fusion by viral protease activity human immunodeficiency virus (hiv) infection in cd + t lymphocytes genetically deficient in lfa- : lfa- is required for hiv-mediated cell fusion but not for viral transmission trafficking and function of the tetraspanin cd function of the cytoplasmic domain of a retroviral transmembrane protein: p e-p e cleavage activates the membrane fusion capability of the murine leukemia virus env protein cell fusion and reprogramming: resolving our transdifferences establishment of a new system for determination of coreceptor usages of hiv based on the human glioma np- cell line the requirements for viral entry differ from those for virally induced syncytium formation in nih t /dtras cells exposed to moloney murine leukemia virus cd -independent human immunodeficiency virus infection involves participation of endocytosis and cathepsin b the supplementary material for this article can be found online at: http://journal.frontiersin.org/article/ . /fmicb. supplementary video | time-lapse video microscopic observation of syncytium formation. hek t cells were transfected by the r-env and gfp expression plasmids. te /mcat cells were stained with the cell tracker orange. one day after transfection, these cells were co-cultured in glass bottom dish and observed by time-lapse microscopy. the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © izumida, kamiyama, suematsu, honda, koizumi, yasui, hayashi, ariyoshi and kubo. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- - sors bg authors: clementi, nicola; criscuolo, elena; diotti, roberta antonia; ferrarese, roberto; castelli, matteo; dagna, lorenzo; burioni, roberto; clementi, massimo; mancini, nicasio title: combined prophylactic and therapeutic use maximizes hydroxychloroquine anti-sars-cov- effects in vitro date: - - journal: front microbiol doi: . /fmicb. . sha: doc_id: cord_uid: sors bg while the sars-cov- pandemic is heavily hitting the world, it is of extreme importance that significant in vitro observations guide the quick set up of clinical trials. in this study, we evidence that the anti-sars-cov activity of a clinically achievable hydroxychloroquine concentration is maximized only when administered before and after the infection of vero e and caco- cells. this suggests that only a combined prophylactic and therapeutic use of hydroxychloroquine may be effective in limiting viral replication in patients. on th march , who director-general characterized covid- as a pandemic. as of th june , , , total cases were confirmed, accounting for , deaths (dong et al., ) . to date, the clinical management of covid- subjects almost exclusively consists of supportive therapy and, in particular, of ventilatory support for the most severe cases. several drugs, some already in clinical trials, are currently being used in order to limit the inflammatory response or to hamper sars-cov- replication (tu et al., ) . regarding the latter, no sars-cov- -specific drugs are currently available and, as a consequence, there is the need for repurposing drugs used in other settings, such as chloroquine (cq) and hydroxychloroquine (hcq) (gao et al., ; gautret et al., ; zhou et al., ) . several studies already demonstrated the in vitro efficacy of cq and hcq, evidencing the latter's higher activity on sars-cov- (colson et al., ; wang et al., ) . importantly, a recent study also modeled the main pharmacokinetic features of hcq trying to infer its lung concentration after different dosing regimens (yao et al., ) . it is therefore important to use this data in order to set up possible scenarios related to the real clinical use of this drug. in this study, we tested hcq against a sars-cov- italian clinical isolate, by using different protocols of drug administration corresponding to its possible prophylactic, therapeutic, and prophylactic/therapeutic use in patients. a single hcq concentration easily reachable in the lung and characterized by high anti-sars-cov- activity was used for the three protocols wang et al., ; yao et al., ) . vero e (vero c , clone e -crl- ; atcc) cells were cultured in dulbecco's modified eagle medium (dmem) supplemented with non-essential amino acids (neaa, x), penicillin/streptomycin (p/s, u/ml), hepes buffer ( mm) and % (v/v) fetal bovine serum (fbs). caco- (human epithelial colorectal adenocarcinoma cells, atcc htb- ) cells were cultured in minimum essential medium (mem) supplemented with neaa ( x), p/s ( u/ml), hepes buffer ( mm), sodium pyruvate ( mm), and % (v/v) fbs. calu- (human lung cancer cell line, atcc htb- ) were cultured in mem supplemented with neaa ( x), p/s ( u/ml), hepes buffer ( mm), and % (v/v) fbs. a clinical isolate hcov- /italy/unisr / (gisaid accession id: epi_isl_ ) was isolated and propagated in vero e cells, and viral titer was determined by % tissue culture infective dose (tcid ) and plaque assay for confirming the obtained titer. all the infection experiments were performed in a biosafety level- (bls- ) laboratory of microbiology and virology at vita-salute san raffaele university, milan, italy. bafilomycin a (bfla ) and hydroxychloroquine (hcq) were obtained from merck. an aliquot ( . ml) of the transport medium of the nasopharyngeal swab (copan's kit utm r universal viral transport medium-copan) of a mildly symptomatic sars-cov- infected patient was mixed with an equal volume of dmem without fbs and supplemented with double concentration of p/s and amphotericin b. the mixture was added to % confluent vero e cells monolayer seeded into a cm tissue culture flask. after h adsorption at • c, ml of dmem supplemented with % fbs and amphotericin b were added. twenty-four hours post-infection (hpi) another ml of dmem supplemented with % fbs and amphotericin b were added. live images were acquired (olympus ckx inverted phase-contrast microscopy) daily for evidence of cytopathic effects (cpe), and aliquots were collected for viral rna extraction and in-house one-step real-time rt-pcr assay ( . / s - ( ) - ). five days post-infection (dpi) cells and supernatant were collected, aliquoted, and stored at − • c (p ). for secondary (p ) virus stock, vero e cells seeded into cm tissue culture flasks were infected with . ml of p stored aliquot, and infected cells and supernatant were collected hpi and stored at − • c. for tertiary (p ) virus stock, vero e cells seeded into cm tissue culture flasks were infected with . ml of p stored aliquot and prepared as above described. p virus stocks were titrated using both plaque reduction assay (pra, pfu/ml) and endpoint dilutions assay (eda, tcid /ml). for pra, confluent monolayers of vero e cells were infected with -fold-dilutions of virus stock. after h of adsorption at • c, the cell-free virus was removed. cells were then incubated for h in dmem containing % fbs and . % agarose. cells were fixed and stained, and viral plaques were counted. for eda, vero e cells ( × cells/ml) were seeded into wells plates and infected with base dilutions of virus stock. after h of adsorption at • c, the cell-free virus was removed, and complete medium was added to cells. after h, cells were observed to evaluate cpe. tcid /ml was calculated according to the reed-muench method. viral genome from supernatant infected cells was extracted using qiaamp viral rna mini kit following manufacturers' instructions. reverse transcription and subsequent amplification were performed using random hexamer primers. the amplicons were sequenced on the illumina miseq ngs platform (illumina, san diego, ca, usa). amplicon purification and quantification were performed by agencourt ampure xp (beckman coulter, villepinte, france) and qubit dsdna assay kit (thermofisher scientific, waltham, ma, usa), respectively. library preparation was performed by using the nextera xt dna library prep kit (illumina, san diego, ca, usa). the library generated was then diluted and sequenced with miseq reagent kit v ( -cycles) (illumina, san diego, ca, usa) on the miseq platform. the quality of raw sequences obtained from miseq run was first checked using fastqc (v . . ) (babraham bioinformatics). the reads were aligned on reference sequence (gisaid accession id: epi_isl_ ) using bwa-mem and rescued using samtools alignment/map (v . ) and bamtofastq. finally, the contigs were generated using spades (v . . ). vero e cells ( × cells/ml) were seeded into wells plates and treated with hcq at different concentrations ( : serial dilutions, spanning from to . µm). cells were pretreated with the drug for h prior to virus infection at • c, followed by virus adsorption ( tcid /ml, pfu/ml, sars-cov- ) for h in the presence of the molecule. then, the cells were washed with pbs and further cultured at • c with the molecule-containing medium for h. then, cytopathic effect (cpe) was assessed using a scoring system ( = uninfected; . to . = increasing number/area of plaques; = all cells infected) to evaluate treated and untreated cells. infection control (score ) was set as % infection inhibition, uninfected cells (score ) as % infection inhibition. the whole surface of the wells was considered for the analysis ( x magnification). cell supernatants were collected for real-time pcr (rt pcr) analysis. all conditions were tested in quadruplicate. vero e cells ( × cells/ml) were seeded into wells plates and treated with µm hcq. in detail, cells were pretreated with the drug for h prior to virus infection at • c, followed by virus adsorption at different concentrations ( . - moi, sars-cov- ) for h in the presence of the molecule. then, the cells were washed with pbs and further cultured at • c with the molecule-containing medium for h. then, the cytopathic effect (cpe) was assessed using the scoring system above described, and results were normalized on virus infection control. cell supernatants were collected for rt-pcr analysis. all conditions were tested in triplicate. cell viability assay was performed using the cell proliferation kit ii (xtt) (roche diagnostics, merck). briefly, the tetrazolium salt , -bis-( -methoxy- -nitro- -sulfophenyl)- h-tetrazolium- -carboxanilide (xtt) is cleaved by viable cells to form an orange formazan dye that can be quantified photometrically at nm. before the assay, vero e , caco- , and calu- cells ( × cells/ml) were cultured in -well plates for h. the culture medium was replaced by medium containing µm hcq following full-time experimental setting, and cells were incubated for h. xtt was added to each well and the plates were incubated for an additional h. the optical density was measured at nm (reference wavelength− nm) using a multiskan go plate reader (thermo scientific instruments). for quantifications, the background levels of media without cultured cells were subtracted. vero e cells ( × cells/ml) were seeded into wells plates and treated with hcq ( µm) at different stages of virus infection. for full-time treatment, cells were pre-treated with the drug for h prior to virus infection at • c, followed by virus adsorption for h in the presence of the molecule. then, cells were washed with pbs, and further cultured at • c with the molecule-containing medium until the end of the experiment. for pre-adsorption treatment, the agent was added to the cells for h at • c before virus infection and maintained during virus adsorption. then, the mixture was replaced with fresh medium without molecule till the end of the experiment. for post-adsorption assays, the drug-containing medium was added to cells only after virus adsorption and maintained until the end of the experiment. bfla ( nm) was tested as control of inhibition of viral infectivity at a phagolysosome level only in a pre-adsorption treatment, alone or in combination with pre-adsorption hcq treatment. uninfected cells were included in all experimental settings to exclude possible drug-toxicity cpe. for all the experimental groups, cells were infected with tcid /ml sars-cov- , and absorption was performed for h at or • c. the full-time experimental setting was figure | hcq dose-response assessment. hcq ec against sars-cov- was obtained by both cpe and rt-pcr analysis on results from full-time experimental setting on vero e cells. mean values and sd (for rt-pcr values) and sem (for cpe values) are reported for all experimental replicates. all conditions were tested in quadruplicate and tested in duplicate in rt-pcr. figure | hcq antiviral effect using different multiplicities of infection (moi). cpe analysis resulted in a statistical difference between treated and untreated cells (****p < . ) when using . moi or less, rt-pcr only from . moi to decrease (****p < . ). performed also using caco- and calu- cells. live images were acquired (olympus ckx inverted phase-contrast microscopy), cpe was assessed as described above and cell supernatants were collected for rt-pcr analysis at hpi. all conditions were tested in quadruplicate. viral rna extraction and real-time rt-pcr viral rna was purified from µl of cell culture supernatant using the qiaamp viral rna mini kit (qiagen), following the manufacturer's instructions. subsequently, the purified rna was used to perform the synthesis of the first-strand cdna, using the superscript tm first-strand synthesis system for rt-pcr (thermo fisher scientific), following the manufacturer's instruction. real-time pcr, using sybr r green dye-based pcr amplification and detection method, was performed in order to detect the cdna. we used the sybr tm green pcr master mix (thermo fisher scientific) the forward primer n f: tta caa aca ttg gcc gca aa, the reverse primer n r: gcg cga cat tcc gaa gaa, and the following pcr conditions: • c for min, cycles of • c for s, annealing at • c for s and elongation at • c for s, followed by a final elongation at • c for min (hirotsu et al., ) . rt-pcr was performed using the abi-prism ht fast real-time instruments (applied biosystems) by using optical-grade -well plates. samples were run in duplicate in a total volume of µl. cpe observed for different experimental settings using hcq and bfla , alone or in combination, were normalized to figure | hcq cpe reduction on veroe cells infected with sars-cov- . cpe was assessed using a scoring system ( = uninfected; . to . = increasing number/area of plaques; = all cells infected) to evaluate treated and untreated cells. infection control (score ) was set as % infection inhibition, uninfected cells (score ) as % infection inhibition. the whole surface of the wells was considered for the analysis ( x magnification). blue gradient indicates the reciprocal of cpe detected in treated cells compared to virus infection control (white color corresponds to % cpe). protection levels are indicated by color gradient: white corresponds to no protection, dark blue shows full protection. bfla treatments are reported as experimental control of virus fusion process inhibition. virus adsorption was performed at and • c. frontiers in microbiology | www.frontiersin.org corresponding virus infection control. rt-pcr results were analyzed calculating delta ( ) ct as the difference between ct values obtained for experimental settings and infection control. then, two-way anova and tukey's multiple comparisons (for caco- and calu- experiments and virus curve for testing hcq susceptibility) or sidak's multiple comparisons (for hcq dose-response curve, time of addiction experiments and xtt cell viability evaluation) test were performed for the evaluation of cpe scoring and ct differences (graphpad prism ). ec was calculated using non-linear regression with least squares regression as a fitting model (graphpad prism ). virus isolation was achieved after < h. at hpi the cytopathic effect (cpe) was already evident on vero e cells. ngs analysis was performed by illumina miseq obtaining the whole genome sequence of the cultured isolate hcov- /italy/unisr / (gisaid accession id: epi_isl_ ). compared to the first italian isolate sequenced (hcov- /italy/cdg / ), that diverged from the hcov- /wuhan/wiv / reference strain at position , , , , and , , two more nucleotide mismatches were identified, g a and c a. g a is located in the ′ utr, while c a is a missense mutation causing the i l variation in the orf a polyprotein, specifically in the region that is part of the nsp membrane domain. no obvious consequence of the two polymorphisms can be drawn based solely on the sequence. different concentrations of hcq were tested on vero e to determine the effective concentration of the drug against sars-cov- in vitro infection (figure ) . ec resulted from both cpe and rt-pcr analysis were . and . µm, respectively. as ec analysis resulted in a low hcq effective dose, it was tested using different viral loads to better define the optimal conditions to complete the preliminary setting for subsequent experiments (figure ) . cpe analysis resulted in a statistical difference between treated and untreated cells (p < . ) when using . moi or less, rt-pcr only from . to decrease (p < . ). xtt assay was performed to determine cell tolerability to hcq treatment, and µm of the molecule tested in full-time treatment resulted in no drug-related toxicity on all three cell lines tested in the study (figure ) . median values for all experimental replicates, tested each one in duplicate in rt-pcr, and % ic range reported with error bars (*p < . ; **p < . ; ****p < . ). all conditions were tested in quadruplicate. frontiers in microbiology | www.frontiersin.org the two molecules were tested using different experimental protocols, and virus adsorption was performed at both and • c. cpe was assessed at hpi (figures , ) . virus infection positive control showed marked effects on cell morphology at • c as well as • c adsorption conditions. hcq was effective in full-time treatment at both adsorption temperatures, and in post-adsorption treatment only when the virus was added to cells for h at • c. the molecules did not show the same degree of protection from cpe in pre-adsorption treatment at both adsorption temperatures, as well as in post-adsorption treatment at • c adsorption. bfla was tested as control of inhibition of viral infectivity at a phagolysosome level in a preadsorption treatment, showing full cpe protection at • c virus adsorption, while only partial protection was observed when the virus was added to cells at • c (figures , ) . no drugrelated cytotoxic effect was observed on uninfected cells, in all experimental settings. cell supernatants of different experimental settings were collected and analyzed by rt-pcr, and results confirmed cpe data analysis (figure ) . in detail, a significant statistical difference of ct was observed with hcq full-time treatment compared to infection control, both at • c (p < . ) and • c (p < . ) virus adsorption, while hcq post-adsorption treatment was effective (p < . ) only when the virus was added to cells at • c. interestingly, bfla addition to hcq preadsorption treatment resulted in a significant statistical difference of ct when compared to infection control at • c virus adsorption. when tested on different cell lines, hcq resulted extremely effective on caco- cells (p < . for the higher tested concentrations, p < . for the minor one), while a non-statistical difference was observed when tested on calu- cells (figure ) . in the lack of sars-cov- -specific drugs, it is extremely important to evaluate the clinical potential of drug-repurposing to face the current pandemic (yao et al., ) . hcq has gained the attention of the scientific and medical community based on previous in vitro data on similar viruses (sars and mers) and on preliminary reports discussing its possible clinical effectiveness in therapy or prophylaxis based on observations performed on vero e -based infection models (colson et al., ; gao et al., ; gautret et al., ) . in this atypical context, in which on-field medicine often anticipates experimental laboratory pre-clinic, there is an urgent need for prompt experiments addressing specific clinical questions. for example, it is not clear what is the best administration regimen to maximize possible hcq anti-viral effectiveness in covid- patients. in this regard, a recent study evaluated the direct anti-sars-cov- effect in vitro and, importantly, modeled its bioavailability at the lung level where it could maximally exert its antiviral activity. based on a dosing regimen of mg given twice daily for day, followed by mg twice daily for more days, it also suggested the more useful drug concentrations to be used in clinically-oriented phenotypic laboratory assays wang et al., ; yao et al., ) . on that basis, we evaluated the hcq antiviral activity when administered before (pre-adsorption), after (postadsorption), or before and after (full-time) virus adsorption to simulate, on vero e cells, its possible prophylactic, therapeutic and prophylactic/therapeutic clinical use. moreover, we first focused our attention on a single concentration ( µm) easily achieved, well-tolerated and endowed with a strong antiviral activity (yao et al., ) . moreover, to speculate on its possible mechanism of action, we also evaluated hcq activity performing virus adsorption at and • c. in fact, at • c the virus enters the cell in a more physiological context while, conversely, at • c virus it can dock to the cell receptor, but its internalization is much more limited. in the prophylactic setting (pre-adsorption), µm hcq did not interfere effectively with the viral replicative cycle neither at • c nor at • c, as evidenced in the cpe and the rt-pcr analysis. limited antiviral activity was also observed in the therapeutic setting (post-adsorption) but, interestingly, a higher hcq antiviral activity was observed at • c, suggesting its predominant sars-cov- interference at the endosomal level (vincent et al., ; hu et al., ) . overall, and most importantly, these results suggest a limited activity of hcq when administered only prophylactically or therapeutically. on the contrary, significant antiviral activity was observed in the prophylactic/therapeutic (full-time) experimental setting both at and • c, as evidenced both by cpe and rt-pcr analyses. this observation allows us to speculate on the need for a combined prophylactic and therapeutic clinical use of hcq to maximize its antiviral effects. however, a possible bias of in vitro studies aimed at evaluating the antiviral activity of putative drugs can be represented by the moi used for testing the inhibitory activity of drugs. that is why we used different moi for testing the inhibitory activity of hcq. our data suggest how using moi higher than . to perform in vitro testing can impact on the hcq inhibitory capability. we also determined, by cpe evaluation and rt-pcr analysis, the hcq ec ( . and . µm, respectively) at h post-infection. moreover, to better translate to the clinic what observed on vero e , hcq activity was assessed also on cells of human origin: the caco- (colorectal adenocarcinoma epithelial-like cells) and the calu- (lung adenocarcinoma epithelial cells). as observed for vero e cells, no hcq direct toxicity was observed for both caco- and calu- . interestingly, a dose-response effect of hcq was appreciated only on caco- cells. on the contrary, no significant reduction of viral rna amount was appreciated on calu- cells. this observation will deserve certainly further investigations focused on both mechanistic aspects of hcq-mediated inhibition of sars-cov- and on the possible interpretation of clinical trial outcomes. in the atypical scenario of an ongoing pandemic, preclinical medical research should be focused on simple and fast observations potentially useful for the prompt set up of clinical trials. however, all possible hcq side effects already known (chatre et al., ; zhou et al., ) should be considered and evaluated also under the light of concerns regarding two clinical trials recently described and retracted (mehra et al., a,b; science aaftao, ) . as an example, our observation could be translated in a clinical study on extremely high-risk categories, such as health care workers, based on the prophylactic administration of hcq followed by its therapeutic use in case of positivity to sars-cov- . whole genome sequence data of hcov- /italy/unisr / have been uploaded on gisaid database (https://www.gisaid. org/) with the following accession id: epi_isl_ . the study involving human participants was reviewed and approved by ospedale san raffaele irb in the covid- biobanking project. the patient from whose samples the virus was isolated provided written informed consent. this manuscript has been released as a pre-print at biorxiv preprint. doi: . / . . . , hu et al. ( ) . nc and nm conceived the study. ec, rd, and rf performed the experiments. nc, nm, ec, mca, and mcl analyzed the data. nc, nm, ec, and rd wrote the manuscript. nc, nm, ld, rb, and mcl revised the manuscript. all authors contributed to the article and approved the submitted version. cardiac complications attributed to chloroquine and hydroxychloroquine: a systematic review of the literature chloroquine and hydroxychloroquine as available weapons to fight covid- an interactive web-based dashboard to track covid- in real time breakthrough: chloroquine phosphate has shown apparent efficacy in treatment of covid- associated pneumonia in clinical studies hydroxychloroquine and azithromycin as a treatment of covid- : results of an open-label non-randomized clinical trial double-quencher probes improved the detection sensitivity of severe acute respiratory syndrome coronavirus (sars-cov- ) by one-step rt-pcr. medrxiv insights from nanomedicine into chloroquine efficacy against covid- hydroxychloroquine, a less toxic derivative of chloroquine, is effective in inhibiting sars-cov- infection in vitro retraction: cardiovascular disease, drug therapy, and mortality in covid- retracted: hydroxychloroquine or chloroquine with or without a macrolide for treatment of covid- : a multinational registry analysis the pandemic's first major research scandal erupts a review of sars-cov- and the ongoing clinical trials chloroquine is a potent inhibitor of sars coronavirus infection and spread remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus ( -ncov) in vitro in vitro antiviral activity and projection of optimized dosing design of hydroxychloroquine for the treatment of severe acute respiratory syndrome coronavirus (sars-cov- ) covid- : a recommendation to examine the effect of hydroxychloroquine in preventing infection and progression the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © clementi, criscuolo, diotti, ferrarese, castelli, dagna, burioni, clementi and mancini. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- - v sq k authors: meyer sauteur, patrick m.; unger, wendy w. j.; nadal, david; berger, christoph; vink, cornelis; van rossum, annemarie m. c. title: infection with and carriage of mycoplasma pneumoniae in children date: - - journal: front microbiol doi: . /fmicb. . sha: doc_id: cord_uid: v sq k “atypical” pneumonia was described as a distinct and mild form of community-acquired pneumonia (cap) already before mycoplasma pneumoniae had been discovered and recognized as its cause. m. pneumoniae is detected in cap patients most frequently among school-aged children from to years of age, with a decline after adolescence and tapering off in adulthood. detection rates by polymerase chain reaction (pcr) or serology in children with cap admitted to the hospital amount – %. although the infection is generally mild and self-limiting, patients of every age can develop severe or extrapulmonary disease. recent studies indicate that high rates of healthy children carry m. pneumoniae in the upper respiratory tract and that current diagnostic pcr or serology cannot discriminate between m. pneumoniae infection and carriage. further, symptoms and radiologic features are not specific for m. pneumoniae infection. thus, patients may be unnecessarily treated with antimicrobials against m. pneumoniae. macrolides are the first-line antibiotics for this entity in children younger than years of age. overall macrolides are extensively used worldwide, and this has led to the emergence of macrolide-resistant m. pneumoniae, which may be associated with severe clinical features and more extrapulmonary complications. this review focuses on the characteristics of m. pneumoniae infections in children, and exemplifies that simple clinical decision rules may help identifying children at high risk for cap due to m. pneumoniae. this may aid physicians in prescribing appropriate first-line antibiotics, since current diagnostic tests for m. pneumoniae infection are not reliably predictive. the clinical entity of "atypical" pneumonia was recognized in the s many years before the etiological agent was established (mccoy, ) . the term separated this entity of pneumonia from classical pneumococcal pneumonia due to its lack of response to available antibiotics and the distinct clinical presentation without typical lobar pneumonia and a less severe disease course. that is why the term "walking pneumonia" has been introduced to denote this mild form of pneumonia. it was in a patient with "atypical" pneumonia in , where mycoplasma pneumoniae was first isolated from sputum in tissue culture by eaton et al. ( ) . at that time, it was believed to be a virus because it was resistant to penicillin and sulfonamides and passed through bacteria-retaining filters. experiments with marine recruits and adult prisoners demonstrated that the socalled eaton agent caused lower respiratory tract infections in humans (chanock et al., a,b) . in , it was first cultured on cell-free medium and classified as m. pneumoniae (chanock et al., ; chanock, ) . today we know that mycoplasmas are prokaryotes that lack a cell wall and represent the smallest self-replicating organisms (figure ) . with a size of , base pairs, the genome of m. pneumoniae is at least five times smaller than that of escherichia coli (himmelreich et al., ) . the absence of a cell wall and the specialized attachment organelle facilitate close contact with the host respiratory epithelium, which supplies the bacterium with the necessary nutrients for its growth and proliferation. mycoplasma pneumoniae causes both upper and lower respiratory tract infections, with community-acquired pneumonia (cap) as the major burden of disease. although m. pneumoniae infections are generally mild and self-limiting, patients of every age can develop severe and fulminant disease (kannan et al., ) . m. pneumoniae can also cause extrapulmonary manifestations that affect almost every organ (narita, ) . in children, m. pneumoniae infections were first reported in when % of children with lower respiratory tract disease were tested positive by a fourfold rise in antibody titers against the eaton agent (chanock et al., ) . to date, it is known that the incidence of m. pneumoniae infections is generally higher in children than in adults (foy et al., ) . this review focuses on the characteristics of m. pneumoniae infections in children, and discusses simple clinical decision rules that may further aid clinicians in identifying patients at high risk for m. pneumoniae cap. mycoplasma pneumoniae is transmitted by respiratory droplets through close contact. the incubation period can be long from up to weeks. outbreaks have been reported within families, schools, universities, institutions, camps, and military bases. family members of index patients with acute respiratory infection and detection of m. pneumoniae in the upper respiratory tract were found positive in % by polymerase chain reaction (pcr) (dorigo-zetsma et al., ) . thereof, % were < years of age and % did not develop any respiratory symptoms. at universities, the largest outbreak within years in the u.s. was observed during september -december , , where a total of cap cases were identified among students, and out of tested cases ( %) were positive for m. pneumoniae by quantitative real-time pcr (centers for disease control and prevention [cdc], ) . outbreaks appear mainly during m. pneumoniae epidemics that occur in - years cycles, in addition to a background endemic pattern (jacobs, ) . the most recent epidemic in europe occurred in - with a peak incidence in finland of / , cases in (polkowska et al., ; jacobs et al., ) . the cyclic occurrence of epidemics may be facilitated by a decreasing herd immunity and different m. pneumoniae genotypes circulating in the human population (jacobs, ) . the two major circulating genotypes, or subtypes, of m. pneumoniae are indicated as subtype and . differences between these subtypes in the amino acid sequence of the major adhesion protein p are believed to play a role in the epidemiology of infections with m. pneumoniae (su et al., ; vink et al., ) . the differences between the -kda p proteins of subtype and isolates were found to be concentrated in two specific amino acid stretches within the protein. these regions are encoded by two dna elements within the p gene, i.e., repetitive elements repmp / and repmp . the repmp / and repmp are not unique to the p gene, but are also found at other sites within the bacterial genome (spuesens et al., ) . homologous recombination events between these repetitive elements, which are similar to each other, but not identical, may form the basis of antigenic variation of the p protein of m. pneumoniae (vink et al., ) . while such recombination events may induce antigenic variation within subtype or subtype strains, m. pneumoniae strains cannot switch from one subtype to the other, as the entire set of repmp elements found in one subtype differs significantly from those found in the other subtype. moreover, changes in the proportion of the two subtypes of m. pneumoniae were not observed between and in europe (jacobs et al., ) . although cap is the major burden of disease, milder clinical presentations of m. pneumoniae respiratory infections may be much more common than cap. these include acute bronchitis and upper respiratory tract infections (esposito et al., (esposito et al., , . m. pneumoniae could be detected by pcr and/or serology in % of non-streptococcal pharyngitis cases (esposito et al., ) . it is estimated that - % of children with m. pneumoniae respiratory infection develop cap and that < % of cap cases are severe enough to require hospitalization (waites and talkington, ) . between and , m. pneumoniae was detected by culture of respiratory specimens and/or a fourfold titer rise in complement fixation test (cft) in - % of radiologically confirmed cap cases in seattle, u.s. (foy et al., ) . in subsequent etiological studies, m. pneumoniae accounted for - % of the isolates identified by pcr and/or serology in children with cap admitted to the hospital (juven et al., ; principi et al., ; baer et al., ; michelow et al., ) . m. pneumoniae was first reported as the most common bacterial cause of cap in children requiring hospitalization in a u.s. multicenter study from to in nashville and salt lake city (jain et al., ) . in this study, m. pneumoniae could be detected by pcr in ( %) out of cases with cap, whereas streptococcus pneumoniae was found in cases ( %). manifest upper and/or lower respiratory tract infections with m. pneumoniae occur at all ages (foy et al., ) . recent observations have indicated that m. pneumoniae has also a relatively high prevalence in the respiratory tract of children < years (principi et al., ; gadsby et al., ) . m. pneumoniae cap, however, was reported to be most frequent among school-aged children from to years of age, with a decline after adolescence and tapering off in adulthood (figure ) (foy et al., ) . this notion was corroborated in the recent cap study in the u.s., where m. pneumoniae was detected significantly more frequent in children ≥ years of age than in younger children ( % vs. %) (jain et al., ) . in addition to the presentation at school-age, children with cap due to m. pneumoniae have been found to present with a significantly longer duration of fever compared with other children with cap (fischer et al., ) . other symptoms that may be associated with m. pneumoniae cap are the absence of wheeze and the presence of chest pain (wang et al., ) . however, there is still a paucity of high quality data regarding clinical signs and symptoms associated with m. pneumoniae infections. a recent cochrane review therefore concluded that figure | detection of m. pneumoniae in community-acquired pneumonia (cap) according to age group. infection was diagnosed by culture of respiratory specimens and/or a fourfold titer rise in complement fixation test (cft). adapted with permission from foy et al. ( ) . the absence or presence of individual clinical symptoms or signs cannot be used to help clinicians accurately diagnose m. pneumoniae in children and adolescents with cap (wang et al., ) . pathogenic effects in the respiratory tract may be caused by m. pneumoniae either directly (by active infection), indirectly (by infection-induced immune mechanisms), or both (narita, ) . m. pneumoniae causes direct injury through the generation of activated oxygen. a potential candidate protein of m. pneumoniae that may be involved in causing direct damage to the respiratory tract is a pertussis toxin-like protein termed community-acquired respiratory distress syndrome (cards) toxin (kannan and baseman, ; becker et al., ) . a recombinant version of the cards toxin has been shown to bind with high affinity to surfactant protein a and to exhibit mono-adp ribosyltransferase and vacuolating activities, which causes disruption of the respiratory epithelium in animal models (kannan and baseman, ) . in addition to the direct damage resulting from infection by m. pneumoniae, the immunological response following infection generates inflammatory reactions that may cause pulmonary and extrapulmonary symptoms. more severe symptoms of cap have been observed in older children and adolescents (waites and talkington, ) . this suggests that the age-dependent magnitude and nature of inflammatory responses in childhood may be a major factor contributing to the development of m. pneumoniae-associated disease, similar to what is observed, e.g., in infectious mononucleosis or rheumatic fever. in fact, the severity of m. pneumoniae cap in children was closely associated with increased concentrations of interleukin (il)- and il- in acute phase serum and pleural fluid samples (narita and tanaka, ) . in addition, it has been demonstrated that cell-mediated immunity contributes to the pathogenesis of m. pneumoniae cap, as it was shown that the severity of cap correlated positively with the size of a cutaneous induration following intradermal injection of m. pneumoniae antigens (mizutani et al., ). this study described patients with cap, of which were children - years of age, diagnosed by a significant rise in antibody titers against m. pneumoniae with cft. the strongest skin reactions were seen in patients with severe cap. mycoplasma pneumoniae and other "atypical bacteria" have long been implicated in the pathogenesis of asthma (atkinson, ) . there are many studies that have addressed this issue in the recent past. in an observational study on children and adults with asthma, m. pneumoniae infection was diagnosed in % of children with asthma ( / ) and was found more frequent in patients with chronic asthma ( %) than in those with asthma exacerbations ( %; p = . ) (bebear et al., ) . the diagnosis of m. pneumoniae infection in this study was performed by pcr and/or serology. another recent study diagnosed m. pneumoniae in children with acute asthma ( %, / ) and refractory asthma ( %, / ), as well as in healthy controls ( %, / ), but did not find significant differences between these three groups (wood et al., ) . the high detection rates reported in this study were obtained using novel diagnostic methods [cards toxin enzyme immunoassay (eia) and cards gene-specific pcr] (wood et al., ) . in a recent taiwanese study (yeh et al., ) , children and adults with m. pneumoniae infection, diagnosed by positive immunoglobulin (ig) m or fourfold igg titer increase, but without prior asthma history were included from to and followed until the diagnosis of asthma or the end of . compared to matched patients without m. pneumoniae infection, the cumulative incidence of asthma was significantly higher in the m. pneumoniae cohort than in the control cohort (p < . ). patients with m. pneumoniae infection were at higher risk of having earlyonset asthma (age at asthma diagnosis < years) and lateonset asthma (age at asthma diagnosis ≥ years). these most recent findings suggested that m. pneumoniae can induce airway inflammation and contribute to incident asthma. interestingly, exposure to recombinant cards toxin resulted in an allergictype inflammatory response and airway hyperreactivity in mice and baboons (hardy et al., ; medina et al., ) . it will be interesting to investigate whether cards toxins induce a similar allergic response during m. pneumoniae infections. apart from the respiratory tract infection, m. pneumoniae can cause extrapulmonary manifestations in almost every organ, including the skin and the hematologic, cardiovascular, musculoskeletal, and nervous systems (narita, ) . these manifestations may be caused either by direct local effects of m. pneumoniae, after dissemination of the bacteria throughout the body, or indirect effects, such as autoimmune reactions. the most frequent manifestations are diseases of the dermatologic and nervous system. skin manifestations occur in up to % of all m. pneumoniae infections, including mostly non-specific exanthems, erythema nodosum, urticaria, stevens-johnson syndrome, and a rare but distinct disorder with prominent mucous membrane involvement denominated as m. pneumoniae-associated mucositis (mpam) (schalock and dinulos, ; meyer sauteur et al., ) . this condition was first described by fuchs ( ) , and therefore also referred to as fuchs syndrome (meyer sauteur et al., ) . a recent review identified patients with mpam at a median age of . years at presentation (range - years, children or young adolescents ≤ years) (meyer sauteur et al., ) . all patients presented with prodromal respiratory symptoms with a median duration of days, and pneumonia was found in chest radiography in %. oral lesions were present in all cases (figure ) , ocular lesions in %, and urogenital lesions in %. there were no skin lesions in %. although % of the patients were admitted to the intensive care unit, no one suffered from long-term sequelae. encephalitis and guillain-barré syndrome (gbs) constitute the most common and severe neurologic manifestations, where m. pneumoniae infection is established in up to and % of patients, respectively (bitnun et al., ; sinha et al., ) . in m. pneumoniae-associated encephalitis, both a direct infection of the central nervous system (cns) and an immune-mediated process have been implied to be involved (narita, ) . because the detection rate of m. pneumoniae by pcr in cerebrospinal fluid (csf) of m. pneumoniae encephalitis patients is relatively low ( - %) (bitnun et al., ; daxboeck et al., ; christie et al., a; domenech et al., ), a significant proportion of the cases is believed to be immune-mediated. this is supported by the finding that various cases with m. pneumoniae encephalitis in which bacterial dna could not be detected in csf had a more prolonged duration of respiratory symptoms before the onset of encephalitis (> - days) (bitnun et al., ; narita and yamada, ; daxboeck et al., ) . these cases indicate that m. pneumoniae encephalitis represents a postinfectious disorder, which manifests after clearance of the bacteria from the cns or respiratory tract by the immune system (meyer sauteur et al., b) . a recent study presented children with m. pneumoniae detected in the respiratory tract or csf by pcr, ( %) of whom had encephalitis ( - , toronto, on, canada) (al-zaidy et al., ) . interestingly, patients in which m. pneumoniae was detectable in the respiratory tract but not in csf showed pulmonary infiltrates on chest radiograph more frequently than patients with positive pcr in csf ( % vs. %). this suggests that pneumonia may be an indicator for a remote inflammatory process in m. pneumoniae encephalitis patients, which was also shown in % ( / ) of children observed during a national surveillance, all with negative pcr in csf ( - , switzerland) (meyer sauteur et al., ) (figure ) . mycoplasma pneumoniae expresses adhesion proteins and glycolipids that share structural homology with a variety of host cells (molecular mimicry) and may induce cross-reactive antibodies (meyer sauteur et al., b) . in children with m. pneumoniae encephalitis, intrathecal antibodies directed against galactocerebroside (galc) were found (christie et al., b; meyer sauteur et al., a) . of note, all these patients had a negative pcr in csf. galc is a major glycolipid antigen in the myelin sheath of both the peripheral and cns neurons (menge et al., ) . in fact, antibodies against m. pneumoniae infection have been found to cross-react with galc in gbs patients (kusunoki et al., ; ang et al., ) . moreover, anti-galc antibodies caused demyelinating neuropathy in rabbits (saida et al., ) and have been associated with demyelination in gbs (ang et al., ) , but also in encephalitis (christie et al., b) and encephalomyelitis (samukawa et al., ) . the detection of intrathecal antibodies against m. pneumoniae and galc may also be regarded as a promising new diagnostic tool for m. pneumoniae-associated cns disease (meyer sauteur et al., b sauteur et al., , b . like many other respiratory pathogens, m. pneumoniae can be carried asymptomatically in the respiratory tract (foy, ) . recent studies have demonstrated that asymptomatic carriage of m. pneumoniae is highly prevalent. detection rates of m. pneumoniae dna in the respiratory tract of healthy children without respiratory symptoms were % in a dutch study ( - , rotterdam, the netherlands) (spuesens et al., ) and % in a u.s. study ( antonio, tx, u.s.) (wood et al., ) . longitudinal sampling of m. pneumoniae-positive asymptomatic children demonstrated that m. pneumoniae can be present in the upper respiratory tract without causing disease, for up to months (spuesens et al., ) . the prevalence of m. pneumoniae in the upper respiratory tract of asymptomatic children varied considerably between years and seasons. for example, asymptomatic carriage rates of % and % were reported in the spring of and the summer of , respectively (spuesens et al., ) . these data suggest that carriage follows an epidemic pattern. it is tempting to speculate that this fluctuation in prevalence is related to the cyclic epidemics of m. pneumoniae infections. apart from m. pneumoniae, children were found to simultaneously carry many pathogens in their nose and throat (spuesens et al., ) . these pathogens include the bacteria s. pneumoniae, staphylococcus aureus, moraxella catarrhalis, and haemophilus influenzae, and the viruses influenza a/b, human metapneumovirus, respiratory syncytial virus, parainfluenzavirus, rhinovirus, coronavirus, bocavirus, and adenovirus. the simultaneous presence of two or more of these pathogens was detected in % of asymptomatic children (spuesens et al., ) . in children with m. pneumoniae cap, co-existence of m. pneumoniae with other pathogens has also been described (juven et al., ; michelow et al., ) , and was recently reported in % of the patients (jain et al., ) . the impact of co-infections in m. pneumoniae cap on disease severity is not yet determined. because the mere presence of m. pneumoniae in the upper respiratory tract is neither indicative nor predictive for respiratory disease, the routine diagnostic procedures to detect acute respiratory infections with m. pneumoniae need to be reconsidered. an overview of diagnostic tests with their advantages and drawbacks is shown in table . current guidelines (bradley et al., ; harris et al., ) recommend pcr and single-sample serological tests to diagnose m. pneumoniae infections. the sensitivity of serological tests depends on the time point of the first serum and on the availability of paired sera for seroconversion to igg and/or rise in antibody titer. specific serum igm emerges within week after initial infection and about weeks before igg (meyer sauteur et al., b) . although specific serum iga arises even earlier than igm, it could be detected only in % of pcrpositive children with symptomatic respiratory tract infection (spuesens et al., ) . cross-reactions with other pathogens and non-infectious disease has been described for cft and particle agglutination assay, but also some eias lack the required sensitivity and specificity (beersma et al., ) . further, it (loens et al., ) ; -epidemiological differentiation of clinical strains on the basis of differences in the p gene by pcr (spuesens et al., ) or in the number of repetitive sequences at a given genomic locus by multiple-locus variable-number tandem repeat analysis (mlva) . -"gold standard": fourfold titer increase as measured in paired sera. high sensitivity, high specificity ad -confirmatory assay . less sensitive and less specific than eia ad -subjective interpretation. adapted from meyer sauteur et al. ( b) . ad, advanced diagnostic test; cft, complement fixation test; csf, cerebrospinal fluid; eia, enzyme immunoassay; ifa, immunofluorescent assay; ig, immunoglobulin; pa, particle agglutination assay; pcr, polymerase chain reaction; rd, routine diagnostic test; repmp, repeated m. pneumoniae dna. largely replaced by eia; for the evaluation of an intrathecal antibody synthesis (granerod et al., ) , either by calculation of an antibody index (reiber, ) or through parallel immunoblotting of simultaneously collected csf and serum samples (monteyne et al., ) . frontiers in microbiology | www.frontiersin.org is intriguing that the detection of igm, as well as igg and iga by eia could not discriminate between the asymptomatic and symptomatic groups of children (spuesens et al., ) . the demonstrated positive serological results in asymptomatic m. pneumoniae pcr-positive children (n = ; igm in %, igg in %, and iga in %) may simply reflect one or more previous encounters with m. pneumoniae and are not necessarily related to the presence of m. pneumoniae in the respiratory tract. thus, it is questionable whether or not a positive result in these tests actually indicates the etiological role of m. pneumoniae in all cases. in that sense, the positive predictive value of these tests may be overestimated, whereas the negative predictive value may be acceptable (bradley et al., ) . the "gold standard" for diagnosis of m. pneumoniae infections is still considered to be a fourfold increase in antibody titer as measured in paired sera (gardiner et al., ) . however, the use of convalescent sera is not useful in clinical practice because it is too time-consuming and does not allow clinicians to initiate treatment protocols in a timely fashion. clinicians therefore need to be aware of the implications and clinical significance of a positive pcr or serology test result. while diagnostic tests may not be reliably predictive for a symptomatic m. pneumoniae infection, the clinical assessment of this entity is being revisited. the british thoracic society guidelines recommend that bacterial pneumonia should be considered in children when there is persistent or repetitive fever > . • c together with chest recession and a raised respiratory rate (harris et al., ) . a chest radiograph should not be considered a routine investigation in children thought to have cap. in fact, although bilateral, diffuse infiltrates are common, none of the radiographic findings associated with m. pneumoniae cap is specific (john et al., ) . a fast-and-frugal clinical decision tree provided a rapid probability estimate of the cause of cap in children ( months- years; - (fischer et al., ) . m. pneumoniae infection was diagnosed in % (n = ) of these children by pcr in respiratory specimens and serology (seroconversion and/or fourfold rise in antibody titer). compared with other children with cap, patients with m. pneumoniae were older and had a longer duration of fever (p < . ). asking the simple question regarding the age of the child and the duration of fever allowed identification of the following group at high risk for cap due to m. pneumoniae: children with cap who have had fever > days and who were > years of age. the score model placed % of all patients with m. pneumoniae infection into the high-risk group (figure ) . these simple rules may further aid physicians in prescribing appropriate first-line antibiotics. in consequence of the diagnostic uncertainty for m. pneumoniae infections, the british thoracic society guidelines suggest empiric macrolide treatment at any age if there is no response to first-line β-lactam antibiotics or in the case of very severe disease (harris et al., ) . the lack of a cell wall makes (eshaghi et al., ) ; europe: the netherlands ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) : % ( / ) (spuesens et al., ), germany ( - : % ( / ) (dumke et al., ), france ( : % ( / ) (peuchant et al., ) , italy ( ): % ( / ) ), scotland ( (ferguson et al., ), switzerland ( - : % ( / ) (meyer sauteur et al., a) , england and wales ( - ) : % ( / ) (brown et al., ) . m. pneumoniae resistant to cell wall synthesis inhibitors such as β-lactam antibiotics. the antibiotics with the best minimum inhibitory concentration values against m. pneumoniae include macrolides, tetracyclines, and fluoroquinolones (waites and talkington, ) . although the latter two have a good in vitro inhibitory effect against m. pneumoniae, tetracyclines may cause teeth discoloration (waites and talkington, ) and fluoroquinolones may affect the developing cartilage in young children (adefurin et al., ) . thus, they are not recommended by current guidelines in young children; the age limit for tetracyclines is ≥ years, while that of fluoroquinolones is adolescence with skeletal maturity (bradley et al., ) . the occurrence of arthropathy due to fluoroquinolones, however, is uncertain, and all musculoskeletal adverse effects reported in the literature had been reversible following withdrawal of treatment (adefurin et al., ) . the protein synthesis inhibitors of the macrolide class have a more favorable side effect profile and are therefore the first-line antibiotics for m. pneumoniae infections in children (bradley et al., ) . although antibiotics are effective against m. pneumoniae in vitro (bebear et al., ) , there is lack of evidence on their in vivo efficacy. observational data indicated that children with cap due to m. pneumoniae have a shorter duration of symptoms and fewer relapses when treated with an antimicrobial agent active against m pneumoniae (mccracken, ; waites and talkington, ) . a recent cochrane review evaluated seven studies on the effectiveness of antibiotic treatment for m. pneumoniae lower respiratory tract infections in children (gardiner et al., ) . however, the diagnostic criteria, the type and duration of treatment, inclusion criteria, and outcome measures differed significantly, making it difficult to draw any specific conclusions, although one trial suggested that macrolides may be efficacious in some cases (esposito et al., ) . it is clear that studies on the efficacy of antibiotics rely on a correct diagnosis of m. pneumoniae infections. given the aforementioned shortcomings of current diagnostic tests, conclusions on the efficacy of antibiotic treatment will have to be reexamined. since , the extensive macrolide use led to an alarming worldwide increase in the prevalence of macrolide-resistant m. pneumoniae (mrmp) strains (bebear et al., ) . resistance is based on specific point mutations in domain v of the s rrna (at positions , , and ), which reduce the affinity of macrolides to the large subunit ( s) of the bacterial ribosome (bebear et al., ) . mrmp has been observed during macrolide treatment as a result of antibiotic selective pressure (cardinale et al., ; chironna et al., ; saegeman et al., ) . to date, macrolide resistance has been detected on a worldwide scale. mrmp had developed in asia (hong et al., ) , where mrmp rates have risen as high as % in china (zhao et al., ) . mrmp has now also been reported from north america and europe (figure ) . the clinical relevance of macrolide resistance in hospitalized children with cap may lie in prolonging the symptoms of the disease (okada et al., ; cardinale et al., ; zhou et al., ) . zhou et al. ( ) found that an increase in mrmp may also have serious clinical consequences in children, leading to more severe radiological findings of cap and even an increase in extrapulmonary manifestations. in this study, hospitalized children with cap due to mrmp developed more often extrapulmonary disease than children with cap caused by macrolide-sensitive strains ( % vs. %; p = . ) (zhou et al., ) . these manifestations included skin diseases and nervous system complications in % and %, respectively, of the mrmp-infected children. serum inflammatory cytokine levels (inf-γ, il- , and ip- ) were higher in patients infected with mrmp than in patients infected with macrolide-sensitive strains (matsuda et al., ) . this suggests that the higher and more persistent inflammatory stimulation by mrmp may increase the possibility of severe lung lesions and extrapulmonary complications. while previous attempts to produce vaccines on the basis of inactivated bacteria resulted in limited efficacy against cap and various adverse effects (linchevski et al., ) , the recent use of recombinant proteins as potential vaccines was found to be promising: the immunization of mice with a immunogenic recombinant protein encompassing the c-terminal part of the p protein (rp ) induced strong mucosal and systemic antibody responses against m. pneumoniae, and reduced lung inflammation in infected mice (zhu et al., ) . another study showed that immunization of guinea pigs with a chimeric protein consisting of rp and the p adhesion protein of m. pneumoniae resulted in a robust antibody response that led to a reduction in bacterial loads in the respiratory tract (hausner et al., ) . the increasing prevalence of mrmp has become a significant issue, since mrmp can potentially cause more severe and even extrapulmonary disease. because symptoms and radiologic features of m. pneumoniae cap seem to be unspecific and current diagnostic procedures cannot discern between carriage and infection in a 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complications occurred in macrolide-resistant mycoplasma pneumoniae pneumonia protective immune responses in mice induced by intramuscular and intranasal immunization with a mycoplasma pneumoniae p c dna vaccine we thank steve gschmeissner (bedford, uk) for performing scanning electron microscopy of the m. pneumoniae strain. the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © meyer sauteur, unger, nadal, berger, vink and van rossum. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- -ud lhkyi authors: fecchi, katia; anticoli, simona; peruzzu, daniela; iessi, elisabetta; gagliardi, maria cristina; matarrese, paola; ruggieri, anna title: coronavirus interplay with lipid rafts and autophagy unveils promising therapeutic targets date: - - journal: front microbiol doi: . /fmicb. . sha: doc_id: cord_uid: ud lhkyi coronaviruses are enveloped, single-stranded, positive-sense rna viruses that can infect animal and human hosts. the infection induces mild or sometimes severe acute respiratory diseases. nowadays, the appearance of a new, highly pathogenic and lethal coronavirus variant, sars-cov- , responsible for a pandemic (covid- ), represents a global problem for human health. unfortunately, only limited approaches are available to treat coronavirus infections and a vaccine against this new coronavirus variant is not yet available. the plasma membrane microdomain lipid rafts have been found by researchers to be involved in the replication cycle of numerous viruses, including coronaviruses. indeed, some pathogen recognition receptors for coronaviruses as for other viruses cluster into lipid rafts, and it is therefore conceivable that the first contact between virus and host cells occurs into these specialized regions, representing a port of cell entry for viruses. recent data highlighted the peculiar pro-viral or anti-viral role played by autophagy in the host immune responses to viral infections. coronaviruses, like other viruses, were reported to be able to exploit the autophagic machinery to increase their replication or to inhibit the degradation of viral products. agents known to disrupt lipid rafts, such as metil-β-cyclodextrins or statins, as well as autophagy inhibitor agents, were shown to have an anti-viral role. in this review, we briefly describe the involvement of lipid rafts and autophagy in coronavirus infection and replication. we also hint how lipid rafts and autophagy may represent a potential therapeutic target to be investigated for the treatment of coronavirus infections. coronaviruses (covs) are a large group of enveloped animal and human viruses, with a single-stranded positive-sense rna genome. the coronaviridae family, to which they belong, includes four genera of covs, indicated as alphacoronavirus (αcov), betacoronavirus (βcov), gammacoronavirus (γcov), and deltacoronavirus (δcov) (cui et al., ; chen et al., ) . αcov and βcov have evolutionary evolved from bats and rodents, and have been responsible for human infections; whereas δcov and γcov derive from avian species (woo et al., ) . αcov and βcov are further divided into subgroups, a- b and a- d, respectively, (graham et al., ; drexler et al., ; cui et al., ; fung and liu, ) (supplementary figure s ) . coronaviruses have been regarded as etiologic agents of both relatively mild and severe respiratory infections/diseases in humans (zhao et al., ; paules et al., ) . hcov-nl , hcov- e, hcov-oc , and hcov-hku cause the common cold and mild upper respiratory diseases in immunocompetent hosts, although some of them can be potentially virulent in infants, young children, and elder individuals. highly pathogenic human coronaviruses, responsible for more severe respiratory diseases, include sars-cov that caused the pandemic outbreak of severe respiratory tract infection (ksiazek et al., ) , mers-cov, responsible for the outbreak in middle eastern countries (zaki et al., ) , and the novel sars-cov- , actually causing a pandemic of severe pneumonia that started in china in december (benvenuto et al., ; zhu et al., ) . coronavirus and coronavirus-like infections have been described also in domestic and wild animals, such as swine [porcine transmissible gastroenteritis virus and porcine epidemic diarrhea virus], cattle (bcov), horses, camels, cats, dogs, rodents, birds, bats, rabbits, ferrets, mink, and various wildlife species (fehr and perlman, ; lin et al., ; banerjee et al., ; cui et al., ; ye et al., ) . although many coronavirus infections are subclinical in animals, however, they cause a range of diseases that can have serious consequences for animal health as well as for the economic losses in livestock. in addition, domestic animals may have been important intermediate hosts for virus transmission from natural hosts to humans (fehr and perlman, ; ye et al., ) . since only limited approaches are available to treat or prevent coronavirus infections , the importance of identifying novel therapeutic targets is evident. viruses have evolved a close interplay with the host cell and the first encounter between the virus and target cell occurs at the plasma membrane. lipid rafts are specialized plasma membrane microdomains involved in important processes of the virus infections and of the host target cells (rosenberger et al., ) . viruses exploit lipid rafts to gain infection of the target cells; therefore, pharmacologic depletion or disruption of lipid rafts may provide a tool to reduce or inhibit viral replication (khandia et al., ) . furthermore, it has been widely demonstrated that lipid rafts play a fundamental role in autophagic machinery: they are associated with autophagosome morphogenesis, either in the initiation or in the maturation phases (matarrese et al., ) . autophagy, a cell process involved in cellular homeostasis, stress, and immune responses to viral infections, is a two-edged process in virus infections, since it may have pro-or antiviral roles (kudchodkar and levine, ; khandia et al., ) . viruses, on the other side, during co-evolution with the host cell, developed mechanisms to usurp/exploit the host autophagic system (jheng et al., ) . this minireview reports on the available knowledge about the interplay between coronaviruses, including the sars-cov- , with lipid rafts and autophagic pathways, in order to focus the attention to novel potential targets to inhibit coronavirus infections. lipid rafts are plasma membrane microdomains ( - nm) enriched in cholesterol, glycosphingolipids, and phospholipids. they are also found in the endoplasmic reticulum (er) (browman et al., ) , in the golgi complex (eberle et al., ) , and on the membrane of endosomes (sobo et al., ) and phagosomes (dermine et al., ) . there are two main types of lipid rafts based on their protein composition: "planar lipid rafts" and "caveolae" enriched by the proteins flotillin and caveolin, respectively (mcguinn and mahoney, ) . both possess a similar lipid composition that confers resistance to solubilization by non-ionic detergents at low temperatures (brown and rose, ) , and the characteristic of being able to be isolated in sucrose gradient (sargiacomo et al., ) . because of their dynamic and heterogeneous structure, they can rapidly assemble and disassemble, changing their composition in response to intra-and extracellular stimuli (simons and toomre, ) . a key component of lipid rafts is cholesterol that is the glue that maintains raft architecture, as demonstrated by the disorganization and disruption of lipid rafts upon depletion of plasma membrane cholesterol by the cholesterol depleting agent methyl-β-cyclodextrin (mβcd) (simons and ehehalt, ; zidovetzki and levitan, ) . these membrane regions play an important role in a variety of cellular functions, but principally they recruit and concentrate several signaling molecules (song et al., ; galbiati et al., ; razani et al., ; patel and insel, ) that, by interacting with caveolin and flotillin, form a sort of signal transduction platform. therefore, lipid rafts are involved in many biological functions including endocytosis, signal transduction, cell communication, and regulation of autophagy (nabi and le, ; shi et al., ) . because of their capacity to cluster into a "phagocytic synapse" several pathogen recognition receptors (toll-like receptors, c-type lectin receptors), lipid rafts are the focus of intense research in the field of infection. in particular, they are involved in several steps along a viral infection, such as virus entry into the host cell (fusion and internalization), viral protein transport, viral assembly, and budding processes (hogue et al., ) . several enveloped (hiv- , influenza viruses, coronavirus, flavivirus) and non-enveloped (sv and rrotavirus) viruses exploit the raft platform to bind to their specific receptors (ono and freed, ; pelkmans, ; li et al., ; takahashi and suzuki, ) . recent studies have implicated lipid rafts in coronavirus entry and egress through a multistep endocytic process, although the detailed mechanism remains to be disclosed (li et al., ) . figure depicts a schematic view of the role of lipid rafts in two paradigmatic human coronaviruses, hcov- e and sars-cov, infections. briefly, the receptors for the two coronaviruses, aminopeptidase n (apn/cd ) and angiotensin converting enzyme (ace ), respectively, are located into lipid rafts and play a fundamental role in the initial step of the virus infection. apn/cd , a zinc-binding aminopeptidase, is expressed in several cell types (endothelial, granulocytic, and monocytic cells; epithelial cells of the kidney; respiratory tract and intestine) and is required also for porcine, canine, and feline figure | schematic representation of the role of lipid rafts in coronavirus infection of the host cells is a multistep endocytic process characterized by a series of complex events tightly regulated in space and time. step entry process of coronavirus into the host cells is initiated by the binding of the spike glycoprotein with the specific receptor (ace , apn/cd ) located into lipid rafts/caveolae. this interaction causes conformational changes of the viral particle, which trigger specific signaling events necessary for the viral entry mechanism. step lipid rafts/caveolae-mediated endocytosis is followed by intracellular trafficking of virus particles in transport vesicles (early and late endosomes). the low ph in late endosomes induces a conformational change in coronavirus that mediates fusion of the viral envelope with the endosomal membrane. step viral genomes are translated in two polyproteins, pp a and pp ab, which encode the non-structural viral proteins that form the replication transcription complex. this complex produces genomic rna as well as multiple subgenomic mrnas encoding structural proteins. translation of mrna encoding for the nucleocapsid proteins occurs in the cytoplasm where the newly synthesized proteins interact with new genomes to form ribonucleoprotein particles. in contrast, matrix, envelope and spike proteins translation occurs into the er. coronavirus uses also the autophagy machinery for replication and has evolved strategies to avoid autophagy-induced lysosomal degradation. step after assembly the progeny viral particles, virus-containing vesicles (smooth-wall vesicles) are budded and released into the extracellular environment through fusion with the plasma membrane (exocytosis). alternatively, we speculate that coronavirus might utilize multivescicular bodies (mvbs) and take advantage of the exosomal pathway for egress. coronavirus recognition. ace , encoded on the x chromosome, is a metalloprotease long known to be a key player in the reninangiotensin system that co-localizes with caveolin- and gm and is expressed on type i and ii pneumocytes, enterocytes, endothelial cells of the heart and kidney, epithelial cells of the kidney, and the testis (hamming et al., ) . sars-cov- , the etiological agent of the covid- pandemic, also binds ace to infect host cells, by similarity to sars-cov, from which it differentiates for point mutations on the spike protein, essential for receptor binding yan et al., ) . by analogy to sars-cov, it can be hypothesized that sars-cov- may also use lipid rafts for its entry into the host cells. the steps following coronavirus entry are not clearly identified. a recent study demonstrated that sars-cov exploits the activity of cathepsin l, an endosomal cysteine protease, to initiate proteolysis and activation of membrane fusion within endosomes (simmons et al., ) . in addition, coronavirus mouse hepatitis virus (mhv) infection is inhibited when early endosome-associated proteins, rab and eea , are down-regulated by sirna (burkard et al., ) and infectious bronchitis virus (ibv) release of the nucleocapsid into the cytoplasm (wang et al., ) occurs through the fusion with endosome-lysosome membranes. these data suggest that coronaviruses exploit the early and late endosome compartment after viral entry, as several other viruses (adenovirus, human papillomavirus, polyomavirus, african swine fever virus, and influenza virus) (lakadamyali et al., ; sánchez et al., ; spriggs et al., ) . recent evidence shows that there is a close interplay between lipid rafts and autophagy during physiological cell function, as lipid rafts can regulate autophagy by interacting with autophagosomes and autophagy-related proteins, such as atg and atg (chen et al., ; garofalo et al., ) . in addition a functional and structural link between lipid rafts and autophagy comes from the evidence that chemical (e.g., mβcd, fumonisin b ) or biological (e.g., sirna inhibition of key enzymes involved in the sphingolipids metabolism) molecules capable of altering the chemical composition or molecular organization of lipid rafts have also a strong impact on autophagy (khandia et al., ) . autophagy is an evolutionarily conserved (from yeast to mammals), selective, and finely regulated process that generates atp and precursors for the synthesis of macromolecules (ktistakis and tooze, ) and is considered as a survival process put in place by cells under stressful conditions, such as pathogen microbial infections. it plays a key role in cellular homeostasis and is responsible for the turnover of cellular organelles (yu et al., ) , as its main function is to remove and recycle non-essential, damaged, or obsolete cellular components, such as whole organelles or macromolecules (mizushima and komatsu, ) . during autophagy, cytoplasmic portions are enclosed into double membrane bound vesicles (autophagosomes) that then fuse with late endosomes/lysosomes, whose contents are degraded by lysosomal proteases. autophagy has been shown to have additional function in innate immunity, by degradation of viruses, or intracellular pathogens, as well as by presenting pathogen components to the immune system (mao et al., ) . autophagy can have a pro-viral role, promoting virus infection and propagation, as well as an antiviral role, essentially depending on the virus strain, on the phase of infection, on the infected cell type, but also on the cellular microenvironment (lennemann and coyne, ) . during co-evolution with their natural hosts, viruses developed the ability to hijack autophagic mechanisms to their advantage by using them for immune escaping, or using autophagosomes as a replicative niche. however, data on autophagy interplay with coronaviruses are scant so far, related on no more than works published between and . in general, it is suggested that covs can interact with some components of the autophagic pathway in an opposite way with a dual effect: utilizing autophagy components to promote viral replication and/or to inhibit degradation of viral products through the autophagic pathway (cong et al., ) . in some coronaviruses, infection results are still contradictory, as in the case of mers-cov (corman et al., ) , which has been reported to induce phosphorylation changes in key kinases, such as akt and mtor, that regulate the early steps of autophagic process (kindrachuk et al., ) , thus stimulating autophagy in infected cells. in contrast, gassen et al. reported that mers-cov activates the skp kinase, consequently reducing beclin (becn ) activity and inhibiting the fusion of autophagosomes with lysosomes, which results in autophagy inhibition (gassen et al., ) . in general, it has been hypothesized that coronaviruses would induce accumulation of autophagic vacuoles to obtain a larger availability of the membrane structure necessary for their replication (gassen et al., ) , which is consistent to the observed co-localization of the rodent coronavirus, mhv, replication complex, with the autophagic proteins lc and apg (prentice et al., ) . likewise, the sars-cov takes advantage of autophagic pathway to replicate and transcribe its own genome, as suggested by inhibition of its replication through inactivation of gsk- (glycogen synthase kinase- ), a serine/threonine kinase that inhibits autophagy, through the mammalian target of rapamycin (mtor) complex (mtorc ) (wu et al., ; zúñiga et al., ) . a very recent study showed that sars-cov- , similar to mers-cov, strongly reduced the autophagic flux in infected cell lines downregulating the ampk/mtorc pathway, altered autophagy-relevant signaling, and also reduced autophagosome/lysosome fusion efficiency (gassen et al., ) . as a result, the sars-cov- virus could take advantage of the autophagy reduction, thus preventing viral product degradation and enhancing double membrane vesicles (dmv) availability, indispensable for their replication. the cell organelle most involved in the dynamic membrane changes is the er. it is therefore not surprising that some coronaviruses, through the insertion of their trans-membrane glycoproteins at the er level, are able to induce er stress. this is the case, for example, of the orf protein produced by porcine epidemic diarrhea virus (pedv), which induced er stressdependent autophagy in different porcine and human cell types inhibit viral entry/endocytosis nomura et al., ; thorp and gallagher, ; choi et al., ; glende et al., ; lu et al., ; guo et al., ; owczarek et al., ; szczepanski et al., ; baglivo et al., ; fantini et al., statins ( (zou et al., ) . in the same vein, mhv was observed to use the host machinery to export vesicular er to originate membranes for the genesis of dmv. as regards the non-structural protein (nsp) of the avian infectious bronchitis virus (ibv), a gammacoronavirus, in addition to providing membranes useful for the replication of the virus, it could prevent its degradation within the lysosomes, effectively escaping the autophagy-based cellular defensive mechanisms. similar properties have also been observed for mhv and sars nsp proteins and prrsv arterivirus nsp , nsp , and nsp (cottam et al., ) . coronaviruses may represent a threat for human and animal health for their potential to cross the species barrier, thus acquiring human virulence, as evidenced by the previous sars-cov and mers-cov outbreaks and by the ongoing covid- pandemic. notwithstanding, treatment and prophylaxis measures to control coronavirus infections are lacking so far, either in humans or in livestock animals. as outlined in this review, lipid rafts and autophagic pathways play a pivotal role in coronavirus infection, being critical for viral entry and replication, as well as for viral release from the host cells. actually, lipid rafts are the focus of intense research in the field of infection, and it is conceivable to consider targeting some lipid raft components in order to inhibit virus infection at the cell level. in particular, lipid raft disruption by cholesterol-depleting agents has been shown to inhibit infection of several microbes by blocking their entry into the host cells (nomura et al., ; thorp and gallagher, ; choi et al., ; riethmüller et al., ; glende et al., ; lu et al., ; guo et al., ; owczarek et al., ; szczepanski et al., ; abu-farha et al., ; baglivo et al., ; fantini et al., ; rodrigues-diez et al., ; table ). in human immunodeficiency virus (ono and freed, ) , herpes simplex virus, and rotavirus infections, mβcd, a widely used raft-disrupting agent, has been shown to affect virus entry, thereby reducing their infectivity (dou et al., ; wudiri et al., ) . in addition, in japanese encephalitis virus and dengue virus infection, disruption of lipid rafts by mβcd has been shown to decrease viral infection acting at both viral entry and intracellular replication step (lee et al., ) . β-cyclodextrins are extensively utilized in pharmaceutical formulations as excipients to enhance solubility, bioavailability, and stability of many drugs (loftsson and brewster, ) . in the light of the above-described antiviral activity, further studies on pharmacokinetic and safety of mβcd could foster its clinical application as an antimicrobial agent in humans. statins, used in human therapy for their ability to inhibit cellular synthesis of cholesterol, have also been reported to have an anti-viral effect, inhibiting infection of flavivirus, such as dengue virus (denv), hepatitis c virus (hcv), west nile virus (wnv), and zika virus (mackenzie et al., ; andrus and east, ; martínez-gutierrez et al., ; españo et al., ) , and their application to inhibition of the coronavirus infection would be worth considering. likewise, the pharmacological modulation of autophagic processes can represent an attractive therapeutic strategy for elimination of the viral pathogen or containment of the infection (rubinsztein et al., ; clark et al., ) . in fact, different drugs described as inhibitors or inducers of the autophagy that control host cell pathways process involved in coronavirus infection, have sparked interest for their potential antiviral activity (shakya et al., ; liu et al., ; xu et al., ; yang et al., ; table ). one of this, chloroquine (cq), and its derivative hydroxychloroquine (hcq), known as antimalarial drugs (o'neill et al., ) , which are able to inhibit autophagy by raising the lysosomal ph (golden et al., ) , have also been evaluated for hiv infection (romanelli et al., ) . very recently, clinicians have paid attention to cq and hcq as a possible treatment of patients infected by the novel emerged sars- cov- (clinicaltrials.gov, a,b,c,d,e,f,g,h,i,j) . this insight is also supported by some recent works, including a recent publication by gao et al. that indicates some positive effects of cq on the course of pneumonia associated with the infection and on the reduction in healing (cortegiani et al., ; gao et al., ; geleris et al., ; yu et al., ) . on the contrary, other studies have highlighted the ineffectiveness of these drugs both in the viremic phase and in respiratory complications (boulware et al., ; mahévas et al., ; rosenberg et al., ) . although some data, both referring to covid- and sars, have highlighted some positive effects of cq and hcq in the evolution of the disease, thus showing their therapeutic potential, at the moment, there are no data that can support the use of these drugs to control the entire pathological process related to sars-cov- (gies et al., ) . in the same vein, rapamycin, also known as sirolimus, an autophagy inducer already used as an immunosuppressant, has been tested, with some success, in the treatment of covid- (nct ) (clinicaltrials.gov, k). these cases may represent a repositioning of the drugs with clinical success in treatment areas beyond their original approved use. according to this, the antiviral in vitro activity of spermidine, niclosamide, and nitazoxanide (known autophagy inducers) vs. sars-cov- was recently reported (shakya et al., ; liu et al., ; xu et al., ; yang et al., ) . thus, a prophylactic approach to covid- using these drugs, which are well tolerated, clinically applied, or fda-approved compounds, would be rational (gassen et al., ) . importantly, treatments for emerging infections by targeting host cell pathways, rather than the infectious agent directly, or to complement antivirals with drugs that enhance host cell resistance mechanisms have thus become an active and promising therapeutic strategy. this strategy is even more important and urgent to be explored in the case of such potentially and suddenly pandemic virus family, as coronaviruses are. kf contributed to the design of this study, drafted the manuscript, and drew figures relative to lipid rafts. sa contributed to the conception of the idea and drafted the manuscript and figure relative to coronavirus description. dp conducted a wide literature search and drafted the manuscript on the lipid rafts section. ei drafted the manuscript on the autophagy section. pm provided autophagy expertise, contributed to draft and revision of the manuscript, and concurred with the final version of the review. mg provided lipid rafts expertise, and contributed to draft and review the manuscript and to the organization of the review. ar conceived the idea, drafted and revised the manuscript, coordinated the activity, and concurred with the final version of the review. all authors contributed to the article and approved the submitted version. the authors gratefully acknowledge the financial support obtained from the italian ministry of health (to mg), grant pe- - , and peretti foundation (to pm), grant . we thank massimo delle femmine for his support with the figures. the supplementary material for this article can be found online at: https://www.frontiersin.org/articles/ . /fmicb. . /full#supplementary-material figure s | taxonomy of covs classification scheme of some representative human (in red) and animal (in black) coronaviruses. abbreviations: fcov, feline coronavirus; tgev, transmissible gastroenteritis virus; prcv, porcine respiratory coronavirus; hcov, human coronavirus; pedv, porcine epidemic diarrhea virus; ro-batcov, rousettus bat cov; phev, porcine hemagglutinating encephalomyelitis virus; mhv, mouse hepatitis virus; bcv, bovine coronavirus; sars-cov, severe acute respiratory syndrome coronavirus; bat sarsr-cov, bat sars-like coronavirus; mers-cov, middle east respiratory syndrome coronavirus; bucov, bulbul coronavirus; pdcov, porcine deltacoronavirus; bwcov, beluga whale coronavirus; ibv, avian infectious bronchitis virus; tcov, turkey coronavirus. the role of lipid metabolism in covid- virus infection and as a drug target use 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and molecular mechanisms isolation of a novel coronavirus from a man with pneumonia in saudi arabia current status of potential therapeutic candidates for the covid- crisis antagonism of the interferon-induced oas-rnase l pathway by murine coronavirus ns protein is required for virus replication and liver pathology a novel coronavirus from patients with pneumonia in china use of cyclodextrins to manipulate plasma membrane cholesterol content: evidence, misconceptions and control strategies porcine epidemicdiarrhea virus orf proteincausesendoplasmicreticulum stress to facilitate autophagy coronavirus nucleocapsid protein facilitates template switching and is required for efficient transcription key: cord- - qi pbz authors: benej, martin; danchenko, maksym; oveckova, ingrid; cervenak, filip; tomaska, lubomir; grossmannova, katarina; polcicova, katarina; golias, tereza; tomaskova, jana title: quantitative proteomics reveal peroxiredoxin perturbation upon persistent lymphocytic choriomeningitis virus infection in human cells date: - - journal: front microbiol doi: . /fmicb. . sha: doc_id: cord_uid: qi pbz experimental data indicate that during persistent infection, lymphocytic choriomeningitis virus (lcmv) may both directly or indirectly modulate regulatory cellular processes and alter cellular functions that are not critical for survival, but are essential for cell homeostasis. in order to shed more light on these processes, two-dimensional differential in-gel electrophoresis ( d-dige) and maldi-tof tandem mass spectrometry were used to determine the proteome response of the hela cell line to persistent lcmv infection. quantitative analysis revealed differentially abundant proteins. functional analysis showed that lcmv-responsive proteins were primarily involved in metabolism, stress, and the defense response. among identified proteins, we discovered significant changes for peroxiredoxins, a family of antioxidant enzymes. decreased amount of these antioxidant proteins correlated with elevation of reactive oxygen species (ros) in infected cells. increased levels of ros were accompanied by changes in the pattern of telomere restriction fragments (trfs) in infected cells and mediated activation of hypoxia-inducible transcription factor- (hif- ) and phosphatidylinositol -kinase (pi k)/akt signaling pathways. moreover, treatment with antioxidants resulted in reduced levels of viral nucleoprotein, indicating a connection between ros-dependent signaling and viral replication. persistent viral infections are currently one of the most important global health problems in the world. understanding how viral persistence is initiated and maintained, and the pathological consequences of ongoing virus replication, is therefore of high importance. the prototypic mammarenavirus, lymphocytic choriomeningitis virus (lcmv), provides a suitable model system for investigation of the mechanisms of persistent viral infection, virushost interactions, and pathogenesis. studies using this viral model have resulted in significant progress in virology and immunology that is applicable to other human microbial and viral infections (oldstone, ; zinkernagel, ) . in addition, lcmv is a neglected human pathogen of clinical significance that may lead to severe disease and consequences after prenatal infection (barton and mets, ; mets et al., ; barton et al., ) , and life-threatening conditions in immunosuppressed individuals (fischer et al., ; palacios et al., ; macneil et al., ) . moreover, several mammarenaviruses, such as the lassa, junin, and machupo viruses, are associated with severe hemorrhagic fever disease with significant morbidity and mortality in humans, representing serious public health concerns in their endemic regions (geisbert and jahrling, ; buchmeier et al., ) . currently, supportive care and ribavirin (nucleoside analogue) are the only treatment options available (damonte and coto, ; parker, ) . therefore, considerable interest focuses on the development of therapeutic strategies against mammarenavirus infection and disease. understanding how viruses and the hosts interact and how their interactions change over time would help in the rational design of targeting compounds. mammarenaviruses are enveloped viruses with a bisegmented negative-strand rna genome and a non-lytic life cycle restricted to the cell cytoplasm. the s segment of the genome encodes the nucleoprotein (np) that encapsidates viral rna and the glycoprotein precursor (gpc). after post-translational modification, gpc yields gp and gp that mediate virus entry. the l segment encodes the viral rna-dependent rna polymerase (l) responsible for viral rna synthesis, and the zincbinding protein (z) important for viral budding (buchmeier et al., ) . mammarenaviruses are able to establish persistent infection in their natural hosts and in a number of mammalian cells in vitro. during persistence, mammarenaviruses continue to replicate and express viral proteins. at the same time, they interfere with normal host homeostasis, resulting in possible disease without the destruction of the infected cell. additionally, they actively suppress the host's immune response in order to avoid recognition and to allow the spread of infection (oldstone, ) . although research on lcmv has led to many insights into viral persistence, the answers to several crucial questions related to the principles, by which persistence is initiated and maintained, are still lacking. a highly suitable tool to study virus-host cell interactions is proteomics, since viral infection fundamentally affects host cell proteins. it does so at many functional levels, such as affecting the cell signaling pathways, cytokine and growth factor production, apoptosis, phagocytosis, protein degradation, and cytoskeletal rearrangement (coiras et al., ) . so far, several large-scale analyses of host proteome (bowick et al., (bowick et al., , (bowick et al., , , kinome (bowick et al., ) , and transcriptome (djavani et al., (djavani et al., , muller et al., ) have been performed to study the consequences of an acute mammarenavirus infection. moreover, a number of studies employed the proteomic approach to identify interacting partners of mammarenavirus proteins in human cells (khamina et al., ; king et al., ; iwasaki et al., ; loureiro et al., ; ziegler et al., ) . on the other hand, none of them has focused on the host cellular response during persistent infection. herein, we analyzed proteomic changes in a human cell line hela upon persistent infection with the mx isolate of lcmv using quantitative gel-based proteomics. our data reveal significant changes in the proteins associated with metabolism, antioxidant activity, protein folding, rna binding, and immune response. many of the differentially abundant proteins have not been reported previously as virus-responsive upon lcmv infection. thus, these proteins represent potential targets for further in-depth investigations. understanding their roles in persistent lcmv infection would reveal more about the complexity and dynamics of interactions between virus and host cells, and would benefit antiviral research. this study thus offers useful basis for further research into the function of human proteins in persistent infection. human cervical carcinoma cells (hela), human alveolar adenocarcinoma cells (a ) (atcc ccl- ), and baby hamster kidney fibroblast cells (bhk- ) (atcc ccl- ), were grown in dulbecco's modified eagle medium (dmem) containing % fetal calf serum (fcs) supplemented with µg/ml gentamicin in a humidified atmosphere with % co at • c. lcmv mx isolate was continuously propagated in persistently infected hela cells (designated hela-mx). the mx isolate represents a persistent form of the virus capable of propagation through cell-cell contacts without releasing infectious virions (gibadulinova et al., ; reiserova et al., ; tomaskova et al., ) . a cells were infected with mx isolate via cell-free extract from persistently infected bhk- cells (bhk-mx) as described in laposova et al. ( ) and subsequently passaged several times to allow virus spread. whole-proteome samples for d-dige analysis were prepared according to standard protocol. briefly, the cells were washed with ice-cold pbs, and µl of lysis buffer ( mm nacl; m tris, ph . ; % triton x- ; and . % sds) were added to the culture, followed by min incubation on ice. the lysed cells were scraped off, transferred to tubes, passaged through an insulin syringe and centrifuged at , × g for min at • c. next, nine volumes of precipitation solution (acetone:methanol : ) were added, and the resulting solutions were incubated overnight at − • c. the samples were then centrifuged at × g for min at • c. obtained pellets were air-dried and resuspended in µl of utc solution ( m urea, m thiourea, % chaps). final protein concentrations were determined using the bradford method (bio-rad). for the preparative gel, µg of each sample were mixed with the utc solution to the final volume of µl. an equal volume of loading/reswelling buffer [ mm dithiothreitol (dtt); % (v/v) immobilized ph gradient (ipg) buffer, ph - nl (ge healthcare), . % destreak reagent (ge healthcare); adjusted with utc solution to the final volume of µl] was added to the mixture. eighteen centimeters ipg strip with non-linear - ph range (ge healthcare) was passively reswelled in µl of the sample mixture overnight. isoelectric focusing was performed using the multiphor ii unit (ge healthcare), with the following -step protocol: ( ) v, h; ( ) v, h; ( ) v, h; ( ) v, h; ( ) v, h; and ( ) v, h. the current limit was set to ma, with a ramping voltage gradient between the steps. after separation in the st dimension, the strip was equilibrated in sds equilibration solution ( m urea; % sds; % glycerol; . mm tris-hcl ph . ) containing . % (w/v) dtt for min; followed by min incubation with sds equilibration solution in the presence of . % iodoacetamide (iaa). the equilibrated strip was placed on top of a % polyacrylamide gel cast between low-fluorescence glass plates (the backing plate being pretreated with bind silane solution; ml % ethanol, . ml mq water, . ml acetic acid and µl of bind silane, h incubation at room temperature) and fixed by adding . % agarose gel dissolved in mq water with a trace of bromophenol blue. separation in the nd dimension was carried out using the protean xl (bio-rad) chamber. the resulting glass-backed d gel was incubated × min in fixing solution ( ml ethanol, ml acetic acid, ml mq water), the solution being replaced between individual fixing steps. total proteins were stained by overnight incubation with sypro ruby protein gel stain (molecular probes) in the dark. the gel was washed × min in wash solution ( ml methanol, ml acetic acid, ml mq water) and × min in mq water prior to scanning. resulting d map of the proteome was visualized by the pharosfx molecular imager (bio-rad), using µm resolution and appropriate laser/filter wavelengths. in the first step, sample ph was adjusted to fit the range of . - . using m tris. fluorescent dyes cy , cy , and cy were used for minimal sample labeling (ge healthcare). fifty micrograms of each sample were labeled with pmol of either cy or cy , in biological triplicates, using the dye-swap technique between individual replicate pairs. for the internal standard, µg of each sample replicate was mixed with pmol of cy per sample. after min of on-ice incubation in the dark, the labeling reaction was stopped by adding / volume of mm l-lysine to the samples. after a short spin, the labeled mixtures were incubated on ice for min in the dark. subsequently, the samples for each replicate gel were mixed in the following manner cy :cy :cy : : , adjusted with utc solution to the final volume of µl and mixed with an equal volume of the loading/reswelling buffer. ipg strips with non-linear - ph range were passively rehydrated in µl of the sample mixtures overnight in the dark. the d separation protocol was identical to that of the preparative gel with three exceptions -(i) % polyacrylamide gel was cast between two low-fluorescence glass plates, not pretreated with bind-silan; (ii) the proteins in the analytical gels were not fixed, but directly scanned (iii) particular emphasis was put on working in the dark. obtained d gels were immediately scanned between two low-fluorescence glass plates using the pharosfx molecular imager at µm resolution and at appropriate excitation/emission wavelengths corresponding to cy , cy , and cy individual channels. quantitative gel analysis was carried out using decyder . software package (ge healthcare). for those proteins discovered to be different in mock-infected and lcmv-infected samples, analysis of significance was conducted using the student's t-test. only significantly different protein spots (p < . ), with an at least . -fold abundance difference (ratio of mock versus infected sample mean normalized spot signals) were regarded as upor down-regulated. these protein spots of interest were chosen for mass spectrometry detection. target spots of interest were excised from the preparative gel using exquest spot cutter (bio-rad) and destained. after reduction and alkylation, the gel plugs were digested overnight with sequencing grade modified trypsin (promega). the digested peptides were extracted with µl of % acetonitrile (merck) containing . % trifluoroacetic acid (tfa) (merck). samples with digested peptides were evaporated to µl in concentrator plus (eppendorf) and purified using µ-c ziptips (merck millipore). subsequently, µl of peptide mixtures and µl of . mg/ml chca matrix in % acetonitrile, . % tfa, and mm ammonium phosphate were pipetted onto µm anchorchip maldi target (bruker). data were acquired on ultraflextreme tof mass spectrometer (bruker) operated by flexcontrol . (bruker) in positive reflector mode. for each position on the target laser shots were summed in - m/z range. next, we selected the most intense precursor ions per sample for fragmentation with signalto-noise threshold . tandem mass spectra were recorded by accumulation of shots in lift mode using lid (laserinduced dissociation) mechanism. laser power was boosted by % without collision gas, and the detector voltage was increased by %. monoisotopic peak lists were generated in flexanalysis . (bruker) by snap algorithm. precursor spectra with signal-tonoise less than ten were removed, and the remaining masses were externally recalibrated. fragment spectra were smoothed using savitzky-golay algorithm (three cycles with . m/z width), the baseline was subtracted with tophat algorithm, and filtering was done on signal-to-noise level five. subsequent processing for protein identification was done through proteinscape . (bruker) interface connected to mascot server . (matrix science). fixed carbamidomethyl cysteine, variable oxidized methionine, single trypsin miscleavage, and appropriate mass tolerances ( ppm for precursor ions and . da for fragments) were specified. queries were done against homo sapiens proteins downloaded from uniprot in april , containing , sequences. protein identifications were accepted if the ion scores of at least two different matched peptides were higher than the probability of identity threshold ( at p < . ). the mass spectrometry proteomics data have been deposited to the proteomexchange consortium via the pride partner repository (vizcaino et al., ) with the dataset identifier pxd and doi: . /pxd . gene ontology annotation was acquired from the gene ontology project using the panther database for biological procedures and molecular function . functional enrichment analysis was performed by fisher's exact test with false discovery (fdr) correction, with fdr p < . considered significant (mi et al., ) . the protein-protein interaction network was analyzed using the string . database with default settings, while the interaction score was set to medium confidence ( . ) (jensen et al., ). equal amounts of proteins from untreated mock-infected and lcmv-infected cells or cells treated with mm n-acetyl-l-cysteine (nac) for h were separated in % sds-polyacrylamide gel, transferred onto a polyvinylidene difluoride membrane (immobilon-p; millipore), and probed with antibodies specific for lcmv np [in-house generated mab m (tomaskova et al., ) ], alpha-enolase (abcam #ab , : dilution), gp /hsp -beta (millipore #abf , : dilution), prdx (invitrogen #pa - , : dilution), phospho-akt (ser ) (cell signaling technology # , : dilution), akt (cell signaling technology # , : dilution), β-actin (cell signaling technology # , : dilution), and alpha-tubulin (abcam [ab ], : , dilution). detection was performed with hrp-or irdye-labeled secondary antibodies visualized with enhanced chemiluminescence or odyssey clx imager system, respectively. densitometric analysis of protein abundance was performed using imagej software (schindelin et al., ) or image studio lite software, respectively. the generation of reactive oxygen species (ros) was assessed using the fluoroprobe -(and- )-chloromethyl- , -dichlorodihydrofluorescein diacetate (cm-h dcfda). cells ( × cells/well) cultured in -well plates were washed with phosphate-buffered saline supplemented with calcium/magnesium (pbs) and incubated with µm cm-h dcfda (molecular probes) in pbs for min in the dark at • c. after replacement of the reactive agents with pbs, , -dichlorofluorescein (dcf) fluorescence was measured at an excitation wavelength of nm and an emission wavelength of nm using the synergy/h microplate reader (biotec instruments). values were corrected for background auto-fluorescence of non-stained cells and normalized to the number of cells assessed by dapi staining. briefly, at the end of the experiment, cells were fixed with ice-cold methanol for min at − • c, washed twice with pbs and then incubated with dapi ( µg/ml) for min in the dark. after a subsequent washing step with pbs, dapi fluorescence was measured in each well using nm excitation and nm emission wavelengths. the genomic dna (gdna) was isolated from hela and hela-mx cells using qiaamp dna mini kit (qiagen) according to manufacturer's instructions. three µg of genomic dna were digested using a mixture of restriction enzymes (hhai, hinf , mspi, haeiii, rsai, alui) as described by mender and shay ( ) . the dna fragments were separated in % agarose gel for h at . v/cm and stained with . µg/ml ethidium bromide solution for min (stained gel served as a loading control). the gel was then incubated for min in denaturation solution ( . m nacl, . m naoh), min in neutralization solution ( . m nacl, . m tris, ph . ) and min in × ssc ( m nacl, . m na-citrate, ph . ). the dna was then transferred to immobilon ny + membrane (emd millipore) with a vacugene xl blotter (ge healthcare) in × ssc and fixed by incubating the membrane at • c for h. the membrane was pre-hybridized for min at • c in hybridization buffer ( × ssc, × denhardt solution, . % sds) and hybridized at • c overnight in the same buffer containing radioactively labeled telomere-specific c-rich probe prepared as described by mender and shay ( ) . the membrane was washed once with wash buffer i ( × ssc, . % sds) for min at • c, twice for min in wash buffer ii ( . × ssc, . % sds) at • c and twice for min at room temperature with wash buffer iii ( . × ssc, % sds). the signal was detected by personal molecular imager fx (biorad). hela and hela-mx cells were seeded into a -well plate at × cells per well and transfected with µg of the luciferase vector (hre-luc) containing hypoxia-responsive elements the next day, using the turbofect transfection reagent (thermo fisher scientific) according to the manufacturer's instructions. cells were co-transfected with ng of prl-tk renilla vector (promega) to normalize for the transfection efficiency. luciferase reporter construct containing three hypoxia response elements (hre; -mers) from the pgk- gene was a gift from navdeep chandel (emerling et al., ) (addgene plasmid # ; rrid:addgene_ ). reporter gene expression was assessed h after transfection using the dual luciferase reporter assay system (promega) and the synergy ht reader with gen software (biotec instruments). luciferase activity was normalized against renilla activity. figure | preparative d gel. a total of µg proteins from each sample of lcmv-infected and mock-infected hela cells were resolved by d page. the protein spots were visualized by sypro ruby protein gel stain. this preparative d-gel was used to cut out protein spots that were differentially abundant in analytical gels. arrows indicate the isolated and identified protein spots with at least . -fold up-regulation or down-regulation. spots are numbered according to table . statistical analyses were performed using graphpad prism software for windows, unless stated otherwise. two-tailed unpaired t-test was used to analyze significant differences between two cell groups and one-way anova was used to determine statistically significant differences between three or more independent groups, with p < . considered significant. to investigate global protein changes in hela cells during persistent infection with the mx strain of lcmv, equal amounts of total proteins prepared from mock-infected and lcmv-infected hela cells were subjected to d-dige analysis. we used three independent biological replicates to generate comprehensive and reliable data (supplementary figure s ) . the separation strength was satisfactory; the average yield per gel comprised approximately protein spots. initial betweengel matching was carried out by manual landmarking, followed by automatic matching and extensive manual evaluation of the matched spots between individual gels in the decyder d . software. only protein spots showing significance (p < . ) and at least . -fold difference in abundance were considered as up-or down-regulated. according to our analyses, the abundance of protein spots was significantly altered after lcmv infection. since decyder does not allow identification of proteins present only in one condition and absent in the other, we complemented gel analysis with a manual qualitative study of protein presence between individual samples. in total, protein spots were present only in one condition (mockinfected or lcmv-infected). these protein spots were marked as "proteins of interest (pois)" and matched against the sypro ruby-stained preparative gel. the proteins of interest, which we were able to map on the preparative gel, were excised and analyzed by maldi-tof tandem mass spectrometry. twenty-four protein spots with non-redundant proteins were successfully identified, including up-regulated and down-regulated proteins (figure ) . detailed information on the identified proteins is provided in table . several proteins were present in more than one spot, namely: heterogeneous nuclear ribonucleoprotein k (hnrnp k; spot and , both more abundant upon infection), mitochondrial kda heat shock protein (hsp ; spot and , both less abundant in cells affected by persistent virus), and keratin, type ii cytoskeletal (ck- ; spot and , which showed opposite changes). these proteins are most likely different post-translational or alternatively spliced variants. proteins present only in one condition were: (a) unique upon lcmv infection -mitochondrial hydroxymethylglutaryl-coa synthase, guanine deaminase, annexin a , and rab gdp dissociation inhibitor beta; (b) unique in the mock-infected cell line -cellular retinoic acid binding protein , prostaglandin e synthase , and eukaryotic initiation factor a-i. apart from proteins discussed in more detail below, other accumulated proteins upon infection included galectin and macrophagecapping protein, and less abundant endoplasmin, gtp-binding nuclear protein ran, peptidyl-prolyl cis-trans isomerase fkbp , and adenine phosphoribosyltransferase. to better understand the implications of the cellular response to lcmv infection, the corresponding biological functions of the differentially regulated proteins were categorized according to the gene ontology database, using the panther classification system. most of the proteins with significantly altered abundance were involved in metabolic processes ( . %; proteins), particularly lipid and glucose metabolism; cellular processes, such as cellular communication and cell cycle ( . %; proteins); immune system processes ( . %; proteins); developmental processes ( . %; proteins); localization ( . %; proteins); and cellular component organization or biogenesis ( . %; proteins) (figure ) . functional enrichment analysis revealed that the most overrepresented were proteins with peroxiredoxin activity (go: ; raw p-value = . e- ; fdr = . e- ) and proteins that play a role in rna binding (go: ; raw p-value = . e- ; fdr = . e- ). furthermore, network analysis was conducted to reveal protein-protein interactions. forty-two associations were observed in the protein network analysis generated by string . database. the network had significantly more interactions than expected randomly (six expected interactions; ppi enrichment p-value < . e- ). such an enrichment indicates that the proteins are at least partially biologically connected. the confidence view of the protein-protein associations is shown in figure , where the strength of the association is indicated by the thickness of the connecting line (confidence score > . ). similar to gene ontology analysis, the most connected proteins have important functions in antioxidant activity, protein folding, and rna binding. to validate the changes in protein accumulation patterns in response to lcmv infection, two identified proteins, alphaenolase (eno ; more abundant) and heat shock protein kda beta, member [hsp b (endoplasmin); less abundant] were selected for western blot analysis. the reason to select hsp b was its important functionality, pinpointed by the string network analysis. figure a shows a prominent although not statistically significant increase in protein abundance of eno and a decrease for hsp b in infected cells. these results are consistent with those observed in the d-dige analysis. the densitometric analysis of three biological replicates revealed hela-mx/hela average ratio of . for eno and − . for hsp b , while alpha-tubulin was used as a loading control ( figure b) . the results were very close to the changes observed in the d-dige analysis, which were . for eno and − . for hsp b . in this study, we observed a decreased amount of peroxiredoxin (prdx ), peroxiredoxin (prdx ), and peroxiredoxin (prdx ) upon lcmv infection. peroxiredoxins (prxs) are members of an ubiquitous family of thiol-specific peroxidases that reduce mainly hydrogen peroxide (h o ) to water with the use of reducing equivalents provided through the thioredoxin system (wood et al., ) . to confirm the alteration in the abundance of peroxiredoxins during lcmv infection, we selected the most affected prdx for western blot analysis. figure a shows an almost identical decrease in the expression of prdx (− . ) in infected cells as we observed in the d-dige analysis (− . ). since one of the major functions of prxs is cellular protection against oxidative stress, we hypothesized that decreased levels of these antioxidant proteins may lead to an elevation of the ros content in infected cells. the measurement of ros generation in mock-and lcmv-infected cells supported our prediction. we revealed significantly higher production of ros ( . -fold) in lcmv-infected cells than in mock-infected cells ( figure b) . interestingly, another antioxidant enzyme, thioredoxin reductase (trxr ), was more abundant in infected cells (table ), yet this seems to be insufficient to counterbalance the decrease in the levels of peroxiredoxins. in order to determine whether the increased ros level described above is unique to the mx-infected hela cells or can also be seen in other cell lines, we decided to use a lung epithelial cells, which are commonly used as an in vitro model system for arenavirus infections. similar to hela cells, we detected a . figure s a) . further, we hypothesized that increased levels of ros may have an effect on dna of the infected cells. telomeric sequences composed of arrays of -ttaggg- repeats are particularly vulnerable to oxidative damage due to stretches of g residues that are susceptible to the conversion to -oxoguanine ( -oxog). accumulation of -oxog in telomeric repeats may affect accessibility of chromosomal ends to telomerase, binding efficiency of telomere-binding proteins, and/or formation of protective secondary structures such as telomeric loops (von zglinicki, ; ahmed and lingner, ) . as a result, increased levels of ros yield changes in the length of telomeric tracts. to test this hypothesis, we isolated genomic dna from control and infected cells and measured the length of telomere restriction fragments (trfs). we observed that the infected cells contained a more heterogeneous population of trfs and a higher proportion of shorter fragments compared with the control cells (figure ). this indicates that the infected cells have a compromised ability to maintain the standard length of telomeres. it has become apparent in recent decades that less-reactive ros, especially hydrogen peroxide, can function as intracellular signaling molecules regulating multiple physiological and pathological cell processes. it has been shown that ros mediate activation of several signaling pathways and transcription factors, including the pi k/akt signaling cascade (lee et al., (lee et al., , and hif (patten et al., ) . to assess biological implications of increased ros in infected cells, we investigated whether lcmv infection affected transcriptional activity of hif- . using a luciferase reporter containing hre, we detected a modest, but significant increase in hif- transactivation in infected hela cells in comparison to control cells ( figure a) . further, we evaluated activation of akt by probing cellular protein lysates for phospho-akt (s ), which is a marker for activated akt. figures b,c , phosphorylation of akt was induced in infected cells, while overall akt levels were similar in both infected and uninfected cells. since we assumed that activation of akt was triggered by redox signaling, we further investigated the effect of antioxidants on infected cells. we detected decreased levels of phospho-akt in lcmv-infected hela cells treated with n-acetyl cysteine. moreover, inhibition of ros accumulation by the antioxidant also decreased the levels of viral np (figures b,d) . in addition, we observed similar impact of antioxidant treatment in lcmvinfected a cells, although the reduction in np levels was less pronounced (supplementary figure s b) . . the signal obtained with anti-alpha-tubulin antibody was used as loading control. presence of np confirms lcmv infection. one representative of three biological replicates is shown. (b) densitometric analysis of protein abundance was performed using imagej software. relative quantity of proteins was calculated as the ratio of the intensity of each signal to the intensity of the related tubulin internal standard. values represent the means of three biological replicates. error bars denote the standard deviations. * * p < . (hela-mx vs. hela). these data suggest that lcmv maintains its replication in persistently infected cells by regulating redox signaling through modulation of local levels of h o and subsequent activation of cellular processes that it uses for its own benefit. viruses and host cells have established complex, dynamic interactions that either facilitate the efficiency of viral infection or defend the host against invading pathogens. these interactions are crucial for the regulation of metabolism and other processes in the host cell and their elucidation is critical for a detailed description of mechanisms of viral pathogenesis, eventually leading to effective treatment. in this report, we investigated the host response to persistent lcmv infection using d-dige-based proteomic approach. as the model system of persistent infection we used human hela cells long-term infected with lcmv mx isolate (over passages). the mx isolate likely represents a lcmv variant that has undergone complex adaptations. it is characterized by reduced accumulation of viral glycoproteins on the surface of . the signal obtained with anti-β-actin antibody was used as loading control. one representative of three biological replicates is shown. densitometric analysis of protein abundance (right) was performed using imagej software. relative quantity of proteins was calculated as the ratio of the intensity of each signal to the intensity of the related β-actin internal standard. values represent the means of three biological replicates. error bars denote standard deviations. * * p < . (hela-mx vs. hela). (b) ros generation was assessed in a microplate reader using dcf fluorescence and normalized to the number of cells measured by dapi staining. the results represent the mean from three independent biological experiments, each done in eight replicates. error bars denote standard deviations. data are presented as relative increase compared to control (hela cells), which was set to . * * * * p < . (hela-mx vs. hela). infected cells and by spreading to uninfected cells mainly through cell-cell contact without the release of infectious viral particles, all of which strongly resembles persistent lcmv neuronal infection in its native host (rodriguez et al., ) . the use of hela-mx cells has several advantages, such as the feasibility of use of a large number of cells for proteomic analysis, reproducibility, and viral persistence resembling a life-long chronic infection. however, in terms of natural lcmv infection, hela cells possess some limitations and one needs to be cautious about generalizing the results. therefore, we performed some of the experiments also on the a cell line, a model of type ii alveolar epithelial cells (supplementary figure s ) . this cell type is the first to be in contact with the virus during primary infection. it has also been shown that a cells are capable of antigen presentation (salik et al., ) . although the data demonstrating chronic infection in humans are not available, the persisting and figure | lmcv-infected cells exhibit changes in the pattern of telomeric restriction fragments. (a) total dna was isolated from uninfected (hela) and infected (hela-mx) cells, digested by a cocktail of restriction enzymes and the resulting dna fragments were separated by % agarose gel electrophoresis. (b) dna was transferred to nylon membrane and hybridized with a radiolabeled telomeric probe. trfs (telomeric restriction fragments), dna ladder (generuler kb dna ladder (thermo scientific). a representative of three independent experiments is shown. reactivated virus may be a source of unexplained complications (often fatal) in immunosuppressed transplant recipients, as well as in developing embryos (peters, ; bonthius, ). thus, a better understanding of cellular processes exploited or subverted by viruses during persistent infection can help develop new strategies to treat mammarenavirus infections in humans. our analysis revealed protein spots differentially accumulated upon persistent lcmv infection in hela cells. proteins were identified with high confidence. remarkably, the abundance of several antioxidant enzymes was shown to be significantly altered. namely, the levels of prdx , prdx , and prdx were . - . times lower in lcmv-infected cells (table ). in addition, functional enrichment analysis, as well as network analysis, pointed out that the antioxidant system was in the center of virusinduced host proteome response (figure ) . the key functions of prxs include cellular protection against oxidative stress and modulation of signaling pathways that use hydrogen peroxide as a second messenger (rhee et al., a,b) . a significant amount of published data suggests that prxs are hydrogen peroxide sensors that play a central role in redox signaling (rhee et al., ; latimer and veal, ; netto and antunes, ) . this is implicated in a number of biological processes, including growth factor signaling, cell differentiation, and cytokine production (hampton and o'connor, ) . regulation of prxs modifies the concentration of h o and thereby facilitates its signaling functions. we indeed confirmed a slight but significant elevation of the ros content alongside the downregulation of prxs by lcmv both in hela and a cells (figure b and supplementary figure s a) . ros, in the form of h o , can modify protein function, and/or structure through the mechanism of cysteine oxidation that influences a number of signaling cascades. for example, ros activate pi k either directly or by inactivating the phosphatase pten via oxidizing cysteine residues within its active site, resulting in an increased activation of akt and modulation of its downstream targets (lee et al., ; connor et al., ) . once activated, akt promotes cell survival, growth, metabolism, and proliferation by phosphorylating various effectors (ersahin et al., ) . in line with these findings, we observed enhanced levels of active akt in lcmv-infected cells. in addition, inhibition of ros by n-acetyl cysteine led to suppressed phosphorylation of akt in infected cells, suggesting that akt becomes activated in a ros-dependent manner (figures b,c) . notably, the treatment with antioxidants also resulted in reduced levels of viral np in hela, as well as in a lcmv-infected cells, suggesting a link between ros-dependent signaling and virus replication (figures b,d and supplementary figure s b ). these findings are in accordance with previous studies that have demonstrated that ros generated in response to lcmv play an important role in virus binding and subsequent virus replication (michalek et al., ) . moreover, it has been shown that the pi k/akt pathway plays an important role in different steps of the life cycle of a variety of viruses. for instance, infection with the new world mammarenavirus junin virus has been shown to induce the pi k/akt pathway, and inhibition of this pathway has resulted in a decreased infectious virus yield because of blocking the recycling of the transferrin receptor engaged in junv cell entry (linero and scolaro, ) . furthermore, pi k/akt pathway inhibition reduced budding and, to a lower degree, lcmv rna synthesis, but not cell entry (urata et al., ) . additionally, the sars coronavirus induced weak activation of akt that was crucial for the establishment of persistence (mizutani et al., ) . in contrast, pi k/akt pathway inhibition could not interfere with the establishment of persistent junv infection in vero cells, and this pathway was not crucial for the maintenance of junv persistence (linero and scolaro, ). data are presented as a relative increase compared to control (hela cells), which was set to . * * * * p < . (hela-mx vs. hela). (b) immunoblot analysis of p-akt (s ), total akt, and viral np with specific antibodies using whole-cell extracts prepared from untreated (−) mock-infected hela cells (hela) and lcmv-infected hela cells (hela-mx) or cells treated (+) with mm nac for h. the signal obtained with anti-β-actin antibody was used as loading control. one representative of at least four biological replicates is shown. (c) densitometric analysis of protein abundance was performed using imagej software or image studio lite software. relative quantity of p-akt was calculated as the ratio of the intensity of each signal to the intensity of the related signal for total akt. values represent the means of six biological replicates. error bars denote the standard deviations. data are presented as relative change compared to control (untreated hela cells), which was set to . * p = . (hela vs. hela-mx), * p = . (hela-mx vs. hela-mx nac), p = . (hela vs. hela nac). (d) densitometric analysis of protein abundance was performed using imagej software or image studio lite software. relative quantity of np was calculated as the ratio of the intensity of each signal to the intensity of the related β-actin or tubulin internal standard, respectively. values represent the means of four biological replicates. error bars denote the standard deviations. data are presented as relative change compared to untreated hela-mx cells, which was set to . * * p = . (hela-mx nac vs. hela-mx). reactive oxygen species also regulate the activation of several transcription factors, including hifs. hif- is the main hypoxiainducible transcription factor responsible for cell and tissue adaptation to low oxygen, by controlling cell metabolism, proliferation and survival, erythropoiesis and angiogenesis (semenza, ) . there is increasing evidence demonstrating that non-hypoxic stimuli can also activate hif- . it has been shown that the pi k/akt/mtor signaling cascade directly increases the expression of hif- encoding gene on the transcriptional and translational levels (dery et al., ) . any aberrant stimulation of this pathway leads to activation of hif- α, even in normoxic conditions. an increased ros production has been reported as essential for increased hif translation through the pi k pathway (iommarini et al., ) and for hif stabilization through the inactivation of prolyl hydroxylases in a nonhypoxic system (diebold and chandel, ) . accordingly, we demonstrated enhanced hif- transactivation in lcmv-infected cells (figure a) , as well as increased expression of eno (figure ) , a typical hif- target gene (semenza et al., ) . interestingly, hsp b expression was reduced by persistent lcmv (figure ) , which has also been previously reported in the case of hypoxia in min cells (bensellam et al., ) . pi k/akt signaling, as well as the hif transcription factors, have in common that they play an essential role in regulating cellular metabolism (dery et al., ; yu and cui, ) . in view of that, we observed that a large number of proteins identified in this study, such as the above mentioned alphaenolase or hydroxymethylglutaryl-coa synthase, are involved in metabolism, as indicated by the go analysis (figure ) . moreover, we have shown previously that the exposure of mx-infected hela cells to chronic hypoxia resulted in a hif-dependent increase of viral replication and enhanced formation of infectious virions (tomaskova et al., ) . in addition, there is accumulating evidence that many viruses, through various mechanisms, affect the hif- pathway, causing multiple downstream effects, such as modifying host cell metabolism, stimulating inflammation, and facilitating viral replication (morinet et al., ; cuninghame et al., ) . some viruses, such as the vaccinia virus and hepatitis c virus impair hif- α prolyl hydroxylation, which leads to its stabilization (nasimuzzaman et al., ; mazzon et al., ) . hepatitis b virus stabilizes hif- α by diminishing the interaction between vhl and hif- α (moon et al., ) . influenza virus a h n inhibits the proteasome and decreases fih- expression, thereby activating the hif- pathway by stabilizing hif- α (ren et al., ) . contrary to downregulated prxs, we observed a . fold increased abundance of cytoplasmic trxr (encoded by txnrd ) in lcmv-infected cells ( table ) . the thioredoxin (trx) system consisting of nadph, trxr, and thioredoxin, is a key antioxidant mechanism in protection against oxidative stress. it regulates protein dithiol/disulfide balance through its disulfide reductase activity (arner, ) . as already mentioned, trx provides electrons to peroxiredoxins to remove reactive oxygen and nitrogen species. trx reductase then reduces oxidized trx back using the reducing nadph equivalents. thus, trx reductase plays a crucial role in regeneration of a catalytically active form of prxs (lu and holmgren, ) . we can, therefore, speculate that elevated levels of trxr can maintain proper accumulation of local h o to ensure that it acts as a signaling molecule and does not cause oxidative damage. based on the abovementioned facts, it is possible that lcmv modulates the delicate interplay inside cells between oxidants and antioxidants, and determines the activity profile for a variety of proteins that maintain persistent infection (such as transcription factors, kinases and phosphatases, cytoskeletal proteins, or metabolic enzymes). on the other hand, elevated ros levels in infected cells can be a double-edged sword, as they can negatively affect genomic stability of host cells. telomeres, the nucleo-protein complexes at the chromosomal ends, are essential parts of the genome providing solutions to both end-protection and endreplication problems (shay and wright, ) . the solution to end-protection problem is provided by a specialized protein complex called shelterin (de lange, ) , in combination with the formation of a secondary structure called the telomeric loop (griffith et al., ) . the end-replication problem is solved by various means. in human germ or stem cells, and most cancer cells (including hela), telomeres are preserved by telomerase, a reverse transcriptase using an rna subunit as a template for extension of an array of -ttaggg- repeats (shay and wright, ) . guanine residues in telomeric repeats are particularly susceptible to oxidation resulting in their conversion to -oxog (oikawa and kawanishi, ) . accumulation of this oxidized purine compromises the telomere maintenance system in a quite complex way, including inhibition of binding of the shelterin components trf and trf , the formation of t-loop, and/or the inhibition of telomerase (ahmed and lingner, ) . thus, increased intracellular levels of ros often result in telomere shortening (von zglinicki, ) , as observed in this study in the case of hela cells infected by lcmv (figure ). furthermore, it was shown previously by the lingner group that telomeres physically interact with prdx and prdx (aeby et al., ) . although the levels of prdx were not substantially affected in hela-mx cells (data not shown), prdx exhibited a . -fold decrease ( table ) . as prdx was shown to interact with telomeres in the s phase of the cell cycle (aeby et al., ) , reduction in its amount in hela cells infected by lcmv may contribute to a detrimental effect of ros on the maintenance of their telomeric repeats. nevertheless, our data suggest that lcmv affects redox signaling and metabolic pathways that support an anabolic program required for efficient virus replication and/or maintenance of persistent infection. the precise mechanism of how lcmv regulates antioxidant-scavenging proteins in order to effectively manage amounts of ros still remains unknown and requires further investigation. although research on lcmv has led to many insights into viral persistence, the answers to several crucial questions about host-lcmv interactions during persistent infection are still lacking. this study analyzed proteomic changes in a human cell line upon persistent infection with lcmv using quantitative gelbased proteomics. we identified modulated host proteins, which play important roles in a number of cellular processes. uncovering their roles in persistent lcmv infection can broaden our understanding of the complex and dynamic virus-host cell interactions and be valuable for antiviral research. we provided experimental evidence that lcmv negatively affects the levels of antioxidant enzymes peroxiredoxins leading to elevated intracellular content of ros. these changes were accompanied by activation of hif- and pi k/akt signaling pathways as well as changes in the profile of telomeric restriction fragments. the treatment with antioxidants resulted in reduced level of viral nucleoprotein, indicating that virus replication and rosdependent signaling are interconnected processes. the datasets generated for this study can be found in the proteomexchange consortium via the pride partner repository, dataset identifier pxd . jt and lt conceived and designed the experiments. mb, md, and jt analyzed the data. jt wrote the first draft of the manuscript. mb, md, and lt wrote the sections of the manuscript. all authors performed the experiments, contributed to the manuscript revision, read, and approved the submitted version. this project was supported by grants / / , / / to jt, / / to tg, / / to lt from the scientific grant agency of the ministry of education of the slovak republic and the slovak academy of sciences, and apvv- - to lt from slovak research and development agency. we would like to thank dr. gabriela flores-ramirez from the department of rickettsiology, institute of virology, bmc, for her technical assistance. the supplementary material for this article can be found online at: https://www.frontiersin.org/articles/ . /fmicb. . /full#supplementary-material figure s | d-dige images of biological triplicates. a µg of each sample were labeled with pmol of either cy or cy dye. preferential binding of the dyes was evaluated using dye-swap technique between individual replicates of the analytical gel. in panels (a,c) the hela protein sample was labeled with cy dye (green channel) while cy labeling (red channel) was used for the hela-mx. panel (b) represents replicate gel no. where the hela sample was labeled with cy and the hela-mx was labeled with cy . intra-and inter-gel variations were normalized according to the internal standard consisting of all analyzed samples in ratio : labeled with cy dye. once normalized, these analytical d gels were used to analyze the differential abundance of proteins between hela and hela-mx samples. figure s | reactive oxygen species production and antioxidant treatment in lcmv-and mock-infected a cells. (a) ros generation was assessed in a microplate reader using dcf fluorescence and normalized to the number of cells measured by dapi staining. the results represent the mean from three independent biological experiments, each done in eight replicates. error bars denote standard deviations. data are presented as relative increase compared to control (a cells), which was set to . * * * * p < . (a -mx vs. a ). 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das, gitishree; sen, sandeep k.; shin, han-seung; patra, jayanta kumar title: endophytes: a treasure house of bioactive compounds of medicinal importance date: - - journal: front microbiol doi: . /fmicb. . sha: doc_id: cord_uid: zq hpjs endophytes are an endosymbiotic group of microorganisms that colonize in plants and microbes that can be readily isolated from any microbial or plant growth medium. they act as reservoirs of novel bioactive secondary metabolites, such as alkaloids, phenolic acids, quinones, steroids, saponins, tannins, and terpenoids that serve as a potential candidate for antimicrobial, anti-insect, anticancer and many more properties. while plant sources are being extensively explored for new chemical entities for therapeutic purposes, endophytic microbes also constitute an important source for drug discovery. this review aims to comprehend the contribution and uses of endophytes as an impending source of drugs against various forms of diseases and other possible medicinal use. plants have served as a source of medicinal bioactive compounds against numerous forms of ailments for centuries. ironically, in recent years, microorganisms associated with plants rather than plants themselves have proved to offer material and products with high therapeutic potential (subbulakshmi et al., ) . endophytes are an endosymbiotic group of microorganisms -often bacteria or fungi -that colonize the inter-and/or intracellular locations of plants (pimentel et al., ; singh and dubey, ) . for these organisms, all or part of their life cycle occurs within their hosts, without causing any apparent symptoms of disease. they are ubiquitous in nature and exhibit complex interactions with their hosts, which involve mutualism, antagonism and rarely parasitism (nair and padmavathy, ) . endophytes are known to enhance host growth and nutrient gain. they may improve the plant's ability to tolerate various types of abiotic and biotic stresses, and enhance the resistance of plants to insects and pests. they produce phytohormones and other bioactive compounds of biotechnological interest (enzymes and pharmaceutical drugs) (joseph and priya, ; parthasarathi et al., ) . researchers have indicated the presence of one or more types of endophytes in every single plant studied to date (strobel and daisy, ) . endophytes can colonize in the stem, roots, petioles, leaf segments, inflorescences of weeds, fruit, buds, seeds and also dead and hollow hyaline cells of plants (hata and sone, ; specian et al., ; stępniewska and kuzniar, ) . the population of endophytes in a plant species is highly variable and depends on various components, such as host species, host developmental stage, inoculum density and environmental condition (dudeja and giri, ) . however, very few studies have exploited these symbiotic groups of organisms and their bioactive metabolites. for the past few decades, it has become evident that the discovery rate of active novel chemical entities is declining. while plant sources are being extensively explored for the discovery of new chemical entities for various therapeutic purposes, endophytic microorganisms play an important role in this search for natural bioactive compounds, with potential use in the health sector and in drug discovery (lam, ) . this review highlights the various sources of endophytes, their secondary metabolites and role as source of drugs. such studies may improve the understanding of endophytes and address the need for new and useful compounds necessary to combat various pathogens associated with human health and other possible medicinal uses. numerous surveys, mostly on the pathogenicity interactions between plants and microorganisms associated with them, have been accomplished. however, after several explanations and studies on the role of microbial diversity associated with various plant species, it was assumed that only a small fraction of the microbes interacting with the plant are pathogenic in nature (andreote et al., ) . most of the microorganisms that inhabit plants play a major role in the plant's health and development, although, sometimes they are neutral (mendes et al., ; philippot et al., ) . it is considered that a single plant species could possess thousands of microbes, categorized as epiphytes (microbial inhabitants of the rhizosphere and phyllosphere; those near or on plant tissue) or endophytes (microbes residing within plant tissues in leaves, roots or stems), depending on their area of colonization in the plant species (oldroyd et al., ; turner et al., ; andreote et al., ) . apart from the disease-causing microorganisms, the presence of other (non-pathogenic) organisms inside the plants was first pronounced by de bary ( ), who detected the presence of microbial cells in the microscopically analyzed plant tissues. however, this observation continued to be unexplored until the definition of endophytes came into existence toward the end of last century. de bary ( ) provided the first definition of an endophyte, as "any organism that grows within plant tissues are termed as endophytes," however, the definition continues to change as per various researchers (wilson, ; hallmann et al., ; bacon and white, ) . petrini ( ) provided the most suitable definition for endophytes, which means any organism that at some part of its life cycle, colonizes the internal plant tissues without causing any type of harm to the host plant. furthermore, due to extensive studies of these groups of microorganisms, the endophytic communities have been divided into different subgroups, such as 'obligate' or 'facultative, ' which are associated with all types of plants (rosenblueth and martínez-romero, ) . endophytes that depend on the metabolism of plants for survival, being spread amongst plants by the activity of different types of vectors or by vertical transmission, are termed obligate endophytes (hardoim et al., ) . whereas, the facultative endophytes are those that live outside the host body during a certain stage of their life cycle and are mostly associated with plants from its neighboring soil environment and atmosphere (abreu-tarazi et al., ) . numerous attempts have been made during recent years to discover the origin of endophytic organisms in different species (hallmann et al., ; mitter et al., ) . initially, researchers choose the rhizosphere or the seed-born microbial communities as the major sources of endophytes. normally, the specific endophytes interaction with various plants and evidence of their strategy of existence and transmission is provided by their genome organization (andreote et al., ) . researchers have reviewed the genome sizes and origins of endophytes by correlating the genome size with the bacterial lifestyle (dini- andreote et al., ) . as per many researchers, the definition of endophytes could be suitable for the hypothesis that they live inside the plant species, where the environment is more stable compared to the soil, where the plant grows. however, there are also some endophytes that only appear in the plant during part of their lifecycle. thus, the endophytic community is made up of organisms from distinct origins, with those with larger genomes likely to live in variable environments, such as soils, while those with smaller genomes are likely to exist in the stable environment and are vertically transmitted (mitter et al., ) . endophytes are associated with plants in various forms, including bacteria (actinomycetes or mycoplasma) or fungi that have been colonized inside the plant tissues. more than genera from phyla of bacterial species have been reported to be associated with endophytes and among them, most of the species belong to the phyla actinobacteria, proteobacteria, and firmicutes (golinska et al., ) . the diversity of endophytic bacteria ranges from gram-positive to gram-negative bacteria, such as achromobacter, acinetobacter, agrobacterium, bacillus, brevibacterium, microbacterium, pseudomonas, xanthomonas etc. (sun et al., ) . bacterial endophytes are diverse in nature and are known to produce different bioactive metabolites that act as antimicrobial and anticancer compounds, for example, with % of them reported from the single genus, streptomyces (berdy, ) . actinomycetes are prokaryotic microorganisms that belong to the phylum actinobacteria and possess mycelium like fungus and forms spores (chaudhary et al., ; barka et al., ) . traditionally, these actinomycetes were considered transitional forms between the fungi and bacteria (barka et al., ) . however, the comparison of actinomycetes to fungi is only superficial because most of their properties are similar to those of bacteria but unlike bacterial cells, the cells of actinomycetes are thin with a chromosome that is organized in a prokaryotic nucleoid and a peptidoglycan cell wall. endophytic actinomycetes are known to produce various chemical entities with unique structures of considerable medicinal importance (gayathri and muralikrishnan, ; singh and dubey, ) . many antimicrobial compounds have been reported from various types of endophytic actinomycetes. streptomyces is one of the dominant genera, which is most commonly isolated as endophytic actinomycetes (zhao k. et al., ; golinska et al., ) . compounds of biological interest isolated from streptomyces sp. include munumbicins (a and b), naphthomycin (a and k), clethramycin, coronamycin, cedarmycin (a and b), saadamycin, and kakadumycins. other active compounds isolated from actinomycetes are paclitaxel extracted from kitasatospora sp. associated with taxus baccata, and tyrosol from emblica officinalis, which is suggested to inhibit food-borne microbes (zhao k. et al., ; gangwar et al., ; golinska et al., ) . mycoplasma species are also reported as plant endophytes. endophytic mycoplasma species were conveyed to be in a symbiotic relationship with some red algae, such as bryopsis pennata, b. hypnoides and also in arcobacte (hollants et al., ) . however, there is no confirmed evidence of its uses, the source of extraction or use against foodborne diseases or other pathogens. fungi are a heterotrophic group of organisms with various life cycles that include symbiotic relationships with a wide variety of autotrophic organisms (dayle et al., ) . endophytic fungi have been classified into two broad groups based on their phylogeny and life history traits. these include the clavicipitaceous, which infect some grasses confined to cool regions and the non-clavicipitaceous endophytes, which are from asymptomatic tissues of non-vascular plants, ferns and allies, conifers and angiosperms and are limited to the ascomycota or basidiomycota group (jalgaonwala et al., ; bhardwaj and agrawal, ) . endophytic fungi produce some of the most broadly used antibiotic and anticancer drugs. penicillenols, extracted from penicillium sp., is cytotoxic to numerous cell lines. taxol, isolated from taxomyces andreanae, is the most effective and successful anticancer drug extracted from endophytic fungi to date. clavatol (torreya mairei), sordaricin (fusarium sp.), jesterone (pestalotiopsis jesteri), and javanicin (chloridium sp.) are all known to possess strong antibacterial and antifungal properties against numerous foodborne infectious agents (jalgaonwala et al., ) . pestacin, isolated from p. microspora, has excellent antioxidant properties. though the endophytes were overlooked for a long time and are considered as pathogen-causing contaminations, many that inhabit inside the plants are often recognized as symbionts, with a distinctive and cherished interaction with the plants with whom they grow (mitter et al., ; berg et al., ) . recent studies have confirmed the occurrence of endophytes by various cultivation-independent assays and by fluorescence in situ hybridization-confocal laser scanning microscopy studies (mitter et al., ; berg et al., ) . cultivation-based techniques, use the recovery and testing of isolates, whereas cultivation-independent techniques screen for variations in the total endophytic communities (menpara and chanda, ) . endophytes can be easily isolated on any microbial or plant growth, such as agar, potato dextrose agar and any nitrogenor carbon-containing media. the most frequent method used to detect and enumerate endophytes involves isolation from surface-sterilized host plant tissue. the main factors that may regulate entophyte colonization within a plant or microbial species, include the genotype of the plant, the growth stage of the plant, the physiological status of the plant, the type of plant tissues, the environmental condition of the soil in which it is grown, the sampling season, the surface sterility, selective media and culture conditions as well as different agricultural practices (gaiero et al., ; golinska et al., ) ecological awareness on the role of endophytes in nature, can also provide the best clues for targeting a particular type of endophytic bioactivity with the greatest potential for bioprospecting (strobel and daisy, ) . endophytes are reported to produce a number of bioactive metabolites in a single plant or microbe which served as an excellent source of drugs for treatment against various diseases and with potential applications in agriculture, medicine, food and cosmetics industries (strobel and daisy, ; jalgaonwala et al., ; godstime et al., ; shukla et al., ) . these secondary metabolites were categorized into various functional groups, alkaloids, benzopyranones, chinones, flavonoids, phenolic acids, quinones, steroids, saponins, tannins, terpenoids, tetralones, xanthones, and many others (figures a,b) (schulz et al., ; strobel and daisy, ; jalgaonwala et al., ; joseph and priya, ; pimentel et al., ; godstime et al., ) . extraction of metabolites from endophytes is affected by various factors, such as the season of sample collection, climatic condition and geographical location . however, with a revolutionary synthetic process that has been developed during the past few years, extraction from plants and other natural sources has now become more feasible, efficient and convenient (hussain et al., ) . the production of bioactive substances by endophytes, has been directly associated with the evolution of the host microorganisms, which may have incorporated genetic information from higher plants, allowing them to better adapt to the host plant and perform some functions, such as protection from various types of pathogens, insects, and grazing animals (strobel, ) . some of the commonly found secondary bioactive compounds from endophytes are described below. taxol (paclitaxol), a complex diterpene alkaloid produced by the endophyte metarhizium anisopliae found in the bark of taxus tree, is one of the most promising anticancer yersinia enterocolitica swine meat and meat products, milk and dairy products joseph and priya, meca et al., frontiers in microbiology | www.frontiersin.org agents developed or synthesized to date (zhang et al., ; visalakchi and muthumary, ; jalgaonwala et al., ) . camptothecin, from nothapodytes foetida is known to have cytotoxic and antifungal properties (joseph and priya, ; han and rahman, ) . huperzine a (hupa), from huperzia serrata, can act as a cholinesterase inhibitor (nair and padmavathy, ) . lignans, such as cathartics, emetics and cholagogue, isolated from endophytic podophyllum hexandrum, are reported to act as anticancer agents (konuklugil, ) . resins, such as etoposide and teniposide extracted from p. emodi, possess strong anticancer activity (konuklugil, ) . compounds such as oxacillin, ampicillin, catechin, gallic acid, and cefalexin are known to possess bactericidal activities (akiyama et al., ) . terpenoids possess antineoplastic, antibacterial, and antiviral effects as well as gastrointestinal stimulation (jalgaonwala et al., ; godstime et al., ) . the endophytic fungus, cytonaema sp., produces triterpenoid helvolic acid with strong antibacterial activity (kumar et al., ) . infectious and parasitic diseases account for approximately half of the deaths worldwide (menpara and chanda, ) . although it is the generation of nano to pico drugs, natural sources have been proven as the best source for drug discovery. medicinal plants and their endophytes are an important source of precious bioactive compounds and secondary metabolites that contribute to more than % of the natural drugs available in the market (singh and dubey, ) . endophytic microorganisms are the storehouse of novel secondary metabolites that can serve as an excellent source of drugs for antiarthritic, antimicrobial, anticancer, antidiabetic, anti-insect, and immunosuppressant activities (jalgaonwala et al., ; godstime et al., ) . to date, only a few plants have been investigated for their endophytic diversity and potential to produce bioactive secondary metabolites. the discovery of novel antimicrobial secondary metabolites and bioactive compounds from different types of endophytic microorganisms is an important alternative to overcome the increasing levels of drugs resistance to various pathogenic microorganisms (godstime et al., ) . there are a number of bioactive compounds, such as camptothecin, diosgenin, hypericin, paclitaxel, podophyllotoxin, and vinblastine, which have been commercially produced by different endophytic fungi present in respective plants and they are of both agricultural as well pharmaceutical importance (joseph and priya, ; zhao et al., a) . these compounds are analogs of various types of phytohormones, essential oils etc. isolated from various endophytes (zhao et al., b; molina et al., ; nicoletti and fiorentino, ) . a detailed list of the endophytes isolated from various sources, their bioactive metabolites and the uses of endophytes as a source of medicine against various diseases is presented in table . plant roots and the gastrointestinal system of animals play a major role in the absorption of various nutrients and thus harbor a large, complex and dynamic group of microorganisms that help to degrade nutrients to more easily absorbed forms (ramirez-puebla et al., ) . the animal gastrointestinal system is inhibited by a number of microorganisms, starting from the archaea to the eukaryotes along with a number of plant-associated bacteria, particularly the endophytes (rosenblueth and martínez-romero, ; parniske, ; ramirez-puebla et al., ) . similarly, various soil types, moisture, plant genotypes, root lysates etc. are the determinants of the root microbiota (doornbos et al., ) . the diversity of microorganisms in healthy humans vary with diet, maintenance of hygiene, hormonal cycles, infections, uptake of medicine, sexual activity, etc. (muzny et al., ; gerber, ) . in this context, various studies associated with the microbiome of humans have been undertaken during recent times. a study from the human microbiome project has also stated the role of the gut microbiome in regulating the host circadian clock, which in mammals is located in the brain (leone et al., ) . these studies have provided evidence that a high-fat diet could alter the microbiome circadian rhythm, thereby suggesting a link between the diet, gut microbiota and obesity (leone et al., ) . furthermore, there are a number of microbiome studies undertaken to find out the associations and diversity of microbiota in both plant and animal systems. longitudinal microbiome studies are undertaken to gain insights into the dynamic behaviors of the microbiota, such as the microbial succession events during maturation of the infant's gut, normal temporal inconsistency in healthy adults, responses to various dietary changes and the use of different antibiotics and in instances of dysbiotic alterations that signal symptomatic diseases. furthermore, the multi-omic analyses that combine information from multiple data sources, such as metabolomes, proteomes and transcriptomes, provide a deep vision into the purposeful changes of the internal microbiome with respect to time. similarly, the computational tools to analyze microbiome time-series data is another area that shows tremendous growth. these techniques could model the inter-individual variability, while automatically capturing commonalities at appropriate levels in the ecosystems (gerber, ) . endophytes are a poorly investigated group of microorganisms capable of synthesizing bioactive compounds that can be used to combat numerous pathogens. these have been dependable sources of bioactive and chemically novel compounds and have proven to be useful for novel drug discovery. biotransformation methods have a wide range of uses, particularly in the production of numerous bioactive compounds, as antimicrobial (vanillin, essential oils), antifungal and antiviral (alkaloids), antioxidant(eugenol), antiinflammatory (cineole) etc. it is imperative to review and highlight the previous successes, ongoing research and latest developments in research associated with endophytic microorganisms to draw the attention of the research community toward this emerging field and possible exploitation of the available sources for their therapeutic uses in various fields, such as the medical, pharmaceutical, food and cosmetics. jkp, sg, gd, and ss wrote the manuscript. h-ss and jkp designed the concept, edited the manuscript. all the authors read and approved the manuscript. this study was supported by the agricultural research center, ministry of food, forestry, and fisheries, republic of korea. endophytic bacteria in long-term in vitro cultivated axenic pineapple microplants revealed by pcr dgge antibacterial action of several tannins against staphylococcus aureus exploring the potential of endophytes from medicinal plants as sources of antimycobacterial compounds exploring interactions of plant microbiomes microbial endophytes taxonomy, physiology, and natural products of thoughts and facts about antibiotics: where we are now and where we are heading unraveling the plant microbiome: looking back and future perspectives a review fungal endophytes: as a store house of bioactive compound diversity and versatility of actinomycetes and its role in antibiotic production angiosperm dna contamination by endophytic fungi: detection and methods of avoidance leipzig: hofmeister's handbook of physiological botany bacterial genomes: habitat specificity and uncharted organisms impact of root exudates and plant defense signaling on bacterial communities in the rhizosphere beneficial properties, colonization, establishment and molecular diversity of endophytic bacteria in legume and non-legume endophytes: exploitation as a tool in plant protection production and genetic improvement of a novel antimycotic agent, saadamycin, against dermatophytes and other clinical fungi from endophytic streptomyces sp. hedaya coronamycins, peptide antibiotics produced by a verticillate streptomyces sp. 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zhang, jingyuan; li, nan; chen, teng; wang, lidong; zhang, fei; mi, lijuan; zhang, jinxia; wang, shuchao; wang, ying; zhou, xintao; zhang, yanyan; li, min; zhang, shoufeng; hu, rongliang title: rapid and sensitive recombinase polymerase amplification combined with lateral flow strip for detecting african swine fever virus date: - - journal: front microbiol doi: . /fmicb. . sha: doc_id: cord_uid: c lszcu african swine fever virus (asfv), the etiological agent of african swine fever (asf), a hemorrhagic fever of domestic pigs, has devastating consequences for the pig farming industry. more than , , pigs have been slaughtered since august in china. however, vaccines or drugs for asf have yet to be developed. as such, a rapid test that can accurately detect asfv on-site is important to the timely implementation of control measures. in this study, we developed a rapid test that combines recombinase polymerase amplification (rpa) of the asfv p gene with lateral flow detection (lfd). results showed that the sensitivity of recombinase polymerase amplification with lateral flow dipstick (rpa-lfd) for asfv was copies per reaction within min at °c. the assay was highly specific to asfv and had no cross-reactions with other porcine viruses, including classical swine fever virus (csfv). a total of field samples were examined using our method, and the agreement of the positive rate between rpa-lfd ( / ) and real-time pcr ( / ) was %. overall, rpa-lfd provides a novel alternative for the simple, sensitive, and specific identification of asfv and showed potential for on-site asfv detection. african swine fever (asf) is a severe viral disease that manifests clinical symptoms of hemorrhagic fever caused by african swine fever virus (asfv) and can result in case fatality rates of up to % in domestic pigs, depending on the virus strain (galindo and alonso, ) . asf, which was first identified by montgomery ( ) in kenya in the s, made its first incursion into europe via two successive entries into portugal (in and again in ) , spreading rapidly throughout western europe and then to south america and the caribbean (michaud et al., ) . it was eventually eradicated by the mid- s, with the exception of sardinia (mur et al., ) . however, since the second major incursion of the disease, initially into georgia in , asf has spread to eastern europe and russia (rowlands et al., ; revilla et al., ) . the virus has continued to spread worldwide, including china. since the first asf case emerged in china in august , more than cases have been recorded in provinces (zhou et al., ) . african swine fever normally presents with non-specific symptoms, including fever, anorexia, vomiting, and diffused hemorrhage in superficial skin. post-mortem examination shows pericardial effusion, kidney enlargement, lymphadenectasis, and darkened and enlarged spleen. all these features are indistinguishable from those seen in classical swine fever virus (csfv) infection (tauscher et al., ; li and tian, ) . at present, no effective treatment or vaccine for asfv is available, and disease control is based mainly on animal slaughtering and strict sanitary measures (cisek et al., ; rock, ) . rapid laboratory diagnosis is important for timely triage and confirmation to control and preventing this disease because of its rapid progression to death and spread. molecular tools based on the detection of the genetic information of asfv have become more widely accepted for asf diagnosis (oura et al., ) . polymerase chain reaction (pcr) and real-time pcr techniques have provided a supportive method to post-mortem asf diagnosis (aguero et al., ; zsak et al., ; fernandez-pinero et al., ) ; however, they cannot be used for field (on-site) detection in pig farms. recently, liu et al. ( ) reported that the improved realtime pcr assay using a universal probe library (upl) probe could be applied to asfv molecular diagnosis under field conditions. due to limitations of the battery-powered realtime pcr instrument, it can process only a moderate number of samples. isothermal recombinase polymerase amplification (rpa) has been successfully used to detect multiple viral pathogens, including infectious bovine rhinotracheitis virus (hou et al., ) , bovine coronavirus (amer et al., ) , ebola virus (yang et al., ) , bovine viral diarrhea virus, or foot-and-mouth disease virus (wang et al., ) . as reports of asfv detection by rpa are limited, we aimed to develop and evaluate a rapid detection tool that combines immunochromatographic strip tests, more commonly referred to as lateral flow devices (lfds), with rpa targeting the conserved asfv p gene. this study was conducted as part of the surveillance of the asf outbreak in china. samples were collected for asf testing and surveillance under the agreement between the ministry of agriculture and rural affairs of the chinese government and farm owners. sample collecting treatment was conducted in accordance with the protocols for viral hemorrhagic fever under the urgent interim guidance for case management established by the world organization for animal health. the protocol for this study was approved by the ethics committee of the military veterinary research, academy of military medical sciences. samples were collected by animal centers for disease control and prevention of jilin province. the viruses were inactivated in the bsl- lab, and the inactivated samples were transferred to the bsl- lab for genomic dna extraction and detection. for standard plasmids, a plasmid extraction kit was used in accordance with the manufacturer's instructions (axygen, united states). for field samples, tissue homogenates were prepared in formaldehyde, and the inactivated virus homogenate was subjected to dna extraction by using a nanomagnetic bead adsorption kit (bailing biotechnology, beijing co., ltd., china). for the whole extraction procedure, lysis, adsorption, and washing were performed, and elution with µl of rnase-free water was conducted to dissolve the dna. the extracted dna was then stored at − • c. the p gene was first amplified in the rpa reaction system comprising the designed primers (labeled biotin) and a -carboxyfluorescein (fitc) labeled-probe, and the expected amplicons were labeled with -fitc and -biotin at the ends. then, the amplified products were recognized by anti-biotin and anti-fitc monoclonal antibodies on the test line, where gold nanoparticles were prefixed (figure ) . the whole genome sequence of a p genotype ii virus-asfv-sy , which was sequenced in our lab (genbank accession number mh )-was used as the reference sequence for rpa primer selection. rpa sense primer ( -aagaagaaagttaatagcagatgccgataccac- ), rpa anti-sense primer ( -biotin-gctcttacatacccttccacta cggaggcaatg- ), and rpa probe primer ( -fam-tgg gttggtattcctcccgtggcttcaaagcaaag[thf]taa tcatcatcgcac-p- ) were designed by using twistdx nfo rpa kits (cambridge, united kingdom) in accordance with the manufacturer's guidelines. taqman pcr was performed to detect asfv p in an mx p multiplex quantitative pcr system (stratagene) in accordance with the manufacturer's instructions. real-time pcr primers for p were used as described before (king et al., ) . the sequences spanning the rpa amplification region were obtained through pcr and cloned into the pmd -t plasmid to generate figure | schematic of rpa-lfd assay. frontiers in microbiology | www.frontiersin.org pmd -p . taqman runs of the experimental samples involved at least three replicates with no-template or no-primer control and the combination of the primers. the pcr conditions were • c for min and cycles of • c for s and • c for s. a standard curve was generated from serially diluted p recombinant plasmids of known copy numbers. a pair of primers and rpa probe primer was designed to specifically detect asfv circulating strains in china. the rpa assay was conducted in a µl system by using a twistamp nfo kit (twistdx tm , cambridge, united kingdom). all the reagents ( nm rpa primers, nm rpa probe, × rehydration buffer, and purified h o) except the template or the sample dna and magnesium acetate were prepared in a master mix, which was aliquoted into each tube of a . ml tube strip containing a dried enzyme pellet. magnesium acetate ( . µl) was pipetted slowly into the tube lids, and subsequently, µl of the sample dna was added to the tubes, the lids were closed, and magnesium acetate was centrifuged into the tubes by using a mini-spin centrifuge. the tubes were immediately used for isothermal amplification. asfv-rpa detection was combined with an lfd (yoshida, co., ltd., china). then, µl of the amplified product was added to the sample pad, and the dipstick was inserted into µl of the sample buffer for - min. a dilution range of to copies per reaction of pmd -p recombinant plasmid was used to evaluate the sensitivity of recombinase polymerase amplification with lateral flow dipstick (rpa-lfd), and the amplicons were evaluated through agarose gel electrophoresis. the reactions were evaluated with prrsv/ch r, csfv/shimen, prv/jl , jev/sa - - , pedv/cv , and pcv b-sd to examine the specificity of the developed rpa-lfd assay. the thermo-reaction procedures were optimized after different primers, probe combinations, and thermo-cycles were run in the real-time pcr system to evaluate the sensitivity of taqman asfv real-time pcr assay. the real-time pcr conditions that yielded the highest amplification efficiency were • c for min and cycles of • c for s and • c for s by using the primer combination reported by king et al. ( ) . the realtime pcr assay was sufficiently sensitive to detect copies per reaction (figure ) . no cross-reactions with prrsv/ch r, csfv/shimen, prv/jl , jev/sa - - , pedv/cv , and pcv b-sd were observed, and a positive signal was detected in asfv-sy and asfv-jl . in this study, copies of the asfv-positive pmd -p plasmid were used as a template to determine the optimal rpa reaction temperature. the reaction mixture was incubated at eight different temperatures ( • c, • c, • c, • c, • c, • c, • c, and • c) for min. the detection result showed that the test line was short at • c and • c, and the test line density did not enhance significantly from • c to • c ( figure a) . in this study, copies of the asfv-positive plasmid were used as a template to determine the optimal rpa reaction time. in figure b , the rpa assay tested positive for asfv in min. the measured dna band density revealed that the dna yield doubled after a reaction time of min and increased slightly after min. the intensity of the dna bands in agarose gel did not significantly change at reaction times of , , , and min. this finding indicated that the rpa reactions during the remaining parts of this study were completed after min. however, the false positive test lines appeared when the reaction time was more than min (invalid result). therefore, min was considered the optimum reaction time for the rpa-lfd assay, and the assay worked from • c to • c. in terms of convenience and safety, • c was chosen as the optimum reaction temperature. the sensitivity results showed that the detection limit of the asfv rpa-lfd assay was copies per reaction of the recombinant plasmid pmd -p . the amplicon in the asfv rpa-lfd assay ( bp) was also tested through subsequent visualization with % agarose gel-electrophoresis after purification, and the sensitivity of rpa based on gelelectrophoresis visualization was copies per reaction (figure ) . in terms of the specificity of asfv rpa-lfd assay, no cross-reactions with other important viruses of pigs were observed (figure ) , and this observation was confirmed by agarose gel electrophoresis. the samples included spleen samples and blood samples of animals (jilin cdc). of these samples, spleen tissues and blood samples were positive for asfv nucleic acid as detected by rpa-lfd. taqman real-time pcr showed that samples were positive. the rpa-lfd detection results were consistent with those of real-time pcr. rpa-lfd revealed that the samples showed a light test line and high cq values. of these samples, one tested sample, which showed with a short test line or no test line, was rotten, and the cq value in real-time pcr was , indicating weakly positive result. although the detection results between the two methods were consistent (table ) , the rpa-lfd assay was more rapid than real-time pcr. in particular, the whole rpa-lfd assay yielded results in min, whereas real-time pcr generated results approximately h later. the average time from symptom onset to outcome in asf is - days; however, the pig farms were usually in rural areas or even in a mountainous area far away from the city laboratory, which required more time for sampling and transportation. once diagnosis and timely treatment of suspected asf pigs is delayed, the risk of asf exposure to other pig farms was increased. in the current outbreak, laboratory testing with taqman real-time pcr is being widely used in affected areas. however, requirements-including a sophisticated thermocycler, complex sample treatment procedures, and expensive reaction instruments-have limited its applications in point-of-care testing (king et al., ) . different isothermal molecule amplification assays, including loop-mediated isothermal amplification assay (lamp) (james et al., ) , polymerase cross-linking spiral reaction (wozniakowski et al., ) , cross-priming amplification method (fraczyk et al., ) , chimeric dna/lna-based biosensor (biagetti et al., ) , and droplet digital pcr (wu et al., ) , have been developed as a rapid, simple, and cost-effective alternative to pcr-based molecule assay. rpa has several advantages. for example, initial heating for dna denaturation is not required, and test conditions ( • c within min) are easily implemented. lamp assay for asfv detection requires a long time ( min) and high temperature ( • c− • c) (james et al., ) . by comparison, rpa assay takes less than min at • c to complete. as such, rpa assay is simpler and more easily used than lamp assay. wang et al. ( ) reported the field validation of real-time rpa for asfv, although this technique can specifically detect asfv plasmid with a detection limit of dna copies per reaction. however, this detection assay is based on an expensive scanner device. detection methods for actual clinical samples have yet to be designed, so the application of this test to clinical samples is limited. gao et al. ( ) developed crosspriming amplification combined with immune-chromatographic strips for the rapid on-site detection of asfv, but its reaction conditions include min at • c and six primers for a reaction system, thereby causing non-specific amplification. the use of lateral flow assays combined with a monoclonal antibody against p protein of asfv can be used to detect p viral and recombinant protein or inactivated culture virus (sastre et al., ) ; however, the sensitivity of the assay is -fold lower than genomic amplification. recombinase polymerase amplification with lateral flow dipstick overcomes the technical difficulties posed by current amplification methods; for example, it operates at a low and constant temperature, does not require the thermal denaturation of templates, and does not rely on an expensive thermocycler (aguero et al., ) . in combination with a commercially magnetic nanobead-based dna extraction kit, this approach can be applied to on-site testing or rapid diagnosis under poorly equipped conditions. rpa is highly resistant to crude samples, suggesting that it can be used for on-the-spot field testing with crude nucleic acid extraction. in an rpa-nfo reaction system, nfo cleaves the primer of the probe at the thf position and effectively deblocks the probe, thereby generating a new hydroxyl group that can act as a primer for polymerase extension; thus, the probe is transformed into an extension primer with an increased specificity of amplification (james and macdonald, ) . the potential defect of rpa-lfd assay for asfv is that it may carry over contaminants in fields because its tube must be opened after amplification is completed. therefore, precautions should be taken. for example, reaction tubes should be carefully opened and closed, gloves should frequently be changed, the progress of pre-and post-rpa amplification should be separated, and reaction time should be shortened if possible. the replacement of dttp with dutp may help prevent such carryover contamination as demonstrated in other nucleic amplified system assays. in summary, we evaluated a rapid rpa-lfd test for the rapid diagnosis of asfv. the superior performance of rpa-lfd for asfv and its consistent detection results with those of real-time pcr indicated its appropriateness for laboratory diagnosis and that it has great potential for on-site testing of asfv circulating in china. the raw data supporting the conclusions of this manuscript will be made available by the authors, without undue reservation, to any qualified researcher. this study was conducted as part of the surveillance of the asf outbreak in china. samples were collected for asf testing and surveillance under the agreement between the ministry of agriculture and rural affairs of the chinese government and farm owners. sample collecting treatment was conducted in accordance with the protocols for viral hemorrhagic fever under the urgent interim guidance for case management established 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droplet digital polymerase chain reaction (ddpcr) for detection and investigation of african swine fever virus development and evaluation of a rapid and sensitive ebov-rpa test for rapid diagnosis of ebola virus disease emergence of african swine fever in china preclinical diagnosis of african swine fever in contact-exposed swine by a real-time pcr assay key: cord- -nnly ju authors: adachi, akio title: grand challenge in human/animal virology: unseen, smallest replicative entities shape the whole globe date: - - journal: front microbiol doi: . /fmicb. . sha: doc_id: cord_uid: nnly ju nan by far the most abundant species in nature is the virus that cannot replicate by itself. viruses, parasitic entities, are found in virtually all unicellular and multicellular creatures. viruses are everywhere. while smallest in size among all species, they can ingeniously replicate, persist, and survive in their individual hosts and/or host populations, and are transmittable among hosts. viruses are sometimes inflicting or fatal for host species and are sometimes inter-species replicons. they keep interacting with their hosts in numerous different manners. unseen viruses thus can reshuffle the whole world through accumulations of their subtle biological effects. in other words, viruses can shape the entire environment around us and are able to directly and/or indirectly influence us by their biologic activities. virology is a multidisciplinary research field and, as an academic discipline of the biology, it extensively analyzes all aspects of viruses derived from every living species by scientific systems/methodologies currently available to us as exemplified and fully described in a series of frontiers special issues designated "research topic" (rt) in the virology section of frontiers in microbiology adachi, , ; miyazaki et al., ; nomaguchi et al., ; berkhout and coombs, ; sato et al., ; adachi and miura, ; dutilh et al., ; sanfaçon, ; yamamoto et al., ) . i wish to emphasize here again that virology is a branch of biological sciences studying the fundamental attributes of a wide variety of unique characteristic viruses. virology concerns biological issues in a broad sense. in the past decade, as many as of , articles approximately have been published and almost rts for specific subjects have been issued or are on the way in the virology section. during this period, we have seen constant and vast increases both in virology-specific, in a narrow sense, articles, and in those giving more conceptual and general knowledge of the biology. notably, the observed advances frequently have been accompanied by remarkable development of novel innovative technologies. in the next decade, concomitant with successful proceeding of the powerful and sharp analyzing systems and/or methods such as computational biology, structural biology, bioinformatics, multi-omics, next-generation sequencing, genome editing, single-cell methodology, and organoid technology (bai et al., ; angermueller et al., ; chen et al., ; dutta et al., ; hasin et al., ; adli, ; artegiani and clevers, ; cheng, ; ibrahim et al., ; rossi et al., ; sbalzarini and greber, ; van dijk et al., ; pickar-oliver and gersbach, ) , virology will certainly continue to contribute much to the progress in all areas of the basic biology from molecular and structural biology to environmental science. in addition, virology will surely represent one of major driving forces to solve a wide variety of practical issues, which are directly or indirectly related to the virus issue itself, i.e., nanotechnology, viral vectors, antiviral drugs, vaccines, gene therapy, interferon therapy, immune therapy, and so on (de clercq, ; guimarães et al., ; szunerits et al., ; de clercq and li, ; athanasopoulos et al., ; grimm and büning, ; singh et al., ; snell et al., ; chambers et al., ; colino et al., ; kaufmann et al., ; ono et al., ; sulczewski et al., ; vetter et al., ; dionne, ; graham et al., ) . it is well-expected from our plentiful experience in the past decade that the virology section, as an outstanding communication platform, plays a critical and central role in various activities of basic and applied sciences. human and animal virology investigates viruses that infect human and all species of animals. upon replication, some viruses are damaging for their hosts to various degrees whereas others rather peacefully co-exist with the hosts. among these viruses, those that cause serious infectious diseases in human and/or animals, are medically, socially, and economically of particular importance, as a matter of course for scientific significance. in fact, our virology section has published numerous articles related to this kind of human and animal viruses and has edited relevant rts in the past decade. on one hand, more general subjects covering various pathogenic viruses and related research areas have been targeted as well. of socially important pathogenic viruses, some are solely tropic for humans, hiv- as an example (hatziioannou et al., (hatziioannou et al., , kamada et al., ; nomaguchi et al., nomaguchi et al., , b , and some like human norovirus are known not to replicate in cultured cells (duizer et al., ; herbst-kralovetz et al., ; ettayebi et al., ; murakami et al., ) . indeed, these viral properties hamper and limit our experimental strategies, and are representing major study projects to overcome in the virology. alternative practical experimental systems, irrespective of being commonly useful and applicable to numbers of viruses or specifically to certain viruses, to circumvent the observed difficulties are definitely required. expert researchers on the viruses have been making every effort to achieve the primary purposes (kamada et al., ; nomaguchi et al., nomaguchi et al., , b soll et al., ; hatziioannou et al., ; ettayebi et al., ; doi et al., ; schmidt et al., ; murakami et al., ) . the trend described above is understandable and wellrecognized by published articles and rts in the human and animal virus field in the virology section. most cited articles and most viewed rts in the human/animal virus field of the section (top , as of february , ) are as follows, respectively: articles, "epidemiological aspects and world distribution of htlv- infection" (gessain and cassar, ) , "pathology of asthma" (kudo et al., ) , "challenges and opportunities in estimating viral genetic diversity from next-generation sequencing data" (beerenwinkel et al., ) , "er stress, autophagy, and rna viruses" (jheng et al., ) , and "zika virus: the latest newcomer" (saiz et al., ) ; rts, "highly mutable animal rna viruses: adaptation and evolution" , "virus discovery by metagenomics: the (im)possibilities" (dutilh et al., ) , "zika virus research" (bueno-marí et al., ), "pathophysiology and epidemiology of virus-induced asthma" (kimura and ryo, ) , and "forefront studies on htlv- oncogenesis" (mahieux and watanabe, ) . the results described above may indicate the research field in which our peers are mostly interested. another point worth mentioning here is the emerging pathogenic viruses such as hemorrhagic ebola virus and pneumonia-causing new coronavirus designated sars-cov- . these viruses are becoming more and more important under today's world environment, as is the case for the influenza virus, a continuous global health threat, that potentially can induce an awful pandemic. it is more and more probable that humans may encounter emerging zoonotic viral pathogens and also re-emerging viruses through extensive inroads into untouched areas. we of course do not know much about the biology, including the ecology, origin, mutation, adaptation, etc., of these new viruses as yet. once transmitted to humans, they readily replicate in and spread among human populations without effective immunity against them. care should be taken not to be infected with the viruses at individual and population levels. infected individuals must be treated properly. in this regard, we virologists always have to prepare the ground for sudden onsets of new emerging diseases. routine collaborative research activities among basic researchers, clinical doctors, and relevant staffs of various expertise underpin the basis for swift responses against them. valid anti-viral strategies can be generated by such well-organized medical teams. viruses can cause acute or chronic infections in human and animal hosts in many mechanistically different ways. while causative viruses for the former do not persist long in individual hosts in general, the latter viruses generate persistent infection and may give rise to latent infection. it is scientifically and practically important and of great interest to determine the underlying mechanisms. elucidating biological and molecular bases for the conflict between viruses and their hosts is therefore one of major missions of current virology. especially, for some pathogenic viruses that have incredibly lengthy interacting period with hosts, or that are prone to readily alter in nature, it is critically important to dynamically investigate their mutations, adaptation, and evolution. hiv- , a representative virus for such category, readily mutates and adapts itself in the course of infection as experimentally revealed by a series of analytical studies (nomaguchi et al., (nomaguchi et al., , a (nomaguchi et al., ,c, (nomaguchi et al., , saito et al., ; yokoyama et al., ; doi et al., ) . here, following seven rts in our virology section are cited as examples of various cases for viral interactions with their hosts: "codon usage and dinucleotide composition of virus genomes: from the virus-host interaction to the development of vaccines, " "host and pathogen mechanisms underpinning viral ecology and emerging infections, " "revealing hiv hiding places: identification and characterization of cellular and tissue reservoirs, " "the interplay between innate immunity and herpesviruses, " "hiv- genetic diversity, " "highly mutable animal rna viruses: adaptation and evolution" , and "the past and the future of human immunity under viral evolutionary pressure" (hurst and magiorkinis, ) . as can be realized by the brief descriptions for the rts and articles published in the rts, there are indeed numerous distinct virus-host interactions to be noted. lastly, regardless of their medical or economic effects on human society, all rna and dna viruses are equally valid targets for extensive scientific studies in today's virology. we aim to publish important articles resulting from those studies that significantly advance our understanding of the viruses and related issues in our virology section. the most noticeable topic in the field of human and animal virology in the next decade would depend upon what happens in the virological world around us. it could be a deadly virus that causes global infection, or, could be some discovery or invention that changes our concept. attention should be paid to endogenized viral elements that affect the hosts in a biologically significant way. the relevant research field is rapidly growing as described recently in our journal (staege and emmer, ; turnbull and douville, ; flynn and moreau, ; moelling and broecker, ) . as for the correct answer to the question above, it is of course unpredictable at the present time but the virological information and biological finding of the first magnitude that move the academic and public communities will surely determine the result. based on enormous accumulations of scientific and statistical data of numerous kinds in various fields, together with orthodox and innovative analytical systems, it is quite expected that future virology can further deepen studies on virus populations and predictive science as well as those on a specific individual virus. from this point of view, i would like to enthusiastically encourage researchers outside the virology field, in addition to those who inside, to submit their representative works to our virology section, if there is a little relevance. virology is multidisciplinary in its marked and essential characteristic as stated above. nevertheless, the bottommost role for current virology in the era of "big data" is to functionally/biologically characterize individual viruses for the future by all available tools. by focusing on viruses, we can study and analyze living replicating creatures in all directions. we the virology section of frontiers in microbiology most welcome the collaborative and integrative studies that impact and stimulate the whole world. join us and share the fruits of virology. aa is a sole contributor to this manuscript and approved its submission. animal model studies on viral infections the crispr tool kit for genome editing and beyond deep learning for computational biology use and application of d-organoid technology nonintegrating gene therapy vectors how cryo-em is revolutionizing structural biology challenges and opportunities in estimating viral genetic diversity from next-generation sequencing data quantitative omics and its application to study virus-host interactions-a new frontier editorial: zika virus research overview of the baculovirus expression system protein bioinformatics databases and resources membrane protein structural biology in the era of single particle cryo-em nanoparticles for signaling in biodiagnosis and treatment of infectious diseases the design of drugs for hiv and hcv approved antiviral drugs over the past years key principles of antiretroviral pharmacology hiv- clones are both tractable to grow in rhesus macaques concomitant enhancement of hiv- replication potential and neutralizationresistance in concert with three adaptive mutations in env v /c /c domains laboratory efforts to cultivate noroviruses editorial: virus discovery by metagenomics: the (im)possibilities disease modeling in stem cell-derived d organoid systems replication of human noroviruses in stem cellderived human enteroids assessing the diversity of endogenous viruses throughout ant genomes epidemiological aspects and world distribution of htlv- infection structurebased vaccine antigen design small but increasingly mighty: latest advances in aav vector research, design, and evolution vaccines, adjuvants and autoimmunity multi-omics approaches to disease a macaque model of hiv- infection hiv- -induced aids in monkeys generation of simian-tropic hiv- by restriction factor evasion lack of norovirus replication and histo-blood group antigen expression in -dimensional intestinal epithelial cells editorial: the past and the future of human immunity under viral evolutionary pressure a new era of virus bioinformatics er stress, autophagy, and rna viruses generation of hiv- derivatives that productively infect macaque monkey lymphoid cells host-directed therapies for bacterial and viral infections pathophysiology and epidemiology of virus-induced asthma forefront studies on htlv- oncogenesis structural biology for virus research viruses and evolution -viruses first? a personal perspective bile acids and ceramide overcome the entry restriction for gii. human norovirus replication in human intestinal enteroids virology as biosystematics: towards understanding the viral infection biology editorial: highly mutable animal rna viruses: adaptation and evolution macaquetropic hiv- derivatives: a novel experimental approach to understand viral replication and evolution in vivo systemic biological analysis of the mutations in two distinct hiv- mt genomes occurred during replication in macaque cells species barrier of hiv- and its jumping by virus engineering hiv- mutates to adapt in fluxing environments natural single-nucleotide variations in the hiv- genomic sa prox region can alter viral replication ability by regulating vif expression levels production of hiv- vif mrna is modulated by natural nucleotide variations and slsa rna structure in sa d prox genomic region natural single-nucleotide polymorphisms in the ' region of the hiv- pol gene modulate viral replication ability generation of rhesus macaque-tropic hiv- clones that are resistant to major anti-hiv- restriction factors gag-ca q d mutation elicits trim -independent enhancement of hiv- mt replication in macaque cells baculovirus as a tool for gene delivery and gene therapy the next generation of crispr-cas technologies and applications progress and potential in organoid research improved capacity of a monkey-tropic hiv- derivative to replicate in cynomolgus monkeys with minimal modifications zika virus: the latest newcomer grand challenge in plant virology: understanding the impact of plant viruses in model plants, in agricultural crops, and in complex ecosystems genomics and computational science for virus research how computational models enable mechanistic insights into virus infection derivation of simian tropic hiv- infectious clone reveals virus adaptation to a new host the role of nanotechnology in the treatment of viral infections type i interferon in chronic virus infection and cancer assisted evolution enables hiv- to overcome a high trim α-imposed genetic barrier to rhesus macaque tropism editorial: endogenous viral elementslinks between autoimmunity and cancer? nanoparticle vaccines against viral infections nanostructures for the inhibition of viral infections related endogenous retrovirus-k elements harbor distinct protease active site motifs the third revolution in sequencing technology understanding modern-day vaccines: what you need to know editorial: perspectives for the next generation of virus research: spearheading the use of innovative technologies and methodologies in silico analysis of hiv- env-gp reveals structural bases for viral adaptation in growth-restrictive cells i would like to thank prof. masako nomaguchi (tokushima university, tokushima, japan) for continuous hot/extensive discussions about virology. i also appreciate ms. fumie nishina (kansai medical university, osaka, japan) and ms. kazuko yoshida (tokushima university) for excellent editorial assistance. the author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © adachi. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- - nhyumxl authors: heegaard, peter m. h.; sturek, michael; alloosh, mouhamad; belsham, graham j. title: animal models for covid- : more to the picture than ace , rodents, ferrets, and non-human primates. a case for porcine respiratory coronavirus and the obese ossabaw pig date: - - journal: front microbiol doi: . /fmicb. . sha: doc_id: cord_uid: nhyumxl the ongoing covid- pandemic caused by infection with sars-cov- has created an urgent need for animal models to enable study of basic infection and disease mechanisms and for development of vaccines, therapeutics, and diagnostics. most research on animal models for covid- has been directed toward rodents, transgenic rodents, and non-human primates. the primary focus has been on the angiotensin-converting enzyme (ace ), which is a host cell receptor for sars-cov- . among investigated species, irrespective of ace spike protein binding, only mild (or no) disease has occurred following infection with sars-cov- , suggesting that ace may be necessary for infection but is not sufficient to determine the outcome of infection. the common trait of all species investigated as covid models is their healthy status prior to virus challenge. in contrast, the vast majority of severe covid- cases occur in people with chronic comorbidities such as diabetes, obesity, and/or cardiovascular disease. healthy pigs express ace protein that binds the viral spike protein but they are not susceptible to infection with sars-cov- . however, certain pig breeds, such as the ossabaw pig, can reproducibly be made obese and show most aspects of the metabolic syndrome, thus resembling the more than % of the critically ill covid- patients admitted to hospitals. we urge considering infection with porcine respiratory coronavirus of metabolic syndrome pigs, such as the obese ossabaw pig, as a highly relevant animal model of severe covid- . in view of the ongoing sars-cov- /covid- pandemic (close to million cases and more than , deaths worldwide at the time of writing; johns hopkins corona resource center, ), there is an urgent need to increase the understanding of basic disease mechanisms, expand treatment possibilities, and identify biomarkers that correlate with the onset, severity, and duration of the disease. this will make it possible to reduce the disease complications for the individual patient and for societies worldwide despite the absence of general immunity frontiers in microbiology | www.frontiersin.org september | volume | article toward covid- , be it created by efficient and comprehensive vaccination with yet to be developed vaccines or by herd immunity caused by the pandemic itself. animal models that faithfully reproduce human covid- (i.e., incorporating the most important disease mechanisms, clinical signs, and response to treatment) are of utmost importance in order to achieve this. as commented by others (callaway, ; cleary et al., ; cohen, ) animal species ranging from mice to non-human primates are actively being investigated in the quest for reproducible and faithful models of covid- . a specific focus has been on the angiotensin converting enzyme type (ace ) that is known to be a receptor for sars-cov (the original - , and necessary for sars-cov to infect its host (li et al., ) . the affinity between the sars-cov spike protein and ace has been found to be a major determinant for the susceptibility of the host to sars-cov infection (wan et al., ) . this suggested ace as a possible host receptor also for sars-cov- . ace was indeed identified as a primary factor in determining cellular uptake of sars-cov- into engineered hela cells expressing ace from different species . the ace from human, bat, pig, and civet but not from mouse, supported cellular uptake of sars-cov- . speciesspecific differences in the ability of ace to bind the sars-cov- spike protein and thus in supporting virus entry was also predicted in silico from the ace -binding site of sars-cov spike protein (luan et al., ; wan et al., ) . in most -but not all -cases, a predicted or experimentally proven high affinity between sars-cov- and the ace protein correlates with susceptibility of the species in question to sars-cov- infection, although this does not always translate into disease ( table ) . as noted above, the predicted inability of mouse and rat ace to bind sars-cov- (wan et al., ) was demonstrated experimentally and, in accord with this, mice have been shown not to support sars-cov- replication and disease development (bao et al., ) . using engineered (transgenic) mice harboring the human ace receptor, virus propagation and development of mild disease has been demonstrated (bao et al., ; table ). in addition to transgenic expression, human ace -expression in mice can be achieved by crispr mediated humanization of the mouse receptor or by viral vector-mediated expression of the human protein (using an engineered adenovirus; cohen, ) . other sars-cov- infection models are based on existing sars-cov models such as the syrian hamster in which sars-cov- has been reported to propagate and cause respiratory disease (chan et al., ) although only "mild sars-cov- infection" was obtained (sia et al., ) . ferrets are well-known models for influenza and also get infected with sars-cov- ; they express a spike protein-binding ace protein, however only moderate disease is obtained (kim et al., ) . also, farmed mink have been observed to be easily infected with sars-cov- but again infections are generally mild (oreshkova et al., ) . non-human primates are an obvious choice based on closeness to humans genetically and a study reporting asymptomatic lung pathology after sars-cov- infection in cynomolgus macaques has appeared (rockx et al., ) . similarly, mild symptoms in rhesus macaques with weight loss yes, mild ( ) yes (pred) ( ) cynomolgus macaque, macaca fascicularis yes ( ) mild ( ) yes (pred) ( ) rhesus macaque, yes ( ) mild ( ) yes ( and signs of pneumonia but no fever were observed following sars-cov- inoculation (munster et al., ; shan et al., ) . such results again stress that a sars-cov- spike protein binding ace receptor is necessary for virus entry into cells but may not be sufficient for obtaining more than mild covid- . the pig is an interesting case, as its ace protein was both predicted (wan et al., ) and demonstrated experimentally to bind sars-cov- spike protein; however sars-cov- neither replicated nor caused disease in pigs after experimental infection (schlottau et al., ; shi et al., ) . thus, currently, no animal model of severe covid- that reproduces the salient clinical and pathological features of the disease has been described. mild symptoms are achievable in permissive species, but no model reproducing severe covid- , characterized by both upper and lower respiratory tract infection, pro-inflammatory cytokine activation, and prolonged disease is available, although such models obviously are of particular interest for investigation of intervention modalities and for biomarker research. no clear correlation between ace polymorphisms and covid- susceptibility can be established (devaux et al., ) and generally, the receptor recognition pattern of coronaviruses is complex and involves one or two domains of the spike protein, and both protein and carbohydrate host receptors (li, ) . receptors include dipeptidyl peptidase (receptor for mers-cov) and aminopeptidase n (apn), a receptor for the porcine respiratory coronavirus (prcv) spike protein. in addition, host proteases are necessary for activating entry through concerted cleavage of the spike protein. specifically, the spike protein of sars-cov- contains a cleavage site for the host protease furin (coutard et al., ) , which is essential for viral infectivity (hoffmann et al., ) . cleavage by furin has to be followed by cleavage by transmembrane protease serine (tmprss ) in order to fully activate the sars-cov- spike protein, which is similar to the mechanisms used by mers-cov (hoffmann et al., ) . observations in sars-cov- -infected human subjects show that the disease spectrum of this infection is exceptionally wide, ranging from those with mild symptoms in the majority of cases (> %) to severe, prolonged disease with high mortality and acute need for intensive care to alleviate symptoms and support survival (typically less than % of cases). a high proportion of the most severe cases occurs in patients with underlying chronic disease conditions ; depending on the region, - % of covid- patients requiring intensive care have at least one underlying co-morbidity [cdc covid- response team (a), ; tian et al., ] . in , covid- patients admitted to new york hospitals, % had two or more comorbidities, the three most prevalent being hypertension ( %), obesity ( %), and diabetes ( %; richardson et al., ) . also, case fatality rates are elevated in patients with pre-existing comorbidities such as cardiovascular disease, diabetes, chronic respiratory disease, hypertension, and cancer ( - % compared to overall . % among approx. , chinese cases; wu and mcgoogan, ) . indeed, patients with type diabetes and metabolic syndrome have been estimated to have up to times greater risk of death when suffering from covid- (bornstein et al., ) . also, age, which is frequently associated with a metabolic syndrome-like state is an important risk factor for severe covid- . in the united states, as registered by mid-march , of , covid- cases % were in the above years age group and this age group also accounted for % of intensive care unit admissions, and % of deaths associated with covid- [cdc covid- response team (b), ]. cytokine storm in the lungs and inflammation are suggested as essential for the escalating and prolonged lung disease observed in severely affected covid- patients, as is also the case for other severe human coronavirus infections like sars and mers (mehta et al., ) . intriguingly, immunosuppressive intervention, as often applied to treat pro-inflammatory cytokine over-activation may, in fact, increase covid- severity, depending on the timing and specificity of the treatment (ritchie and singanayagam, ) . this corresponds to observations on infections with animal coronaviruses such as prcv (jung et al., ) . since the majority of severe covid- cases are characterized by having underlying chronic conditions, animal models incorporating such pre-existing conditions are needed to reproduce severe covid- . while other aspects of the infection may be studied in any susceptible animal model, including genetically modified animal models, the study of onset, development and resolution of severe covid- in the background of existing disease may only be performed in an animal model that can reliably reproduce such disease states. there have been at least two, independent, but unsuccessful attempts to infect pigs with sars-cov- (schlottau et al., ; shi et al., ) . however, a range of other coronaviruses do infect pigs (saif, ; saif and jung, ; yang et al., ) , including porcine epidemic diarrhea virus (pedv) and porcine respiratory coronavirus (prcv). as mentioned above, prcv utilizes another cellular receptor than sars-cov- for the spike protein mediated cell entry, namely apn (delmas et al., ) . however, as the pulmonary pathology of prcv infection in pigs resembles sars in humans prcv infection of pigs was previously suggested as a model to examine sars (jung et al., ) . immunosuppression by corticosteroids (jung et al., ; zhang et al., ) or by co-infection with the immunosuppressive porcine respiratory and reproductive syndrome virus (prrsv; renukaradhya et al., ) were both demonstrated to exacerbate disease after prcv infection into a sars-like condition, with lower respiratory tract infection and pro-inflammatory cytokine activation. the pathogenesis due to prcv infection also shows many of the same features as seen with sars-cov- infection (saif and jung, ) , including a similar tissue tropism (upper and lower respiratory tract) and lung cellular tropism (type and type pneumocytes), and overlapping, although milder, clinical signs and lesions (fever, atypical pneumonia and interstitial pneumonia). aspects lacking in non-complicated prcv disease in comparison to covid- include acute respiratory distress syndrome (ards), multiple organ failure and diffuse alveolar damage, as well as blood inflammatory responses and pulmonary fibrosis (saif and jung, ) . thus, respiratory disease due to non-complicated prcv infection in pigs is a mild, self-limiting disease, similar to covid- in most sars-cov- infected individuals (see above). in line with this, we suggest that severe covid- may be faithfully reproduced in prcv-infected pigs co-affected by an underlying, chronic condition such as obesity associated metabolic syndrome. in addition to being an important comorbidity in the exacerbation of covid- (see above), this syndrome has the dual characteristics of a pro-inflammatory state and immunosuppression (andersen et al., ) ; both of these states are speculated to promote severe covid- (ritchie and singanayagam, ) . we would like to emphasize that in addition to closely mirroring human physiology, anatomy, immunology and food preferences, a number of pig breeds have been extensively documented to be an excellent model for human diet-induced obesity including the spectrum of inflammation-related co-morbidities associated with this condition (metabolic syndrome; sturek et al., ) . this is especially true for the ossabaw pig that has been described as having the greatest capacity of any known, terrestrial mammal to produce and store fat (brisbin and mayer, ) . we, and others, have documented that high energy and high fat diets result in the rapid occurrence of obesity in ossabaw pigs (sturek et al., ) . this reproducibly leads to a metabolic syndrome state with clinical signs such as hypertension, high fasting blood glucose, and dyslipidemia, with more advanced end-points such as pre-diabetes, non-alcoholic steatohepatitis (nash), and cardiovascular disease routinely achieved. these conditions are accompanied by signs of a systemic inflammatory state with increased circulatory biomarkers of inflammation (rødgaard et al., ) . as described above, the pig is the natural host of a number of well-described coronavirus infections with various tropisms and virulence. none of these viruses are human pathogens. prcv has been demonstrated, both by intranasal and intratracheal instillation, to lead to respiratory disease in pigs, although with low morbidity and mortality (saif, ) . as also described above, the outcome of the infection was worsened by immunosuppression with dexamethasone (jung et al., ; zhang et al., ) . we hypothesize that disease severity will increase in obese ossabaw pigs infected with prcv compared to pigs of normal weight, and hence will constitute a useful model for severe covid- in humans at risk due to metabolic syndrome associated comorbidities, including aged individuals. with the added benefit of being a well-described pig-specific virus (with no rigorous biosafety demands), we suggest that the obese pig affected by the metabolic syndrome will constitute a highly human-translatable animal model having the potential to significantly facilitate and accelerate sars-cov- /covid- research. both basic disease mechanisms and the efficacy and safety of therapeutic drug candidates for treatment of covid- patients from high risk groups can potentially be studied in this model as can biomarkers for severe covid- ; such markers are of high importance to identify individuals facing severe disease progression to allow early treatment decisions. in summary, there is a compelling need for animal models for covid- that accurately reproduce severe covid- , as these human cases are currently non-treatable and have resulted in very severe societal measures having detrimental economic and political consequences worldwide. while a range of animal species are indeed permissive for sars-cov- infection, only mild or no disease is obtained upon infection. we suggest that a relevant and valid model of severe covid- should preferentially incorporate the most common comorbidities of severe covid- , such as metabolic syndrome, type diabetes, and hypertension, also associated with age. we propose that the persistent inflammation that is part of the metabolic syndrome increases the risk of a cytokine storm during coronavirus infection linking an aberrant metabolic state to severe covid- . non-human primates may be highly relevant as models for sars-cov- infection, however developing primate comorbidity models will be expensive and time-consuming. pig models are highly relevant to human medicine because they closely mimic human anatomy, physiology, and immunology. furthermore, they develop robust metabolic syndrome and cardiorespiratory disease upon diet-induced obesity. intranasal inoculation of prcv in pigs with robust metabolic syndrome such as obese ossabaw miniature pigs, may increase the severity of disease, similar to patients with metabolic syndrome having severe covid- . this type of animal model could accelerate 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acute diarrhea syndrome coronavirus): an update three years after its discovery cytokine responses in porcine respiratory coronavirus-infected pigs treated with corticosteroids as a model for severe acute respiratory syndrome clinical course and risk factors for mortality of adult inpatients with covid- in wuhan, china: a retrospective cohort study a pneumonia outbreak associated with a new coronavirus of probable bat origin conflict of interest: ms and ma are cofounders of corvus biomedical, llc.the remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © heegaard, sturek, alloosh and belsham. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- -mxt stat authors: saraya, takeshi; kurai, daisuke; ishii, haruyuki; ito, anri; sasaki, yoshiko; niwa, shoichi; kiyota, naoko; tsukagoshi, hiroyuki; kozawa, kunihisa; goto, hajime; takizawa, hajime title: epidemiology of virus-induced asthma exacerbations: with special reference to the role of human rhinovirus date: - - journal: front microbiol doi: . /fmicb. . sha: doc_id: cord_uid: mxt stat viral respiratory infections may be associated with the virus-induced asthma in adults as well as children. particularly, human rhinovirus is strongly suggested a major candidate for the associations of the virus-induced asthma. thus, in this review, we reviewed and focused on the epidemiology, pathophysiology, and treatment of virus-induced asthma with special reference on human rhinovirus. furthermore, we added our preliminary data regarding the clinical and virological findings in the present review. more than different types of viruses, such as human rhinovirus (hrv), human metapneumovirus (hmpv), respiratory syncytial virus (rsv), and human parainfluenza virus (hpiv), are known to cause acute respiratory illness (ari; tsukagoshi et al., ) . we recently reported the issue of "virus-induced exacerbation in asthma and chronic obstructive pulmonary disease" (kurai et al., a) , however, among these causative viruses, hrv is now recognized to have a major impact on asthma pathogenesis (fujitsuka et al., ) . from this perspective, we reviewed the literature regarding the epidemiology of hrv-induced asthma in adults, together with preliminary epidemiological data obtained at our institution. hrv belongs to the genus enterovirus and family picornaviridae (turner and couch, ) . hrv possesses a single strand positive-sense rna (ssrna) genome of approximately . kb. the viral capsid is composed of four viral proteins (vp - ) which are assembled into protomers, resulting in a small icosahedral structure with a diameter of about - nm (turner and couch, ) . genetically, hrv is classified into three species; hrv-a, -b, and -c (simmonds et al., ) . furthermore, these species of hrv have more than genotypes (andries et al., ; arakawa et al., ; kiyota et al., kiyota et al., , . molecular epidemiological studies suggest that the dominant species are hrv-a and -c, while hrv-b is relatively rarely detected (arakawa et al., ; kiyota et al., ) . in particular, the vp and vp proteins have variations in their amino acid sequences, accounting for the large number of viral serotypes (turner and couch, ) . the host receptor for hrv in respiratory epithelial cells is the intracellular adhesion molecule (icam- , cd ) for the major hrv serotypes (hrv-a and -b), or low-density lipoprotein receptor (ldlr) for the other minor hrv serotypes. the receptor for hrv-c is not yet known. it has been suggested that the optimal temperature for replication of hrv is relatively cool ( - • c), which would limit infections to the upper airway; however, large or medium sized airways lower in the respiratory tract are now also considered cool enough for hrv replication, in spite of the higher temperature of the lung parenchyma ( • c; mcfadden et al., ) . therefore, hrv is potentially a causative agent of more severe ari such as bronchiolitis and pneumonia (turner and couch, ; watanabe et al., ; smuts et al., ; arakawa et al., ) , and may be associated with virus-induced asthma linsuwanon et al., ; fujitsuka et al., ; smuts et al., ) . hrv might therefore be involved in various aris and additional respiratory complications (kiyota et al., ) . lieberman et al. ( ) reported that the detection of any virus include hrv, the sensitivity rates for nasopharyngeal swab ( . %) was superior than that of oropharyngeal swab ( . %), respectively. the common cold is the third most common primary diagnosis in office visits (hsiao et al., ) , and this disease is generally selflimiting, usually lasting up to days (fashner et al., ) . among the general population, hrv infection causes common colds at a frequency of - % (makela et al., ; van gageldonk-lafeber et al., ) . tyrrell et al. ( ) reported that intranasal www.frontiersin.org inoculation with either hrv serotypes , , and , coronavirus type e, or rsv in healthy volunteers induced patterns of symptom development which were not substantially different from each other. however, individual signs or symptoms occurred earliest in hrv infections, then in coronavirus, and lastly in rsv, appearing up to days after inoculation, which demonstrated the long incubation periods of rsv in volunteers (tyrrell et al., ) . hrv has been implicated in patients with acute otitis media, exacerbation of chronic obstructive pulmonary disease, common cold, and lower respiratory tract infections in neonates, the elderly and immunocompromised. arruda et al. ( ) researching the frequency and natural history of hrv infections in adults during autumn, demonstrated that the first symptom noticed most often was sore throat ( %) in hrv culture-or pcr-positive patients, and stuffy nose in hrv-negative patients ( %), using nasal wash specimens. respiratory symptoms typically develop after - days after inoculation in studies, and uncomplicated hrv infections usually peak - days after inoculation. the median duration of hrv colds is week, but up to % last more than weeks (gwaltney et al., ; rotbart and hayden, ) . it should be noted that in illness caused by hrv, viral shedding occurs naturally for up to days, but predominantly over a - days period. hrv-a type (hrv- ), a major group virus commonly used for experimental human infection, and hrv-a type (hrv- ), which has been used in animal models of hrv infection, are closely related. grunberg et al. ( ) reported that experimental hrv- infection via nasal inhalation leads to a transient decrease of fev . in patients with asthma, and this decreased lung function was correlated with enhanced cold symptoms and / or airway hyperresponsiveness. contoli et al. ( ) demonstrated that type iii interferon (ifn-λ) production levels in ex vivo cell cultures derived from bronchial epithelial cells (becs) and macrophages obtained from asthmatic patients, were lower than in those derived from healthy controls. furthermore, deficient interferon-λ production was correlated with hrv viral load, severity of clinical symptoms and fev . . message et al. ( ) demonstrated that the severity of intranasally inoculated hrv-induced clinical illness in asthmatic subjects was correlated to virus load and lower airway virus-induced inflammation. on the other hand, demore et al. ( ) reported that no difference in clinical symptoms, and patterns of viral shedding, was noted between subjects with persistent allergic asthma and healthy subjects after experimental infection with hrv. these different results after experimental hrv infection in individual studies in asthmatic patients and healthy subjects might be dependent on the severity of the asthma of those subjects who enrolled in the studies. indeed, in several reports, neither defective ifn induction by hrv, nor increased hrv replication was observed in primary human becs derived from subjects with well controlled asthma (lopez-souza et al., ; bochkov et al., ; sykes et al., ) . a few animal models for rhinovirus infection have been showed because a major group of hrv (i.e., hrv- ) did not bind mouse icam- . only a minor group of hrv (i.e., hrv- b) infected the mouse. in this regard, bartlett et al. ( ) generated a transgenic balb/c mouse expressing a mouse-human icam- chimeric receptor for hrv- infection. this study also showed asthma exacerbation model by intraperitoneally sensitized with ovalbumin with aluminum hydroxide followed by intranasal inoculation of hrv- b or uv-inactivated hrv- b. although data regarding virus respiratory infections (vris) as precipitators of asthma attacks in adults are less clear, nicholson et al. ( ) reported that vris are as commonly linked to exacerbations in adults as they are in children (johnston et al., ; fujitsuka et al., ) . this study showed that viruses were detected in % of clinical exacerbative episodes with a decrease in peak expiratory flow rate (pefr) of ml/minute or more, and the most commonly identified virus was hrv, followed by coronaviruses and parainfluenza viruses (nicholson et al., ) . thus, the virus most commonly detected in asthma exacerbations appears to be hrv. although hrv is well known as the most frequent cause of the common cold, the implications of hrv infection vary according to respiratory diseases. table shows the frequency of hrv infection in various adult respiratory diseases such as exacerbation of asthma (nicholson et al., ; atmar et al., ; tan et al., ) , common cold (makela et al., ; van gageldonk-lafeber et al., ) , exacerbation of copd (seemungal et al., ; rohde et al., ; tan et al., ; beckham et al., ; papi et al., ; hutchinson et al., ; ko et al., ; mcmanus et al., ; kherad et al., ; dimopoulos et al., ; perotin et al., ) , community acquired pneumonia (jennings et al., ; johnstone et al., ; johansson et al., ; lieberman et al., ; fry et al., ; wootton et al., ; luchsinger et al., ; takahashi et al., ; huijskens et al., ) , exacerbation of idiopathic pulmonary fibrosis (wootton et al., ) , and asymptomatic infection (fry et al., ) . the risk of exacerbations of asthma in adults is elevated after children return to school, and around december th (the christmas holiday in westernized countries), and this is likely to be due to social interactions with children at these times. prospective monitoring studies using reverse transcription polymerase chain reaction (rt-pcr) indicate that as many as % of acute asthma exacerbations in children, and about % in adults, were associated with the presence of upper respiratory tract (urt) infections. corne et al. ( ) found that the detection rates of hrv in asthmatic ( . %) and healthy participants ( . %) were similar, but the lrt symptoms were significantly more severe and longer lasting in the asthmatic group than in the healthy group based on one definition of urt and lrt symptoms ( table ; johnston et al., ) . there is no common antigen across all strains of hrvs; therefore, no reliable diagnostic method for hrv infection has been established using hrv antigens or hrv-specific antibody. although viral culture is the conventional method for hrv detection, culture methods are not practical in clinical settings for the detection of hrv, because of its slow growing character and requirement for specific culture conditions. furthermore, the diagnostic capability of molecular amplification techniques frontiers in microbiology | virology exacerbation of asthma - nicholson et al. ( ) , tan et al. ( ) , atmar et al. ( ) common cold - makela et al. ( ) cited and adapted from johnston et al. ( ) . such as nucleic acid sequence-based amplification and rt-pcr is superior to those of culture methods (loens et al., ) . experimental hrv infections have been shown to lead to a longlasting excessive airway narrowing in volunteer subjects with asthma (cheung et al., ; grunberg et al., ) . of note, rhinovirus, unlike influenza and other viruses, causes minimal cytotoxicity (fraenkel et al., ) , and the amount of epithelial damage does not correlate with the severity of the symptoms. hrv infection can cause additive or synergistic effects in exacerbation of asthma via the influx of additional inflammatory cells in the airways with preexisted inflammation, resulting in airway cholinergic hyperresponsiveness (nagarkar et al., ) , as an allergic response. the effects of hrv infection such as enhanced contractility of airway smooth muscle (asm) cell and impaired relaxation to cholinergic or β-adrenaergic agonists are attributed solely to binding of the virus to its host receptor icam- on the asm cell surface. this proasthmatic-like effect was recognized even in the situation of complete inhibition of viral replication in vitro, but not in the setting of pretreatment of asm with neutralizing antibody directed against for icam- (grunstein et al., ) . thus, the hrv attachment to icam- itself can affects the contractility of asm cells in the absence of any cytopathic effects, and chun et al. ( ) reported that a cells infected with hrv in vitro produced a higher value of il- and rantes than those of rsv or adenovirus. in addition, only the combination of hrv with der f (house dust mites antigen) acted synergistically to induce il- production. these findings are the reason why the hrv can be a major pathogen for acute exacerbation of asthma. we present a schema for pathogenesis in hrv associated asthma exacerbations (figure ) , which requires the following steps, ( ) hrv attachment to airway epithelial cells, ( ) an innate immune response which leads to epithelial damage, ( ) infection-related airway remodeling. when rt-pcr is used to either supplement or replace conventional culture techniques, viruses have been found in approximately one half to three quarters of adults experiencing an acute wheezing episode (jackson and johnston, ) , and the majority ( %) of viruses identified were hrvs (nicholson et al., ) . however, the evidence is weak, and mechanisms are poorly understood. initially, hrv-a and -b attach to airway epithelial cells via icam- or ldlr (kennedy et al., ) . the receptor or receptors for the recently identified group hrv-c have yet to be clarified. hrv-infected becs secrete a wide range of cytokines and chemokines such as il- , il- , ccl /rantes (regulated on activation, normal t cell expressed and secreted), cxcl /il- , gm-csf, and cxcl /interferon-inducible protein (ip- ; jackson and johnston, ; proud, ) , which induce neutrophilic, lymphocytic, and eosinophilic inflammation together with airway hyperresponsiveness and airway remodeling (wark et al., ; proud, ) . clearance of viral pathogens begins with interferon secretion, and the underproduction of these factors has been postulated to lead to viral-induced exacerbations. there are three types of interferons, based on the receptors they bind: type i (ifn-α/β), type ii (ifn-γ), www.frontiersin.org and type iii (ifn-λ). hrv infection induced epithelial expression of mrna for both type i and type iii ifns, and it has been suggested that impaired epithelial production of ifn-β and ifnλ in asthmatic subjects may contribute to viral exacerbations of asthma (wark et al., ; contoli et al., ) . contoli et al. ( ) showed significant inverse correlations between ex vivo production of ifn-λ and severity of symptoms, bronchoalveolar lavage viral load and airway inflammation, and a strong positive correlation with reductions in lung function during in vivo infection. genome-wide association studies showed that single nucleotide polymorphisms involve in various diseases. interferon-λ polymorphisms may effect on the incidence of hrv infection (russell et al., ) . message et al. ( ) reported virus load in asthmatic subjects as being related to increased lower airway inflammation, and in turn increased lower airway inflammation being related to increased symptoms, reductions in lung function, and increases in bronchial hyperreactivity. these data suggest a causal role for hrv infection in the pathogenesis of asthma exacerbations. investigating virus-allergen interactions, durrani et al. ( ) demonstrated that another mechanism that increased expression and cross-linking of the high-affinity ige receptor, fcεri, on plasmacytoid dendritic cells is associated with reduced hrv-induced ifn-α and ifn-λ secretion, and allergic asthmatic children have significantly reduced hrv-induced ifn-α and ifn-λ production after cross-linking of fcεri. type , or inducible, nitric oxide synthase (inos) is the major nos isoform found in epithelial cells and can generate substantial amounts of nitric oxide (no). the no molecules both inhibit the replication of hrv in airway epithelial cells, and suppresses hrv-induced cytokine production (proud, ) . although the measurement of fractional no concentration in exhaled breath (feno) may be used to support the diagnosis of asthma (dweik et al., ) , however, increasing of feno seems to be not always correlated with viral load during the period of hrv infection (sanders et al., ) . other factors such as allergy, allergen exposure, tobacco smoke, particulates, ozone, stress, and infections such as sinusitis commonly contribute to exacerbations of asthma. grainge et al. ( ) reported that repeated bronchoconstriction in asthma promotes airway remodeling, and there is now clear evidence that airway remodeling begins in early childhood, and can be present even before clinical diagnosis of asthma is established (pohunek et al., ) . increasing evidence regarding hrv-induced wheezing or exacerbation of asthma raises the possibility that hrv infections could contribute to the initiation and subsequent progression of airway remodeling, which involves multiple factors such as increased epithelial release of mucin ac (mu ac), activin a, amphiregulin, matrix metalloproteinase (mmp ), epidermal growth factor (egf), fibroblast growth factor (fgf), and vascular endothelial growth factor (vegf). hrv infection upregulates production of muc ac from epithelial cells, which leads to airflow obstruction in asthma (hewson et al., ) . activin a is a member of the tgf-β superfamily and amphiregulin, a member of the egf family, alters repair processes (leigh et al., ) . both activin a and amphiregulin have been linked to subepithelial basement membrane thickening in asthma. mmp appears to have important roles in asthma exacerbation and airway remodeling (sampsonas et al., ) . expression of vegf and its receptors is increased in asthmatic subjects, and vegf is the major proangiogenic activator in asthmatic airways (feltis et al., ; simcock et al., ) . kuga et al. ( ) reported that . % of adult asthmatic patients with common cold suffered an asthma attack, and common cold was significantly associated with acute exacerbations of asthma. they also stated that hrv infection might be important as the virus was detected by rt-pcr in throat gargles (kuga et al., ) . virus-induced exacerbation of asthma is a critical issue for the general physician. however, among asthmatic patients with exacerbative status, distinguishing between those patients which have vris, and those who do not, is difficult. furthermore, epidemiological data regarding adult asthma exacerbations have been sparsely reported. to investigate the prevalence of vri in exacerbations of adult asthma in both hospitalized or not-hospitalized patients, characterization of clinical and radiological findings was performed. a prospective observational cohort study was conducted at kyorin university hospital, tokyo, japan from august to august (kurai et al., b) . all patients with respiratory symptoms associated with exacerbation of asthma were included, and samples were collected by nasopharyngeal or oropharyngeal swab, and subjected to a pcr method to detect common respiratory viruses. the patients who were enrolled consisted of hospitalized (n = ) or not-hospitalized patients (n = ; table ). in these two groups, the subject's backgrounds were similar for age, sex, smoking rates, and duration of illness, however, the measured value of spo was significantly lower in hospitalized patients ( ± . %) than in non-hospitalized patients ( . ± . %). the incidence of vri was significantly higher in the former group ( . %, n = ) than in the latter group ( . %, n = ; p = . ). in the latter group, influenza virus alone was detected in both patients. furthermore, all hospitalized patients ( %, n = ) had wheezing or severe exacerbation based on the ats (american thoracic society)/ers (european respiratory society) statement (reddel et al., ) , whereas, among nonhospitalized patients, only nine patients ( %) were considered as having a severe exacerbation (p < . ), and patients ( . %) had wheezing (p < . ). these findings suggested that virus infection was certainly associated with the hypoxemia and / or wheezing which resulted in a severe or serious asthma attack, based on the japanese guidelines (ohta et al., ) or the ats/ers statement (reddel et al., ) . previous studies using ohta et al. ( ) , † † defined by reddel et al. ( ) . ** p < . , *** p < . . all data are presented as (mean ± sd). pcr-based viral diagnostics found that viral respiratory infections were detected in up to % of exacerbations of asthma in children and about % of exacerbations in adults (nicholson et al., ; johnston et al., ) , which is similar to our results. serum inflammatory or allergic markers are not different between the hospitalized and non-hospitalized patients ( table ) . in hospitalized patients, the viruses identified were hrv (n = ), hmpv (n = ), and rsv (n = ). at the time of admission, the virus-positive group (n = ) had significant lower values of spo ( . ± . %) than those of the virus-negative group (n = , spo : . ± . %, p < . ), and for the patients whose data are available, the frequency of hypercapnea (paco torr) was significantly higher in the virus positive group ( . %, n = ) than in the virus negative group ( %; p = . ; table ). the mechanisms for hypercapnea in virus infected individuals have not been elucidated. however, cheung et al. ( ) reported that hrv infection causes long lasting excessive airway narrowing in response to methacholine in asthmatic subjects. we speculated that smooth muscle might have a role in exaggerated airway narrowing in virus positive asthmatic patients, as described by king et al. ( ) . interestingly, the incidence of ground glass opacities (ggo) on high resolution computed tomography seemed to be higher for virus-positive hospitalized patients than for virus-negative patients, but it did not reach statistical significance. for example, figure a shows a patchy ggo with thickening of interlobular septa in a -year-old woman who was admitted during an asthma attack induced by hrv-a. figure b also shows ggo in a -year-old man with an asthma attack caused by hrv-c infection. these ggo in both patients could only be detected in hrct, not in chest x-ray. these results suggested that hrv was the major cause of virusinduced asthma, and was possibly involved in lower airway or lung parenchyma features, appearing as ggo. viral infection significantly exaggerated the respiratory status (low spo and hypercapnea) when compared to that of virus-negative asthma exacerbative patients at the time of admission. indeed, in recent years, hrv has been recognized as a common cause of hospital admission, both as an agent of bronchopneumonia and through exacerbation of chronic pulmonary conditions, even in the elderly over years of age (pierangeli et al., ) . curiously, after initiation of treatment with intravenous steroid, both the virus-positive and -negative groups had no significant difference in duration of respiratory failure, wheezing, days in hospital, and even in the time required for steroid treatment. no established treatment for prevention of hrv-induced asthma is available, and we describe the exploratory interventions as follows. inhaled corticosteroid (ics) is the main drug for regular asthma therapy. ics treatment improved airway hyperresponsiveness in asthmatic patients experimentally challenged with hrv, however, ics treatment did not reduce accumulation of inflammatory cells, except for eosinophils in bronchial epithelium (grunberg et al., ) . double-stranded rna (dsrna), a viral product and a ligand for the toll-like receptor- (tlr ), upregulates the expression of inflammatory chemokines in airway epithelial cells. matsukura et al. ( ) reported that treatment of beas- b cells with fluticasone propionate significantly and dose-dependently inhibited dsrna-induced expression of ccl , cxcl , and cxcl protein and mrna. to confirm the effect on ssrna, such as that of hrv, would need further studies. leukotriene receptor antagonist was prescribed in asthmatic patients with or without ics. montelukast treatment did not improve asthma control or cold symptom scores when hrv were experimentally inoculated into mild asthmatics, or healthy subjects (kloepfer et al., ) . it is uncertain whether leukotriene receptor antagonist treatment is effective in the reduction of asthma symptoms associated with hrv infection. zambrano et al. ( ) reported that high serum ige levels in mildly asthmatic children with experimental hrv infection may be associated with enhanced lower respiratory symptoms and elevation of inflammatory markers, such as nasal eosinophil cationic protein and expired nitric oxide, than those of healthy subjects and/or low ige asthmatic patients. the prevalence of asthma was closely associated with the serum ige levels standardized for age and sex (burrows et al., ) , and airway hyperresponsiveness appears to be closely linked to the allergic diathesis, as reflected by the serum total ige level (sears et al., ) . omalizumab, an anti-ige monoclonal antibody, was indicated in inadequately controlled moderate-to-severe persistent allergic asthma patients who were treated with high dose ics. durrani et al. ( ) showed that the ige receptor fcεri is inversely associated with ifn-α and ifn-λ secretion when plasmacytoid dendritic cells derived from allergic asthmatic children were challenged with hrv. omalizumab downregulates fcεri expression on dendritic cells (prussin et al., ) , which may reduce exacerbation of asthma associated with increased production of ifns, through fcεri. no drugs are clinically used in hrv infection, although several drugs have been tried for treatment and prevention of hrv infection. these drugs are summarized in a review (jacobs et al., ) . ifns had a potential protective role in viral induced asthma (cakebread et al., ; gaajetaan et al., ) . becker et al. ( ) showed that exogenous ifn-α, ifn-β, ifn-λ , and ifn-λ inhibited hrv replication in becs from healthy donors. macrolides are known to possess anti-inflammatory and immunomodulatory actions extending beyond their antibacterial activity in pulmonary inflammatory disorders (takizawa et al., ; min and jang, ) . erythromycin inhibits hrv infection by reducing icam- expression on the surface of human tracheal epithelial cells, and modulates inflammation by suppressing the production of proinflammatory cytokines (suzuki et al., ) . yamaya et al. ( ) reported that the mucolytic drug ambroxol hydrochloride, antibiotic drug of levofloxacin (yamaya et al., b) , and bronchodilators (tiotropium, tulobuterol, and procaterol) for asthma or copd (yamaya et al., (yamaya et al., , a (yamaya et al., , may have a beneficial effect in hrv infection, by inhibiting hrv replication and partly reducing icam- expression and acidic endosome production, via the inhibition of nf-kappab activation (yamaya, ) . we reviewed the previous reports regarding hrv-induced asthma exacerbations, together with our results from an institutional prospective study. hrv is a major pathogen for asthma exacerbations, and certainly associated with more serious clinical conditions such as hypoxemia or hypercapnea in hospitalized patients. further accumulation of evidence of virus-induced asthma for multidisciplinary assessment would be helpful for physicians in recognizing the condition or understanding the pathogenic mechanisms. two groups of rhinoviruses revealed by 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hehe; yi, yi; cheng, hao; wang, shaowen; zhang, qin; qin, qiwei; li, pengfei title: specific aptamer-based probe for analyzing biomarker mcp entry into singapore grouper iridovirus-infected host cells via clathrin-mediated endocytosis date: - - journal: front microbiol doi: . /fmicb. . sha: doc_id: cord_uid: ev pq biomarkers have important roles in various physiological functions and disease pathogenesis. as a nucleocytoplasmic dna virus, singapore grouper iridovirus (sgiv) causes high economic losses in the mariculture industry. aptamer-q -complexed major capsid protein (mcp) in the membrane of sgiv-infected cells can be used as a specific molecular probe to investigate the crucial events of mcp endocytosis into sgiv-infected host cells during viral infection. chlorpromazine blocks clathrin-mediated endocytosis, and mcp endocytosis into sgiv-infected cells decreased significantly when the cells were pretreated with chlorpromazine. the disruption of cellular cholesterol by methyl-β-cyclodextrin also significantly reduced mcp endocytosis. in contrast, inhibitors of key regulators of caveolae/raft-dependent endocytosis and macropinocytosis, including genistein, na(+)/h(+) exchanger, p -activated kinase (pak ), myosin ii, rac gtpase, and protein kinase c (pkc), had no effect on mcp endocytosis. the endocytosis of the biomarker mcp is dependent on low ph and cytoskeletal actin filaments, as shown with various inhibitors (chloroquine, ammonia chloride, cytochalasin d). therefore, mcp enters sgiv-infected host cells via clathrin-mediated endocytosis, which is dependent on dynamin, cholesterol, low ph, and cytoskeletal actin filaments. this is the first report of a specific aptamer-based probe used to analyze mcp endocytosis into sgiv-infected host cells during viral infection. this method provides a convenient strategy for exploring viral pathogenesis and facilitates the development of diagnostic tools for and therapeutic approaches to viral infection. iridoviruses are large icosahedral cytoplasmic dna viruses with circularly permuted, terminally redundant, double-stranded dna genomes (qin et al., ) . the family iridoviridae includes six genera: ranavirus, iridovirus, chloriridovirus, lymphocystivirus, megalocytivirus, and decapodiridovirus (chinchar and duffus, ) . singapore grouper iridovirus (sgiv) was first isolated from the grouper epinephelus tauvina. it belongs to the genus ranavirus and currently causes high economic losses in the mariculture industry (qin et al., ; xiao et al., ; liu et al., ) . understanding the pathogenesis of sgiv is necessary to develop effective therapies against it (yu et al., a) . viral infection begins with its attachment to the host cell membrane, and it then enters the cell via specific endocytosis. in the host cell, the sgiv is transported to the replication site, where the viral genes are expressed (seisenberger et al., ) . many sgiv structural genes and non-structural genes have already been studied and are related to viral replication, pathogenesis, and host cell immunity (chinchar et al., ; chinchar and duffus, ) . during infection, modifications appear in the host cell membranes (verdaguer et al., ; abós et al., ; seeger and mason, ; yu et al., a) , which can potentially be used as important biomarkers of infection. such biomarkers can be used to develop diagnostic tools and therapeutic approaches to virus infection (yildirim et al., ; ashcroft, ) . membrane proteins account for about % of the total cellular proteins and have important roles in various physiological functions (shangguan et al., ) . knowledge of these biomarkers will extend our understanding of viral pathogenesis. however, little is yet known about the mechanisms underlying the entry of these biomarkers into host cells. to address this limitation, we used aptamers to investigate the crucial events of biomarker endocytosis into sgiv-infected host cells during viral infection. aptamers are selected by the systematic evolution of ligands with the exponential enrichment technology (selex) (ellington and szostak, ) . aptamers selected against different targets are synthetic oligonucleotides with different sequences and fold into distinct three-dimensional structures, binding their targets with high specificity and affinity (yu et al., b) . although they resemble antibodies in this regard, aptamers have properties that make them more useful than antibodies, such as their ease in synthesis and modification, high reproducibility, and stability. based on these excellent qualities, aptamers are excellent molecular probes for pathogen diagnostics and therapeutics (li et al., wolter and mayer, ; kaur et al., ; zhou et al., ) . for example, aptamer a was selected against the coat protein of red-spotted grouper nervous necrosis virus (rgnnv) and was successfully used to develop an aptamer-based enzyme-linked apta-sorbent assay (elasa) for the rapid and sensitive detection of rgnnv infection (zhou et al., , . aptamers are also used as highly specific molecular probes in pathogen pathogenesis studies (jin et al., ; yu et al., a) . they have been widely used to identify membrane protein biomarkers, such as alkaline phosphatase placental-like (dua et al., ) , stress-induced phosphoprotein ( van et al., ) , and protein tyrosine kinase (shangguan et al., ) . according to several reports, after specifically binding to biomarkers in the cell membrane, some aptamers are actively internalized into the target cell. consequently, they can be used as delivery vehicles for the targeted delivery of drugs (sun and zu, ) . for example, mcnamara et al. ( ) reported the cell-type-specific delivery of small interfering rnas (sirna)-aptamer chimeras, which significantly increased the therapeutic efficacy of the sirna against cancer cells. because aptamers are easy to synthesize, they have great potential utility in targeted therapies. however, little is yet known about the mechanisms of entry of these cell biomarkers into host cells. in a previous study, the aptamer q , which targets whole grouper-iridovirus-infected cells, was successfully generated with cell-selex (li et al., ) . aptamer q has high binding affinity ( . nm) and recognizes sgiv-infected cells with high specificity. the target protein of aptamer q on the membrane of sgiv-infected host cells was identified as the major capsid protein (mcp) of the virus (yu et al., a) . mcp is the predominant structural protein of iridovirus particles and comprises about %- % of the iridovirus particle polypeptides. it is used as a marker to identify and classify iridoviruses (lin et al., ) . mcp plays an important role in iridovirus pathogenesis, including in the formation of the iridovirus particle, the induction of the immune responses, virus-host cell interactions, antigen recognition, and the transcription of viral genes (fu et al., ) . after specifically binding to mcp in the cell membrane, aptamer q is actively internalized into the target cells, indicating that mcp is transported by the vesicular transport system in the host cell during viral infection. therefore, aptamer q can be used as a specific probe with which to investigate the trafficking mechanism and endocytotic pathway of mcp in host cells during sgiv infection (wickner and schekman, ; yu et al., a) . briefly, aptamer q first binds to the target protein (mcp) on the cell surface, and the q -mcp interaction activates the cellular signaling pathways, which internalize q -mcp through one of several endocytotic mechanisms. these include clathrin-mediated endocytosis, caveolae/raft-dependent endocytosis, macropinocytosis, and non-clathrin-caveolae/raftdependent endocytosis (wang et al., ; huang et al., ) . in this study, an aptamer (q )-based specific probe was used to investigate the trafficking mechanism and endocytotic pathway of mcp in host cells during sgiv infection. grouper spleen (gs) cells were grown in leibovitz's l- medium (gibco, grand island, ny, united states) containing % fetal bovine serum (gibco) at • c. the grouper iridovirus strain (sgiv-gx) used in this study was isolated from diseased hybrid grouper (epinephelus fuscoguttatus♀ × e. lanceolatus♂) figure | aptamer cy -q specifically bound to sgiv-infected cells. (a) lscm showed the specific binding of cy -q to sgiv-infected cells but not to normal gs cells. (b) flow cytometry results confirmed that compared with the control group, in which aptamer cy -q ( nm) was incubated with normal gs cells, cy -q specifically bound to sgiv-infected cells. p < . was considered statistically significant (**p < . ). figure | aptamer cy -q was used to characterize the entry process and entry kinetics of mcp into sgiv-infected cells. (a) entry process and entry kinetics detected with flow cytometry. after aptamer cy -q was incubated with sgiv-infected cells at • c for different periods ( , , , , , or min) , no significant change in fluorescence was detected with flow cytometry, indicating that aptamer cy -q only bound to the cell plasma membrane, without endocytosis. when cells were transferred to • c to initiate mcp entry into sgiv-infected cells, a time-dependent increase in the fluorescent signal was detected in the cell cytoplasm with flow cytometry, which peaked at min. (b) cell-specific internalization of the q -mcp complex was detected with lscm. after the cells were incubated at • c for h, the fluorescent signal was observed inside the sgiv-infected cells, confirming the endocytosis of the q -mcp complex. the control group of sgiv-infected cells treated with cy -library showed no fluorescence. figure | inhibitors had no effect on cy -q binding to sgiv-infected cells. after sgiv-infected cells were pretreated with each inhibitor at the safe working concentration for h, the cells were incubated with cy -q ( nm) at • c for h and then collected for flow-cytometric analysis. there was no statistically significant difference between the fluorescence signals of sgiv-infected cells pretreated with or without the inhibitors. bars with different letters are significantly different (p < . ) based on one-way anova. in guangxi province, china, propagated in gs cells, and stored at − • c in the laboratory . grouper spleen cells were cultured to % confluence in -well plates or -mm glass-bottom dishes (cellvis, hangzhou, china; catalog number d - - -n) at • c for h. after the cells were infected with sgiv-gx at a multiplicity of infection (moi) of at • c for h, they were then washed with phosphate-buffered saline (pbs: mm na hpo · h o, mm kh po , mm nacl, % nan ) and collected for further use (yu et al., a) . the aptamer q was generated against sgiv-infected cells with cell-selex in our previous study (li et al., ) . aptamer q was synthesized by life technologies, and the end of aptamer q was labeled fluorescently with cy (cy -q ). chlorpromazine (cpz), methyl-β-cyclodextrin (mβcd), nystatin, genistein, dynasore, (n-ethyl-n-isopropyl)-amiloride (eipa), ml- , nsc , rottlerin, ipa- , chloroquine (cq), ammonia chloride (nh cl), cytochalasin d (cytod), and nocodazole were purchased from sigma-aldrich (st. louis, mo, united states). all reagents were stored and dissolved to specific concentrations according to the manufacturer's instructions. to optimize the safe working concentration of each inhibitor, its cytotoxicity was evaluated as described previously . gs cells ( × ) were cultured in a -well plate (corning, new york, united states) for h at • c, and then incubated with different concentrations of each inhibitor ( µm cpz, µm dynasore, mm mβcd, µm genistein, µm eipa, µm ipa- , µm ml- , µm nsc , µm rottlerin, µm cq, mm nh cl, or µm cytod) at • c for h. untreated gs cells were used as the control group. cck- solution ( µl; beyotime, shanghai, china) was added to the cells, and they were incubated at • c for h. the absorbance at nm was measured with an enzyme-linked immunosorbent assay plate reader (thermo, waltham, ma, united states), according to the manufacturer's instructions. the results of three independent experiments are presented as means ± standard deviations (sd). the working concentration of each inhibitor used in this study caused no significant cytotoxicity. a flow cytometer (beckman moflo xdp, brea, ca, united states) was used to analyze the specific interaction and internalization of the q -mcp complex (li et al., ; yu et al., a) . gs cells ( × ) were cultured to % confluence in -well plates for h and then infected with sgiv-gx (moi = ). at h postinfection, the sgiv-infected gs cells were incubated with cy -q ( nm) at • c or • c. after the mixtures were washed three times with pbs, they were resuspended in µl of pbs for flow-cytometric analysis, in which , events were counted. normal gs cells incubated with aptamer cy -q ( nm) were used as the control. the results are presented as the means ± sd of three independent experiments. for live-cell fluorescent imaging, × gs cells were cultured in -mm glass-bottom dishes (cellvis, hangzhou, zhejiang, figure | clathrin-mediated endocytosis is involved in mcp entry into sgiv-infected cells. after sgiv-infected cells were pretreated with the inhibitor cpz at the safe working concentration for h, they were incubated with aptamer cy -q ( nm) at • c for h. the culture supernatants were then removed, and the cells were washed twice with l- and shifted to • c to initiate the entry of mcp. the internalization of the q -mcp complex was analyzed with flow cytometry and lscm. normal gs cells incubated with cy -q ( nm) were used as the negative control; sgiv-infected cells without inhibitor but incubated with cy -q ( nm) were used as the positive control. (a) compared with the control group, cpz significantly reduced the fluorescent signal inside the sgiv-infected cells. (b) entry efficiency of the q -mcp complex decreased significantly to . % with µm cpz and to . % with µm cpz, indicating the dose-dependent inhibitory effect of cpz on mcp endocytosis. p < . was considered statistically significant (**p < . ). china) and infected with sgiv-gx (moi = ) for h. the sgiv-infected cells were then incubated with aptamer cy -q ( nm) at • c or • c. after the cells were washed with serum-free phenol-red-free medium, their fluorescence was detected with laser scanning confocal microscopy (lscm; nikon, tokyo, japan). normal gs cells incubated with aptamer cy -q ( nm) were used as the control. we analyzed the effects of various inhibitors on the binding of aptamer cy -q to its target protein, mcp, in the membranes of sgiv-infected cells with flow cytometry. gs cells ( × ) were cultured in -well plates at • c for h and then infected with sgiv-gx (moi = ) for h. after the safe working concentration of each inhibitor was applied to the cells for h, the sgivinfected cells were incubated with aptamer cy -q ( nm) in the presence of each inhibitors for h at • c. the culture supernatants were then removed, and the cells were washed twice with pbs and collected for flow-cytometric analysis ( , events). normal gs cells incubated with cy -q ( nm) were used as the negative control; sgiv-infected cells with no inhibitor treatment were incubated with cy -q ( nm) and used as the positive control. the results are presented as the means ± sd of three independent experiments. the mcp entry efficiency was assessed with the protocol described by huang et al. ( ) , with some modifications. gs cells were cultured to % confluence in -well plates or mm glass-bottom dishes at • c for h and then infected figure | mcp endocytosis depends on dynamin. dynamin is essential for clathrin-coated vesicle formation and membrane budding in the late stage, and dynasore is a specific inhibitor of dynamin. (a) the fluorescent cy signal inside the sgiv-infected cells was significantly reduced in the dynasore-treated cells. (b) entry efficiency of the q -mcp complex was greatly and dose-dependently reduced in the presence of dynasore (to . % with µm dynasore, to . % at µm dynasore). p < . was considered statistically significant (**p < . ). with sgiv-gx (moi = ) for h. after pretreatment with each inhibitor at the safe working concentration for h, the sgiv-infected cells were incubated with aptamer cy -q ( nm) in the presence of each inhibitor for h at • c. the cultured cells were then transferred to • c to initiate mcp endocytosis into the sgiv-infected host cells. the internalization of the q -mcp complex was analyzed with flow cytometry and lscm. normal gs cells incubated with cy -q ( nm) were used as the negative control; sgiv-infected cells without any inhibitor were incubated with cy -q ( nm) and used as the positive control. entry efficiency was calculated with the following equation: entry efficiency = (x − control )/(control − control ) × %, where x is the flow-cytometric results for each sample of inhibitor-pretreated sgiv-infected cells incubated with cy -q ( nm) at • c; control is the flow-cytometric result for sgiv-infected cells incubated with cy -q ( nm) at • c, and control is the flowcytometric result for sgiv-infected cells incubated with cy -q ( nm) at • c. the results of the mcp entry assay for each inhibitor are presented as the means ± sd of three independent experiments. the experimental data in the study are expressed as means ± se. intergroup differences were analyzed with one-way analysis of variance (anova), with the spss . statistical software (ibm, armonk, ny, united states). a p value < . was considered statistically significant. confocal fluorescence imaging showed that the cy -labeled aptamer q (cy -q ) specifically bound to the surfaces of sgivinfected cells at • c, but not to normal gs cells. when cy -q was incubated with sgiv-infected cells at • c, the fluorescent signal was only visible around the cell plasma membrane, but not inside the cells (figure a) . the specific binding properties of aptamer cy -q were confirmed with a flow-cytometric analysis ( figure b) . as reported previously, aptamer q recognizes sgiv-infected cells with high specificity and affinity, and the protein detected on the target cell membrane is the viral mcp. therefore, aptamer q can be used as a specific probe to investigate the trafficking mechanism and endocytotic pathway of mcp in host cells during sgiv infection (yu et al., a) . the kinetics of the entry of the q -mcp complex into sgivinfected cells were analyzed with flow cytometry (figure a) and lscm (figure b) . the active internalization of mcp was blocked at • c (li et al., ) . as shown in figure a , after incubation with sgiv-infected cells at • c for different periods ( , , , , , or min) , no significant change in fluorescence was detected with flow cytometry, indicating that aptamer cy -q only bound to the cell plasma membrane, but endocytosis did not occur (figure a) . when the cells were transferred to • c, the entry of the q -mcp complex into the sgiv-infected cells was rapidly initiated. there was a time-dependent increase in the fluorescent signal within the cell cytoplasm, which peaked at min (figure a) . the cellspecific internalization of the q -mcp complex was also detected with lscm ( figure b) . after incubation at • c for h, the fluorescent signal was observed inside the sgiv-infected cells, indicating the endocytosis of the q -mcp complex. the control group of sgiv-infected cells treated with cy -library showed no fluorescence ( figure b ). relative to the control group of normal gs cells, the gs cells incubated with the safe working concentration of each inhibitor in l- medium retained their normal figure | caveolae/raft-dependent endocytosis is not involved in mcp entry during sgiv infection. genistein is a tyrosine kinase inhibitor that blocks caveolae/raft-dependent endocytosis. (a) lscm showed that even at a high concentration ( µm), genistein did not significantly alter the fluorescent signal in sgiv-infected cells pretreated with genistein. (b) there was no significant difference in the entry efficiency of mcp in sgiv-infected cells pretreated with or without genistein. genistein did not block mcp endocytosis. ns indicates not statistically significant. growth, and the cell viability rate exceeded %, indicating that the working concentrations of the inhibitors used in this study caused no significant cytotoxicity (supplementary figure s ). after pretreatment with each inhibitor at its safe working concentration for h, sgiv-infected cells were incubated with cy -q ( nm) at • c for h and then collected for flow-cytometric analysis. the fluorescent signals did not differ significantly in the sgiv-infected cells pretreated with or without each inhibitor, indicating that the inhibitors did not interfere with the binding of cy -q to its target on the plasma membranes of sgiv-infected cells (figure ) . according to our previous studies, the q aptamer ultimately accumulates around the nucleus after it is efficiently internalized into sgiv-infected cells (li et al., ; yu et al., a) . therefore, the effective inhibitory agents could reduce the endocytosis of q and cy signals in target sgiv-infected cells. chlorpromazine (cpz) prevents the assembly of coated pits on the cell surface, so different concentrations of cpz were used to analyze the role of the clathrin-mediated endocytotic pathway in the entry of the q -mcp complex into sgivinfected cells. as shown in figure a , cpz significantly reduced the fluorescent signal inside the sgiv-infected cells compared with that in the control group of sgiv-infected cells incubated with cy -q without cpz. the entry efficiency of the q -mcp complex also decreased significantly to . % at when treated with µm cpz and to . % with µm cpz, confirming the dose-dependent inhibitory effect of cpz on q -mcp entry (figure b) . dynamin is a cellular gtpase that is essential for clathrin-coated vesicle formation and membrane budding in the late stage. dynasore is a specific inhibitor of figure | mcp endocytosis is independent of macropinocytosis. macropinocytosis inhibitors eipa, ipa- , ml- , nsc , and rottlerin were used to analyze the role of macropinocytosis in the endocytosis of mcp by sgiv-infected cells. (a) lscm showed that even at high inhibitor concentrations ( µm eipa, µm ipa- , µm ml- , µm nsc , and µm rottlerin), there was no significant difference between the fluorescent signals in the sgiv-infected cells pretreated with or without the macropinocytosis inhibitors. (b) entry efficiency of mcp did not differ significantly between the sgiv-infected cells pretreated with or without the macropinocytosis inhibitors. therefore, mcp entry into sgiv-infected host cells is independent of macropinocytosis. figure | mcp endocytosis is ph dependent. upon internalization by endocytosis, the incoming substances are usually trafficked by the ph-dependent endosomal system. cq and nh cl inhibit endocytosis by preventing endosomal acidification, so they were used to assess whether a low endosomal ph interferes with mcp endocytosis. (a) lscm showed that both cq and nh cl reduced the internalization of the q -mcp complex by sgiv-infected cells. efficiency of mcp endocytosis was significantly reduced in the presence of cq (to . % with µm cq, to . % with µm cq) (b) or nh cl (to . % with mm nh cl, to . % with mm nh cl) (c) (**p < . ). dynamin and rapidly blocks the formation of clathrincoated vesicles (abban et al., ) . therefore, we used different concentrations of dynasore ( and µm) to confirm the role of dynamin in the entry of the q -mcp complex into sgiv-infected cells. the fluorescent signal inside the sgiv-infected cells was significantly reduced in the dynasore-treated cells (figure a) , and the entry efficiency was greatly and dose-dependently reduced in the presence of dynasore (to . % with µm dynasore, to . % with µm dynasore) ( figure b) . therefore, mcp entered the sgiv-infected host cells via clathrin-mediated dynamin-dependent endocytosis. figure | mcp endocytosis is dependent on cytoskeletal actin filaments. cellular uptake characteristics are related to the actin cytoskeleton, which is disrupted by cytod. therefore, cytod was used to analyze the role of the actin cytoskeleton in mcp endocytosis. (a) lscm showed that the disruption of actin dynamics by cytod significantly reduced the fluorescent signal inside sgiv-infected cells. (b) efficiency of mcp endocytosis decreased significantly to . % with µm cytod and to . % with µm cytod, demonstrating that the disruption of the actin cytoskeleton significantly inhibited mcp endocytosis (**p < . ). the inhibitor mβcd depletes cholesterol from the cell plasma membrane, and cholesterol plays a critical role in caveolae/raft-dependent endocytosis (huang et al., ) . therefore, we used mβcd to analyze the role of cholesterol in the entry of the q -mcp complex into sgiv-infected cells. as expected, mβcd ( mm) significantly reduced the fluorescent signal inside the sgiv-infected cells, and most fluorescence was observed outside the mβcd-treated sgivinfected cell membranes ( figure a) . the entry efficiency of the q -mcp complex also decreased significantly to . % after treatment with mm mβcd and to . % with mm mβcd ( figure b ). genistein is a tyrosine kinase inhibitor that blocks caveolae/raftdependent endocytosis (nabi, ) . therefore, we tested whether genistein blocks the entry of the q -mcp complex. as shown in figure , even at a high concentration ( µm), genistein did not block the entry of the q -mcp complex into sgiv-infected cells. therefore, the entry of mcp into sgiv-infected host cells is dependent on cholesterol and membrane fluidity, but does not involve caveolae/raft-dependent endocytosis. as well as clathrin-and caveolae/raft-dependent endocytosis, macropinocytosis has drawn increasing attention (wang et al., ) . macropinocytosis inhibitors, including the na + /h + exchanger inhibitor eipa, the pak inhibitor ipa- , the myosin ii inhibitor ml- , the rac gtpase inhibitor nsc , and the protein kinase c (pkc) inhibitor rottlerin, were used to analyze the role of macropinocytosis in the entry of the q -mcp complex into sgiv-infected cells. as shown in figure , even at high concentrations ( µm eipa, µm ipa- , µm ml- , µm nsc , and µm rottlerin), the macropinocytosis inhibitors did not block the entry of the q -mcp complex into sgiv-infected cells (figure ) . therefore, mcp enters sgivinfected host cells independently of macropinocytosis. to assess whether low ph interferes with the entry of the q -mcp complex, sgiv-infected cells were pretreated with various concentrations of chloroquine (cq) or ammonia chloride (nh cl), which act as inhibitors of endosomal acidification (wang et al., ) . as shown in figure a , both cq and nh cl reduced the internalization of the q -mcp complex into sgivinfected cells. the entry efficiency was greatly reduced in the presence of cq (to . % with µm, to . % with µm) ( figure b ) or nh cl (to . % with mm nh cl, to . % with mm nh cl) ( figure c) . therefore, mcp endocytosis during sgiv infection is dependent upon a low endosomal ph. to determine the role of actin filaments in mcp endocytosis, the chemical inhibitor cytod was used to inhibit the elongation of actin filaments (wang et al., ) . as shown in figure , the disruption of the actin dynamics by cytod significantly reduced the fluorescent signals inside the sgiv-infected cells. the entry efficiency also decreased significantly to . % with µm cytod and . % with µm cytod. therefore, mcp endocytosis into sgiv-infected cells was inhibited by the inhibitor cytod, indicating that mcp endocytosis during sgiv infection is dependent on cytoskeletal actin filaments. during viral infection, modifications appear in the host cell membranes and are transported by the vesicular transport system of the host cell (wickner and schekman, ; yu et al., a) . these modifications are associated with viral replication and host cell immunity and play critical roles in various physiological functions (abós et al., ; chinchar and duffus, ) . studying the entry pathways of modifications is important in understanding viral pathogenesis and the development of diagnostic tools and therapeutic approaches to the very early critical steps of viral infection. because aptamers specifically recognize and bind their target molecules, they are excellent molecular probes for various biological applications, such as pathogen detection, disease diagnosis, and viral pathogenesis research (sun and zu, ; kaur et al., ) . aptamer q recognizes sgiv-infected cells with high specificity and affinity, and its target protein on the target cell membrane is sgiv mcp. the specific interaction between q and mcp activates cellular signaling pathways, which results in the active internalization of the q -mcp complex via endocytotic mechanisms (yu et al., a) . therefore, aptamer q can be used as a specific probe to investigate the trafficking mechanism and endocytotic pathway of mcp in host cells during sgiv infection. as demonstrated with cy -q , the entry of mcp into sgiv-infected cells was rapidly initiated at a temperature of • c and was followed by a time-dependent increase in the fluorescent signal inside the cells. this signal accumulated around the nucleus. the fluorescence intensity reached its maximum within min, indicating that the entry of all m in the sgiv-infected cell membrane was completed within min. the endocytotic pathways predominantly include clathrinmediated endocytosis, caveolae/raft-dependent endocytosis, macropinocytosis, non-clathrin-caveolae/raft-dependent endocytosis, and some other still poorly characterized mechanisms (huang et al., ) . clathrin-mediated endocytosis is the classical endocytotic mechanism and also the bestcharacterized pathway. some viruses are reported to invade their host cells via clathrin-mediated endocytosis, such as betanodavirus (huang et al., ) , sgiv (wang et al., ) , and african swine fever virus (hernaez and alonso, ) . during clathrin-mediated endocytosis, clathrin is assembled on the plasma membrane to form clathrin-coated pits (ccps), which invaginate to form clathrin-coated vesicles containing the internalized material. cpz is a cationic amphiphilic agent that causes the misassembly of ccps, so it was used to determine the role of the clathrin-mediated endocytosis pathway in the entry of the q -mcp complex into sgiv-infected cells. in this study, the entry efficiency of the q -mcp complex decreased significantly and dose-dependently after cpz treatment. therefore, we conclude that mcp endocytosis during sgiv infection is dependent on clathrin-mediated endocytosis. another well-characterized endocytotic pathway is caveolae/raft-dependent endocytosis, which is an alternative to clathrin-mediated endocytosis. in this mechanism, the extracellular substance first binds to the specialized membrane domain called the "caveola, " which is composed of caveolin and associated with high levels of cholesterol. the signal cascade caused by the activation of tyrosine kinases ultimately results in the slow but efficient internalization of the substance (doherty and mcmahon, ) . some viruses, such as the tiger frog virus (guo et al., ) , infectious spleen and kidney necrosis virus (isknv) (guo et al., ) , and coronaviruses (nomura et al., ) , invade cells via caveola-dependent endocytosis but not clathrin-mediated endocytosis. as reported previously, cholesterol plays a critical role in caveolae/raft-dependent endocytosis (huang et al., ) . in this study, mβcd was used to deplete cholesterol from the plasma membrane of the host cell. as expected, mβcd significantly reduced the cholesterol during the entry of the q -mcp complex into sgiv-infected cells. however, the effect was insufficient to conclude that the internalization of the q -mcp complex is dependent upon caveolae-mediated endocytosis. according to the latest reports, cholesterol is also an essential factor in clathrin-mediated endocytosis because it regulates the fluidity of the plasma membrane, which may affect the formation of clathrin-coated vesicles (huang et al., ) . some viruses, including rhesus rhadinovirus (zhang et al., ) , betanodavirus (huang et al., ) , japanese encephalitis virus (das et al., ) , and chikungunya virus (hoornweg et al., ) , are reported to invade their host cells through cholesterol-dependent clathrin-mediated endocytosis. therefore, whether the entry of sgiv mcp into its host cells involves caveolae/raft-dependent endocytosis warranted clarification. caveolae/raft-dependent endocytosis not only is sensitive to cholesterol depletion but also regulated by reversible phosphorylation (nabi, ) . genistein is a natural flavonoid compound with various bioactivities and is used in the treatment of tumors (banerjee et al., ) , diabetes (behloul and wu, ) , and skin diseases (toutfaire et al., ) . genistein also acts as protein tyrosine kinase inhibitor. previous studies of tiger frog virus (guo et al., ) and isknv (guo et al., ) have shown that genistein regulates and blocks caveolar budding during caveolae/raft-mediated endocytosis through reversible phosphorylation. therefore, we used genistein to determine whether the entry of sgiv mcp into its host cells is dependent on caveolae/raft-dependent endocytosis. as shown in figure , genistein treatment, even at a high concentration ( µm), did not block the entry of the q -mcp complex into sgiv-infected cells. therefore, we conclude that the entry of sgiv mcp into its host cells is dependent upon cholesterol but does not involve caveolae/raft-dependent endocytosis. macropinocytosis is an endocytotic pathway that has recently drawn increasing attention. it plays a critical role in the cellular uptake of agents guggenheim et al., ) , the exchange of extracellular vesicles (such as exosomes and microvesicles) between cells (costa-verdera et al., ), cellular immune defenses (hohn et al., ) , and viral entry (huang et al., ) . both na + /h + exchanger activity and the functional actomyosin network are important in macropinocytosis, and many studies have reported that macropinocytosis requires signaling events and activated protein kinases such as pak , pkc, and gtpase rac . for example, the entry of sgiv into gs cells (wang et al., ) and of echovirus into polarized caco- cells (krieger et al., ) depends on pak and pkc and is associated with macropinocytosis. therefore, several inhibitors were used to analyze the role of macropinocytosis in mcp endocytosis during sgiv infection in this study. ml- was used to antagonize the phosphorylation of myosin light chain kinase, to regulate myosin ii activity, which blocks the functional actomyosin network involved in macropinocytosis. nsc was used to inhibit rac activity, rottlerin to inhibit pkc activity, and ipa- to inhibit pak activity in the host cells (huang et al., ) . however, even at high concentrations, these macropinocytosis inhibitors caused no statistically significant changes in the entry efficiency of mcp. these results suggest that macropinocytosis is not involved in mcp endocytosis. as reported previously, cellular uptake characteristics are also associated with the cytoskeleton (al soraj et al., ; he et al., ) . for example, cell-penetrating peptides (cpps) have been used as vectors for the cellular delivery of therapeutic drugs. they enter cells directly across the plasma membrane and also gain access via endocytosis pathways. al soraj et al. ( ) found that the disruption of the actin cytoskeleton with cytod inhibited cpp entry in the hela and a epithelial cell lines. by contrast, disruption of the actin cytoskeleton reduced the cellular uptake of dextran by hela cells but increased its uptake by a cells. this indicates that the actin cytoskeleton both promotes and interferes with endocytosis. to determine the role of the actin cytoskeleton during mcp endocytosis, cytod was used to destroy the actin cytoskeleton by inhibiting the elongation of actin filaments. disruption of the actin cytoskeleton significantly inhibited mcp endocytosis. after internalization, incoming substances are usually trafficked by the ph-dependent endosomal system, which is responsible for molecular sorting, recycling, degradation, storage, processing, and transcytosis. cq and nh cl inhibit endocytosis by preventing endosomal acidification (guo et al., ) . using these inhibitors, we demonstrated that the endocytosis of the q -mcp complex is ph dependent. in summary, in this study, we used a highly specific molecular probe, the aptamer q , to investigate the crucial events of mcp endocytosis into the host cell during sgiv infection. mcp entry into the host cell during sgiv infection is via clathrin-mediated endocytosis and is dependent on dynamin, cholesterol, low ph, and the actin cytoskeleton, but not on caveolae/raft-dependent endocytosis or macropinocytosis. this is the first time that a specific aptamer-based probe has been used to demonstrate the endocytosis of mcp during sgiv infection. the strategy developed in this study should be applicable to other types of viral infection, thus providing a convenient strategy for exploring viral pathogenesis and facilitating the development of diagnostic tools for and therapeutic approaches to viral infection. this work not only contributes greatly to our understanding of iridovirus pathogenesis but also provides an ideal molecule probe for exploring the behavior of dna viruses in living cells. all datasets generated for this study are included in the article/supplementary material. pl, qq, and qz conceived and designed the experiments. qy, ml, siw, and xw performed the main experiments. hx and shw cultured the cells and performed flow cytometry analysis. yy and hc contributed to the reagents, materials, and analyzed the tools. all authors reviewed the manuscript. we would like to thank the light of the western china visiting scholars program and outstanding chinese and foreign youth exchange program of china association for science and technology. the supplementary material for this article can be found online at: https://www.frontiersin.org/articles/ . /fmicb. . /full#supplementary-material figure s | identification of the safe working concentrations of the inhibitors with a cell viability assay. relative to the control group of normal gs cells, gs cells incubated with the working concentration of each inhibitor in l medium retained their normal growth, and 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authors: chang-liao, wan-ping; lee, an; chiu, yu-han; chang, hui-wen; liu, je-ruei title: isolation of a leuconostoc mesenteroides strain with anti-porcine epidemic diarrhea virus activities from kefir grains date: - - journal: front microbiol doi: . /fmicb. . sha: doc_id: cord_uid: qqhivgkq swine grown under commercial conditions are vulnerable to environmental exposure to several viruses, which may cause infectious diseases and spread easily and rapidly, resulting in significant economic losses in animal husbandry. previous studies have suggested that probiotics seem to be a new and promising alternative to vaccinations to protect animals against potential viral infections. in this study, we used the vero cell culture model of infection to study porcine epidemic diarrhea virus (pedv). we screened lactic acid bacteria (lab) with anti-pedv potential from kefir grains, which are starter cultures used to ferment milk into kefir. twenty-nine lab strains were isolated and identified as enterococcus durans, lactobacillus kefiri, lactococcus lactis, and leuconostoc mesenteroides, according to s ribosomal rna (rrna) and rpoa gene sequence analyses. the anti-pedv activities of the lab intracellular extracts were compared, and the intracellular extracts of ln. mesenteroides showed higher anti-pedv activities than that of the other species. among the ln. mesenteroides strains, a strain designated ypk showed a higher growth rate than that of the other strains and was further evaluated for its anti-pedv activity. the results showed that the intracellular extracts of ln. mesenteroides ypk possessed in vitro prophylactic, therapeutic, and direct-inhibitory effects against pedv in the vero cell model. the expression levels of type interferon (ifn)-dependent genes, including myxovirus resistance (mx ) and interferon-stimulated gene (isg ), were significantly increased after treatment with intracellular extracts of ln. mesenteroides ypk for h. such expression suggests that the anti-pedv activity of ln. mesenteroides ypk could be attributed to its up-regulatory effect on the expression of mx and isg genes. these results suggested that ln. mesenteroides ypk has the potential to provide some levels of host protection against pedv infections. in order to increase swine and poultry production, it is common to raise animals in high-density populations. swine grown under commercial conditions are vulnerable to environmental exposure to several viruses. some viruses may cause infectious diseases, which are spread easily and rapidly cause significant economic losses in animal husbandry. vaccination is one of the most efficient strategies to prevent viral diseases and control infections and is the current industry standard. efficacious vaccines have been developed and applied successfully for the prevention of several infectious viral diseases in swine, such as porcine parvovirus (ppv), foot-and-mouth disease virus (fmdv), porcine circovirus type (pcv ), and transmissible gastroenteritis virus (tgev; meng, ; gerdts and zakhartchouk, ) . however, some of the vaccination methods require the operator to handle each animal, induce the stress on the animal, and are time-consuming and costly (marangon and busani, ) . although efficacious vaccines are available to reduce the impact of the infectious viruses mentioned above, unfortunately, substantial challenges remain in obtaining safe and efficacious vaccines for a variety of newly emerging and re-emerging viruses, such as african swine fever virus and porcine epidemic diarrhea virus (pedv; lee, ) . pedv is a member of the genus alphacoronavirus in the family coronaviridae of the order nidovirales and has emerged as a significant pathogen causing lethal watery diarrhea, vomiting, and dehydration in nursing piglets. highly pathogenic strains of pedv have mortality rates of - % in neonatal piglets, which has resulted in huge economic losses to the swine industry worldwide (koonpaew et al., ; wang et al., ) . accumulated evidence indicates that pedv encodes defensive mechanisms to evade virus recognition by host pattern recognition receptors (prrs) present on antigen-presenting cells, inhibit interferon (ifn) induction, and antagonize ifn signaling and antiviral effector machinery (hao et al., ; koonpaew et al., ) . pedv has been studied extensively and some vaccines have been developed against pedv, but the efficacy of these vaccines in the field remains questionable (wang et al., ) . probiotics, which are live microorganisms that when administered in adequate amounts confer a health benefit on the host, have long been used as feed additives because of their abilities to normalize gut microbiota, boost the immune system, prevent diarrhea, and improve feed conversion efficiency (fontana et al., ; alonso and guarner, ) . the effect of probiotics is achieved mainly through the intervention on gut microbiota, which increases the levels of beneficial bacteria and decreases the pathogenic populations in the gastrointestinal tract (liu et al., ; yadav and shukla, ) . in addition to the microbiota-modulatory properties, recent studies showed that the immunomodulatory activity of probiotics is another important mechanism of action of probiotics for the inhibition of pathogens (llewellyn and foey, ; azad et al., ) . those immunoregulatory probiotics provide host protection against pathogenic infections by modulating innate and adaptive antiviral immune responses (villena et al., ) . several probiotic strains, most of them belonging to lactobacillus and bifidobacterium genera, have been shown to perform antiviral activities (villena et al., ; arena et al., ) . these antiviral activities could be mediated by the immunomodulatory properties of probiotics because it was observed that administration of probiotics induced the expression of ifn and interferonstimulated genes (isgs), which are crucial components of the ifn responses and play a key role in establishing an antiviral state for virus clearance and restriction of spread (zelaya et al., ; villena et al., ; arena et al., ; eguchi et al., ) . if probiotics have antiviral activity, it seems to be a new and promising alternative to vaccinations to protect animals against potential viral infections (al kassaa et al., ) . kefir is an acidic and mildly alcoholic fermented milk product that is believed to have many beneficial activities, such as hypocholesterolemic activity, antibacterial and antifungal activities, antitumor activity, immunomodulatory activity, and quickening of wound healing (bourrie et al., ) . traditionally, kefir is produced from milk fermented with a mixed microflora confined to a matrix of discrete kefir grains, which are a combination of bacteria and yeasts in a symbiotic matrix (marshall and cole, ) . various bacteria and yeasts have been identified in kefir grains. the bacteria present in kefir grains may include acetobacter, bifidobacterium, lactobacillus, lactococcus, leuconostoc, oenococcus, and streptococcus, while the yeasts present in kefir grains may include candida, kluyveromyces, and pichia (lin et al., ; wang et al., ; bourrie et al., ) . numerous bacterial strains with specific properties, such as hypocholesterolemic effect, antiallergenic effect, immunoregulatory effects, and antipathogenic activities, have been isolated from kefir grains (prado et al., ) . however, to the best of our knowledge, following a thorough review of the relevant literature, there has been no study to isolate bacterial strains with antiviral activities from kefir grains. in the present study, we used the vero cell culture model of infection by pedv to screen lactic acid bacteria (lab) with anti-pedv activities from kefir grains. the anti-pedv activities of the lab strains were evaluated in prophylactic, therapeutic, and direct-inhibitory models. the strains with anti-pedv activities were further studied to define their effects on the expression of type ifn-dependent genes, including '- '-oligoadenylate synthetase (oas ), myxovirus resistance (mx ), and interferon-stimulated gene (isg ) in vero cells. a total of lab strains were isolated from kefir grains according to the method described by lin et al. ( ) . the lab isolates were cultured in de mann, rogosa, sharpe (mrs) broth (oxoid, basingstoke, uk) at °c for h without shaking. the bacterial concentrations were measured by optical density readings at nm or by traditional colony counting methods (vinderola et al., ) . for colony morphological observation, the lab isolates were cultivated on mrs agar plates (oxoid) or blood agar plates frontiers in microbiology | www.frontiersin.org (merck, darmstadt, germany) at °c for h and then observed. for cell morphological observation, the lab isolates were cultured in mrs broth (oxoid) at °c for h without shaking. the bacterial cells were harvested by centrifugation at , g for min at °c, stained with gram stain (sigma-aldrich, st. louis, mo, usa) according to the manufacturer's instructions, and observed under a microscope. unstained bacteria were observed under a phase-contrast microscope. molecular identification of the bacterial isolates was performed by the methods described by weisburg et al. ( ) and naser et al. ( ) . genomic dna of the bacterial isolates was isolated using the dneasy blood & tissue kit (qiagen inc., valencia, ca, usa). the standard s ribosomal rna (rrna) gene primers and rna polymerase α subunit gene rpoa primers were used for pcr to amplify the s rrna gene and rpoa gene sequences of the bacterial isolates, respectively (weisburg et al., ; naser et al., ) . the resultant pcr products were sequenced by an automatic sequencing service provided by genomics biotech inc., (new taipei city, taiwan). the nucleotide sequences of the s rrna and rpoa genes were aligned using the national center for biotechnology information's basic local alignment search tool (blast). for the determination of biochemical characteristics, the carbon source use profiles of the lab isolates were determined using an api ch system (biomerieux, inc., marcy l'etoile, france) according to the manufacturer's instructions. for the determination of physiological characteristics, the lab isolates were cultured at °c, °c, ph . , or in the presence of % ethanol according to the methods described by lin et al. ( ) . before the anti-pedv activity assay, the cytotoxicity of the lab isolates on the african green monkey kidney cell line vero was evaluated according to the method described by mosmann ( ) . the vero c cells were purchased from the bioresource collection and research center of food industry research and development institute (bcrc, hsinchu, taiwan) and were routinely grown at °c in a humidified atmosphere of % air and % co in dulbecco's modified eagle's medium (dmem, gibco, grand island, ny, usa) supplemented with % fetal bovine serum (fbs; moregate biotech., queensland, australia). for evaluation of cytotoxicity, a . -ml aliquot of the -h culture of each bacterial strain was centrifuged at , g for min at °c. the bacteria were collected, washed twice with sterile phosphate-buffered saline (pbs; . m, ph . ), resuspended in . ml of sterile pbs, and sonicated for min with an ultrasonicator (model xl, misonix, farmingdale, ny). the sonicated bacteria were fractioned into intracellular extracts and cell-wall pellet fractions by subsequent centrifugation at , g for min at °c. the intracellular extracts were harvested, and the cytotoxicity on vero cells was determined using the -( , -dimethylthiazol- -yl)- , diphenyltetrazolium bromide (mtt) assay according to the method described by mosmann ( ) . briefly, vero cells were seeded at a density of . × cells/well on a -well plate in μl of dmem. after incubating at °c for h, μl of the bacterial intracellular extracts were added into each well and incubated at °c for another h. after washing twice with sterile pbs, the cells were incubated with μl of mtt ( mg/ml in pbs) at °c for h. after the incubation, the medium was removed, and μl of dimethyl sulfoxide (dmso) were added into each well to dissolve the formazan crystals. the absorbance was measured at nm using a microplate reader (victor , perkinelmer inc., waltham, ma, usa), and percentages of cell metabolic activity were calculated as follows: absorbance of sample % / where the absorbance of sample is the absorbance of cells treated with test sample and the absorbance of control is the absorbance of cells treated with dmso. the pedv taiwan pintung strain was isolated in early from the intestinal homogenate of a -day-old suckling pig in taiwan and adapted to vero cells as previously described by chang et al. ( ) . viral infection and propagation were performed in vero cells according to the method described by chang et al. ( ) . before the anti-pedv activity screening experiments were conducted, the viral titers of pedv were adjusted to fifty-percent tissue culture infective dose (tcid )/ml, and the intracellular extracts of bacterial isolates were prepared as described above. vero cells were seeded at a density of × cells/well on a -well plate in μl of modified postinoculation (pi) medium containing dmem (gibco, grand island, ny, usa) supplemented with tryptose phosphate broth ( . %), yeast extract ( . %), and μg/ml of trypsin. after incubating at °c for h, μl of the bacterial intracellular extracts were added into each well and incubated at °c for another h. after washing the cells twice with pi medium, μl of pi medium containing tcid /ml of the pedv was added into each well and incubated at °c for h. after h of incubation, the supernatants were replaced by fresh pi medium and the cells were maintained at °c for h. after washing twice with sterile pbs ( . m, ph . ), the cell metabolic activity was determined using the mtt assay as described previously. effects of lab intracellular extract, cell-wall fraction, and extracellular supernatant against pedv a . -ml aliquot of the -h culture of each lab strain was centrifuged at , g for min at °c. the resultant extracellular supernatants and bacterial cells were harvested separately. the bacterial cells were washed twice with sterile pbs, resuspended in . ml of sterile pbs, and sonicated for min with an ultrasonicator (model xl, misonix, farmingdale, ny, usa). the sonicated bacterial cells were fractioned into intracellular extracts and cell-wall pellet fractions by subsequent centrifugation at , g for min at °c. the extracellular supernatants, intracellular extracts, and cell-wall fractions of each bacterial strain were harvested, and the anti-pedv activities were evaluated as described above. vero cells were seeded on a -well plate and incubated at °c for h. afterward, the cells were washed with pi medium, and μl of pi medium containing tcid /ml of pedv were added into each well and incubated at °c for h. after h of incubation, the supernatants were replaced by μl of fresh pi medium and μl of the bacterial intracellular extracts. the cells were maintained at °c for h. after washing twice with sterile pbs, the cell metabolic activity was determined by mtt assay as described previously. . glyceraldehyde -phosphate dehydrogenase (gapdh) was chosen as an internal control, and all relative gene expression levels were normalized to gapdh by the comparative c t method. the primers used for the relative quantification are provided in table . the data were analyzed using spss version software (ibm spss, new york, ny, usa). one-way analysis of variance (anova) followed by duncan's multiple range test was used to detect the differences among the means of the different treatment groups, and a value of p less than . was considered significant. student's t-test was used to detect the differences between the treatment and control groups, and a value of p less than . was considered significant. each experiment was conducted in triplicate, and all results were expressed as means ± standard deviations. twenty-nine lab strains were isolated from kefir grains. according to the s rrna and rpoa gene sequence analysis, three isolated strains belong to the species enterococcus durans, isolated strains might belong to the species lactobacillus kefiri, five isolated strains might belong to the species lactococcus lactis, and five isolated strains might belong to the species leuconostoc mesenteroides ( table ). the in vitro prophylactic effects of the lab strains on pedv were evaluated in the vero cell model. vero cells were pretreated with the intracellular extracts of lab for h. after the removal of the bacterial intracellular extracts, the vero cells were infected with pedv. if the intracellular extracts of lab possessed an in vitro prophylactic effect against pedv, the bacterial pretreated vero cells would be infected with less pedv and thus would show higher cell metabolic activity than the un-pretreated cells. the in vitro prophylactic effects of the bacterial intracellular extracts of different lab species against pedv were compared, and human ifn-α b was used as a positive control. vero cells pretreated with ifn-α b prior to pedv infection showed a significantly higher cell metabolic activity and less cytopathic effects than the un-pretreated cells (figures , ) , indicating that ifn-α b possessed an in vitro prophylactic effect against pedv. among the cells pretreated with the intracellular extracts of different lab species, the cells pretreated with the intracellular extracts of ln. mesenteroides showed significantly higher cell metabolic activity than those pretreated with the intracellular extracts of the other lab species (figure ). in addition, vero cells pretreated with the intracellular extracts of ln. mesenteroides also showed less cytopathic effects than the un-pretreated cells (figure ) , indicating that ln. mesenteroides possessed an in vitro prophylactic effect against pedv. the in vitro prophylactic effects of the five strains of ln. mesenteroides isolated from kefir grains on pedv were further compared with each other. as shown in figure , the metabolic activity of vero cells pretreated with the intracellular extracts of ln. mesenteroides, regardless of which strain, were similar to those pretreated with ifn-α b (p > . ) but were significantly higher than the un-pretreated cells (p < . ), indicating that all the ln. mesenteroides strains isolated from kefir grains possessed in vitro prophylactic effects against pedv. in vitro prophylactic effects of ln. mesenteroides ypk intracellular extract, cell-wall fraction, and extracellular supernatant against pedv among the ln. mesenteroides strains, a strain designated ypk showed a higher growth rate than the other strains (data not shown) and was further evaluated for its basis of anti-pedv activity. we compared the in vitro prophylactic effects of intracellular extracts, cell-wall fractions, and extracellular supernatants of ypk against pedv. as shown in figure , the metabolic activity of vero cells pretreated with the intracellular extracts and extracellular supernatants of ypk were similar to each other and were significantly higher than those of un-pretreated cells or those pretreated with the cell-wall fractions of ypk (p < . ), indicating that both of the intracellular extracts and extracellular supernatants of ypk possessed in vitro prophylactic effects against pedv. vero cells were infected with pedv for h, and then the remaining pedv was removed. the pedv-infected cells were treated with the intracellular extracts of ypk or ifn-α b for h, and then the metabolic activities of vero cells were determined. as shown in figure , pedv-infected vero cells treated with the intracellular extracts of ypk for h showed higher cell metabolic activity than the untreated cells or those treated with ifn-α b (p < . ), indicating that the intracellular extracts of the ypk strain possessed an in vitro therapeutic effect against pedv. vero cells were co-incubated with pedv and the intracellular extracts of ypk or ifn-α b for h. after removal of the pedv and intracellular extracts of ypk , the vero cells were incubated for h, and then the metabolic activities of the vero cells were determined. the metabolic activities of the vero cells treated with the intracellular extracts of ypk and pedv simultaneously were significantly higher than those treated with pedv alone or those treated with pedv and ifn-α b (figure ; p < . ). these results suggested that the intracellular extracts of ypk possessed an in vitro direct-inhibitory effect against pedv in vero cells. in order to elucidate the antiviral mechanisms of the ypk strain, the expression levels of type ifn-dependent genes, including oas , mx , and isg , were quantified in vero cells after , , or h of treatment with the intracellular extracts of ypk . as shown in figure , ifn-α b, which served as the positive control, significantly increased the expression levels of oas , mx , and isg genes in vero cells at and h. the intracellular extracts of ypk also significantly increased the expression levels of mx and isg genes but did not affect the expression levels of the oas gene in vero cells at h. however, the expression levels of oas , mx , and isg genes in vero cells did not differ between the untreated and ypk -treated groups at h. according to the s rrna and rpoa gene sequence analysis, ypk exhibited . and . %, respectively, identity with ln. mesenteroides (table ) , and its s rrna and rpoa gene sequences were deposited in the ncbi genbank database under accession number mt and mt , respectively. macroscopic observation showed that ypk exhibited a smooth and grayish white colony morphology and did not possess hemolytic capacity on blood agar plate. the cells of ypk appeared purple after gram staining, indicating the strain ypk was gram positive. microscopic observation showed the cells of ypk were observed as spherical or lenticular forms. analysis on the basis of phenotypic (including gramstain-positive, catalase-negative, nonmotile, and asporogenous) and physiological characteristics (including growth at or °c but not growth at ph . or in % ethanol) indicated that ypk was closely related to species ln. mesenteroides. to further confirm the identification of ypk with the species ln. mesenteroides, the biochemical characteristics of ypk were compared with those of ln. mesenteroides subsp. cremoris atcc , ln. mesenteroides subsp. dextranicum atcc , and ln. mesenteroides subsp. mesenteroides atcc by using the api ch system. analysis of carbon all data are expressed as mean ± sd (n = ). bars marked with a star or double stars mean that they are significantly different from the control (cells treated with pbs) at the or % confidence level, respectively. source utilization profiles indicated that both ypk and ln. mesenteroides subsp. dextranicum atcc grew on six out of carbohydrates, including n-acetyl glucosamine, d-fructose, d-glucose, d-mannose, saccharose, and d-trehalose. distinct variation was observed between ypk and ln. mesenteroides subsp. cremoris atcc in the metabolism of the sugars d-fructose, d-mannose, and d-trehalose. additionally, distinct variation was observed between ypk and ln. mesenteroides subsp. cremoris atcc in the metabolism of the carbohydrates amygdaline, l-arabinose, cellobiose, esculine, d-galactose, β-gentiobiose, d-lactose, maltose, α-methyl-d-glucoside, d-raffinose, ribose, d-turanose, and d-xylose. therefore, the carbon source use characteristics of ypk were similar to those of ln. mesenteroides subsp. dextranicum. according to the results of microscopic observations, biochemical characteristics, and the s rrna and rpoa gene sequence analysis, the features of ypk were consistent with those of ln. mesenteroides subsp. dextranicum, as described in bergey's manual of systematic bacteriology (vos et al., ) . the vero cell line is one of the most commonly used cell lines for pedv isolation and propagation (koonpaew et al., ) . in this study, we used a vero cell culture model to evaluate the in vitro prophylactic effects of lab against pedv. four lab species, including e. durans, lb. kefiri, lc. lactis, and ln. mesenteroides , were isolated from kefir grains, and the in vitro prophylactic effects of the intracellular extracts of these four species against pedv infection in vero cells were compared. among these four lab species, the intracellular extracts of ln. mesenteroides showed a higher in vitro prophylactic effect against pedv than the other species did (figure ) . in addition to the in vitro prophylactic effect, the intracellular extracts of ln. mesenteroides ypk also possessed in vitro therapeutic effect and in vitro direct-inhibitory effects against pedv in vero cells (figures - ) . vero cells have a major deletion in the type ifn gene cluster, which results in ifn deficiency (desmyter et al., ; koonpaew et al., ) . although vero cells do not secrete type ifns when infected by viruses, they still have the type ifn receptors and respond normally to type ifns. therefore, vero cells were widely used to compare virus-mediated ifn antagonism specific to the ifn signaling pathway (simmons et al., ) . in the in vitro prophylactic and therapeutic models, the intracellular extracts of ln. mesenteroides ypk did not directly interact with pedv by physical contact. therefore, the in vitro prophylactic and therapeutic effects of the intracellular extracts of ln. mesenteroides ypk against pedv in vero cells seem not be attributed to the direct interaction of bacterial components or metabolites with virus. since the ifn pathway is crucial in initiating viral resistance, we suggest that the in vitro prophylactic and therapeutic effects of the intracellular extracts of ln. mesenteroides ypk against pedv in vero cells could be attributed to its effect on the ifn signaling pathway in vero cells. stimulation of innate immune responses by probiotics could be one of the mechanisms responsible for the protection provided by probiotics against viral infection. other proposed mechanisms include inhibition of virus adsorption and penetration into cells as a result of direct interaction of probiotics and virus, competition between probiotics and virus for epithelial cell receptors, and secretion of metabolites with antiviral activity (mousavi et al., ; biliavska et al., ) . previous studies have shown that specific probiotic bacteria bind to and inactivate rotaviruses and vesicular stomatitis viruses, which lead to blocking of the virus adsorption on the cell (salminen et al., ; kanauchi et al., ) . besides the direct interaction between probiotics and viruses, specific probiotic bacteria could interact with epithelial and mucosal cells and compete with pathogens for attachment to cell receptors, thereby preventing invasion into the cells by a virus (fernandez-duarte et al., ; mousavi et al., ) . in addition, specific probiotic bacteria could synthesize some antiviral metabolites, such as lactic acid, hydrogen peroxide, or bacteriocins (al kassaa et al., ) . in the present study, the intracellular extracts of ln. mesenteroides ypk possessed an in vitro direct-inhibitory effect against pedv in vero cells (figure ) . future studies will be aimed at identifying the mechanisms of the in vitro direct-inhibitory effect of the intracellular extracts of ln. mesenteroides ypk against pedv in vero cells. numerous mechanisms for the immunomodulatory properties of probiotics have been proposed. some extracellular polysaccharides produced by specific probiotic bacteria possess immunomodulatory activities, which induce an increase in the expression of ifn-α, ifn-β, and the antiviral factors mx and rnase l in porcine intestinal epithelial cells (kanmani et al., ) . beside extracellular polysaccharides, some cellular components, such as dna and bacterial cell-wall components, including peptidoglycan, s-layer proteins, teichoic acids, capsule, and pellicle, as well as other released peptides could modulate the innate antiviral immune response (quinteiro-filho et al., ) . in the present study, pretreatment of vero cells with the extracellular supernatants or intracellular extracts of ln. mesenteroides ypk for h before infection with pedv showed higher cell metabolic activities than those of un-pretreated cells, indicating that both of the intracellular extracts and extracellular supernatants of ln. mesenteroides ypk possessed in vitro prophylactic effects against pedv in vero cells. however, pretreatment of pedv cells with the cell-wall fractions of ln. mesenteroides ypk for h before infection with pedv did not impede pedv replication. according to these observations, we suggested that the immunomodulatory activity of ln. mesenteroides ypk seems not to rely on the structural cell components. future studies should be aimed at assessing the molecular mechanism(s) responsible for the observed effects. type ifns exert their antiviral activities though the induction of hundreds of isgs (lenschow et al., ) . classical isgs responsible for inhibition of viral infection include oas , mx , and isg (schoggins, ) . oas , which belongs to the oas enzyme family, is activated by double-stranded rna binding, catalyzes the formation of ′- ′ oligoadenylates to activate cellular rnase l, which in turn, degrade cellular and viral rna (choi et al., ) . mx is a dynamin-like gtpase that appears to target viral nucleocapsids, resulting in the inhibition of viral rna polymerase activity, effectively blocking both transcription and replication of the virus (schoggins, ) . isg is a small, ubiquitin-like molecule that has numerous antiviral functions, including inhibition of virus release, isgylation of both viral and host proteins, and immunomodulatory cytokinelike properties in its unconjugated form (schoggins, ) . in the present study, we determined the effects of intracellular extracts of ln. mesenteroides ypk on the expression levels of oas , mx , and isg genes in vero cells and found that treatment of vero cells with the intracellular extracts of ln. mesenteroides ypk did not affect the expression levels of the oas gene but significantly increased the expression levels of mx and isg genes h after treatment (figure ) , indicating that the anti-pedv activity of the intracellular extracts of ln. mesenteroides ypk could be attributed to its up-regulatory effect on the expression of mx and isg genes in vero cells. according to the results of microscopic observations, biochemical characteristics, and the s rrna and rpoa gene sequence analysis, ypk was identified as ln. mesenteroides subsp. dextranicum. ln. mesenteroides are commonly associated with foods, such as fermented dairy products (e.g., cheese, yogurt, and kefir), fermented vegetables (e.g., sauerkraut and kimchi), and fermented meats (holland and liu, ) . the long history of safe consumption of ln. mesenteroides in traditional fermented foods has led to the conclusion that it is generally regarded as safe (gras; flórez et al., ) . several ln. mesenteroides strains are reported to have anti-listerial, antiviral, or immunomodulatory activities (seo et al., ; shao et al., ) . since the production of exopolysaccharides and bacteriocins are important properties of ln. mesenteroides, the probiotic characteristics of ln. mesenteroides could be attributed to their production of exopolysaccharides and bacteriocins. several studies demonstrated that some bacteriocins produced by ln. mesenteroides have anti-pathogenic activities (stiles, ; holland and liu, ; arakawa et al., ) , and some exopolysaccharides produced by ln. mesenteroides showed antiviral and immunomodulatory activities (nácher-vázquez et al., ; mahdi et al., ) . in our study, we found that the intracellular extracts of ln. mesenteroides ypk possessed in vitro prophylactic, therapeutic, and direct-inhibitory effects against pedv in vero cells, which occur in part through the up-regulation of mx and isg expression in vero cells. to the best of our knowledge, based on a thorough review of the relevant literature, this scientific report is the first of anti-pedv potential for ln. mesenteroides. future research will be conducted to evaluate the protection efficiency of ln. mesenteroides ypk against pedv infections in piglet infectious challenge models. the lab strain ypk isolated from kefir grains was genotypically and phenotypically characterized as belonging to ln. mesenteroides subsp. dextranicum. ln. mesenteroides subsp. dextranicum ypk displayed in vitro prophylactic, therapeutic, and direct-inhibitory effects against pedv in vero cells via up-regulation of mx and isg expression in vero cells. these findings suggest that ln. mesenteroides subsp. dextranicum ypk has a potential to acts as an antiviral agent for protection against pedv infections. the datasets presented in this study can be found in online repositories. the names of the repository/repositories and accession number(s) can be found in the article/supplementary material. j-rl, w-pc-l, and h-wc contributed to conception and design of the study. w-pc-l, al, and y-hc carried out the experiments and did the data analysis. w-pc-l and j-rl wrote the first draft of the manuscript. all authors contributed to the manuscript revision, read and approved the submitted version. the corresponding author takes primary responsibility for communication with the journal and editorial office during publication. antiviral potential of lactic acid bacteria and their bacteriocins linking the gut microbiota to human health production of a bacteriocin-like inhibitory substance by leuconostoc mesenteroides subsp. dextranicum m isolated from mongolian fermented mare milk, airag immunobiosis and probiosis: 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beneficial effects against rotavirus infection viability of probiotic (bifidobacterium, lactobacillus acidophilus and lactobacillus casei) and nonprobiotic microflora in argentinian fresco cheese bergey's manual of systematic bacteriology investigation of microorganisms involved in biosynthesis of the kefir grain emerging and re-emerging coronaviruses in pigs s ribosomal dna amplification for phylogenetic study recent systems biology approaches for probiotics use in health aspects: a review nasal priming with immunobiotic lactobacillus rhamnosus modulates inflammation-coagulation interactions and reduces influenza virus-associated pulmonary damage this research was conducted using funds partially provided by grants from the ministry of science and technology (grant nos. most - -b- - and most - -b- - ) and academia sinica (grant nos. as-kpq- -itar-td and as-kpq- -itar-td ). key: cord- -w spqqt authors: huan, yuchen; kong, qing; mou, haijin; yi, huaxi title: antimicrobial peptides: classification, design, application and research progress in multiple fields date: - - journal: front microbiol doi: . /fmicb. . sha: doc_id: cord_uid: w spqqt antimicrobial peptides (amps) are a class of small peptides that widely exist in nature and they are an important part of the innate immune system of different organisms. amps have a wide range of inhibitory effects against bacteria, fungi, parasites and viruses. the emergence of antibiotic-resistant microorganisms and the increasing of concerns about the use of antibiotics resulted in the development of amps, which have a good application prospect in medicine, food, animal husbandry, agriculture and aquaculture. this review introduces the progress of research on amps comprehensively and systematically, including their classification, mechanism of action, design methods, environmental factors affecting their activity, application status, prospects in various fields and problems to be solved. the research progress on antivirus peptides, especially anti-coronavirus (covid- ) peptides, has been introduced given the covid- pandemic worldwide in . alexander fleming discovered lysozyme in , and this discovery marked the birth of modern innate immunity. since then, antibiotics and antimicrobial peptides (amps) have been discovered. a total of , amps have been reported in the antimicrobial peptide database (apd ) updated on august , . different types of amps have the following commonalities: their number of amino acid residues is between and (average: . ), and almost all amps are cationic (average net charge: . ). however, several anionic amps also exist, and they have several acidic amino acids like aspartic acid and glutamic acid (malkoski et al., ; schittek et al., ; lai et al., ) . the anti-microbial resistance of microorganisms is becoming increasingly serious with the abuse of antibiotics in medicine, agriculture and animal husbandry, especially in developing countries. research from kenya has detected substantial amounts of antibiotic residues in edible meat (ayukekbong et al., ) . the prevalence of vancomycin-resistant enterococcus (vre) and methicillin-resistant staphylococcus aureus (mrsa) is increasing in clinical medicine, so the countermeasures are urgently needed to address these bacterial infections. however, from the http://aps.unmc.edu/ap/ perspective of pharmaceutical companies, the development of new antibiotic drugs results in low profit. thus, replacing antibiotics has become a consideration in the pharmaceutical, agricultural, animal husbandry, and food industries. research on amps is continuously developing and considerable amounts of data on amps have been stored in amp databases. however, the mechanism of amps remains incompletely understood, and further work needs to be performed to determine the relationship between different physicochemical properties to obtain low-cost and highly safe amps with remarkable antimicrobial effects and the specificity and a high capacity for synergies of amps should also be further developed (lazzaro et al., ) . the diversity of natural amps causes difficulty in their classification. amps are classified based on ( ) source, ( ) activity, ( ) structural characteristics, and ( ) amino acid-rich species (figure ). the sources of amps can be divided into mammals (human host defense peptides account for a large proportion), amphibians, microorganisms, and insects according to statistical data in apd . the amps found in oceans have also attracted widespread attention. mammalian antimicrobial peptides are found in human, sheep, cattle, and other vertebrates. cathelicidins and defensins are the main families of amps. defensins can be divided into α-, β-, and θ-defensins depending on the position of disulfide bonds (reddy et al., ) . human host defense peptides (hdps) can protect human from microbial infections but show different expressions in every stage of human growth. for example, cathelicidin ll- , a famous amp derived from the human body, is usually detected in the skin of newborn infants, whereas human betadefensin (hbd- ) is often expressed in the elderly instead of the young (gschwandtner et al., ) . hdps can be identified in many parts of the body such as skin, eyes, ears, mouth, respiratory tract, lung, intestine, and urethra. besides, amps in human breast milk also play an important role in breastfeeding because it can decrease the morbidity and mortality of breastfeeding infants (field, ) . what's interesting is that casein (peptide derived from β-casein - aa), identified in colostrum, shows different levels in preterm human colostrum and term human colostrums . dairy is an important source of amps, which are generated through milk enzymatic hydrolysis. several amps have been identified from α-lactalbumin, β-lactoglobulin, lactoferrin, and casein fractions, and the most famous peptide obtained is lactoferricin b (lfcinb) (sibel akalın, ) . furthermore, whether the amps derived from dairy products can be used for dairy preservation is also an interesting subject to develop. in addition to antimicrobial activity, hdps, such as cathelicidins and defensins, also affect immune regulation, apoptosis, and wound healing (wang, ) . antimicrobial peptides from amphibians play an important role in the protection of amphibians from the pathogens that have induced the global amphibian population decline (rollins-smith, ). frogs are the main source of amphibian amps and the most famous amp from frogs is magainin; the skin secretions of frogs from genera xenopus, silurana, hymenochirus, and pseudhymenochirus under the pipidae family are rich in amps (conlon and mechkarska, ) . furthermore, cancrin, which has an amino acid sequence of gsaqpykqlhkvvnwdpyg, has been reported as the first amp from the sea amphibian rana cancrivora (lu et al., ) . this marks a broader source of amps of amphibians. figure | classification of antimicrobial peptides. (semreen et al., ) . myticusin-beta is an immune-related amp of mytilus coruscus and a promising alternative to antibiotics (oh et al., ) . moreover, ge , known as pardaxin, is a marine amp and the ge -based vaccine has shown the ability to enhance antitumor immunity in mice (huang et al., ) . the activity of amps can be divided into categories according to the statistics of the adp database. these categories can be summarized as antibacterial, antiviral, antifungal, antiparasitic, anti-human immunodeficiency virus (hiv), and anti-tumor peptides (figure ). antibacterial peptides account for a large part of amps and have a broad inhibitory effect on common pathogenic bacteria, such as vre, acinetobacter baumannii, and mrsa in clinical medicine and s. aureus, listeria monocytogenes, e. coli in food and salmonella, vibrio parahaemolyticus in aquatic products. many natural and synthetic amps like nisin, cecropins and defensins have shown good inhibition activity to gram-positive bacteria and gram-negative bacteria. in recent research, amps p (yirkirrffkklkkilkk-nh ) and p (syerkinrhfktlkknlkkk-nh ), which are designed based on aristicluthys nobilia interferon-i, inhibit mrsa and show a low cytotoxicity . antifungal peptides are a subclass of amps that address fungal infections with enhanced drug resistance. many afps have shown excellent anti-fungal activities against common pathogenic fungi, such as aspergillus and candida albicans in clinical medicine, yeast, filamentous fungi (e.g., aspergillus flavus), mold in food and agriculture. except for brevinin, ranatuerin, cecropins, many synthetic peptides also show good antifungal activity. for example, aurh , derived from aurein . , can effectively treat c. albicans infection, which has a lethal rate up to % (madanchi et al., ) . aflatoxin, which is a carcinogen produced by a. flavus, is harmful to the human body. many afps can inhibit the growth of a. flavus. for example, an afp with a sequence of fpshtgmsvppp can inhibit the growth of a. flavus md . a total of antifungal peptides isolated from lactobacillus plantarum te and their mixture can reduce a. flavus spore formation in fresh maize seeds (muhialdin et al., ) . moreover, two chemically synthesized radish amps show a good inhibitory effect against different yeast species, such as zygosaccharomyces bailii and zygosaccharomyces rouxii (shwaiki et al., ) . viruses cause serious harm to human life and huge economic losses to the animal husbandry. the covid- , which is the recent outbreak, has caused great loss of lives and properties. furthermore, foot-and-mouth disease virus, avian influenza virus (aiv), and hiv are long-term threats to human life. so, it is extremely urgent to solve these problems, and antiviral peptides provide new ways. antiviral peptides show a strong killing effect on viruses mainly by ( ) inhibiting virus attachment and virus cell membrane fusion, ( ) destroying the virus envelope, or ( ) inhibiting virus replication (jung et al., ) (shown in figure ). a recent report has shown that amp epi- mediates the inactivation of virus particles and has good inhibitory activity against foot-and-mouth disease virus . moreover, infectious bronchitis virus (ibv) is the pathogen of infectious bronchitis and the inoculation of swine intestinal amp (siamp)-ibv mixed solution remarkably reduced the mortality of chicken embryos compared with the ibv infection group, showing the good inhibitory activity of siamp on ibv (sun et al., ) . anti-hiv peptides are a subclass of anti-viral peptides. the most important examples of these peptides include defensins (including αand β-defensins, which have different mechanisms), ll- , gramicidin d, caerin , maximin , magainin , dermaseptin-s , dermaseptin-s , siamycin-i, siamycin-ii, and rp (madanchi et al., ) and antiviral peptide fuzeon tm (enfuvirtide) has been commercialized as an anti-hiv drug (ashkenazi et al., ) . due to the global spread of the covid- (figure a ), the antiviral peptides against the coronavirus will be discussed in more detail. coronaviruses (covs) belong to the family coronaviridae; they are enveloped viruses with a positive-sense single-stranded rna genome and have a helical symmetry (franks and galvin, ) . covs, including severe acute respiratory syndrome cov (sars-cov) and middle east respiratory syndrome coronavirus (mers-cov) (mustafa et al., ) , and the recent outbreak of covid- have caused serious threats to human life and property. covs can cause lifethreatening respiratory diseases and the viral particle is formed by spike glycoprotein (s), the envelope (e), the membrane (m), and the nucleocapsid (n) (vilas boas et al., ) . it should be noted that their infectivity requires viral spike (s) protein. fusion inhibitor peptides combine with the s protein to interfere with its folding and prevent infection. besides, the s domain of the sars-cov s protein contains heptad repeat hr and hr sequences. peptide hr (hr : sltqinttlldltyemlslqqvvkalnesyidlkel) and its lipid-binding peptide is highly similar or even identical to the near-membrane portion of s protein ferredoxin, which interferes with refolding into post-fusion fusion-catalyzing domains (fds) (du et al., ; park and gallagher, ) . according to recent research, the lipopeptide ek c , derived from ek (sldqinvtfldleyemkkleeaikkleesyidlkel), is the most effective fusion inhibitor against covid- s proteinmediated membrane fusion . homology modeling and protein-peptide docking showed that temporin has potential therapeutic applications against mers-cov (marimuthu et al., ) . two amps from the non-structural protein nsp of sars-cov, k , and k , can inhibit sars-cov replication (ke et al., ) . furthermore, rhesus thetadefensin (rtd- ) treated animals have a marked reduction in mortality in the presence of sars-cov while the peptide alone shows airway inflammation and the one possible mechanism of action for rtd- is immunomodulatory (wohlford-lenane et al., ) . in general, amps against coronavirus can be roughly classified as i) peptides derived from hr , hr and rbd subunits of the spike protein, ii) peptides derived from other amps, iii) peptides derived from non-structural protein (mustafa et al., ) . furthermore, molecular docking analysis indicated that peptides were employed to disrupt the interaction between covid- and ace (angiotensin-converting enzyme ) to inhibit covid- entrance in cells ( figure b ) (souza et al., ) . finally, it should be noted that this therapy lacks clinical trials and the main method of animal experiments is an intranasal administration. this reminds us that nasal drug delivery (ndd) is a potential therapy for amps as anti-coronavirus drugs. besides, the antiviral database avpdb includes numerous antiviral peptides. parasitic protozoa can cause diseases in human and animals through a variety of routes, including animal-to-person or person-to-person contact, water, soil, and food (chalmers et al., ) . and with the increase in parasite drug resistance, the need for new treatments has increased. antiparasitic peptides show their killing effect on parasites which cause diseases such as malaria and leishmaniasis (mangoni et al., ; rhaiem and houimel, ) and amps like cathelicidin, temporins-shd show high inhibition activity against parasites (abbassi et al., ) . in recent research, epi- , a marine synthetic amp, can remarkably inhibit trichomonas vaginalis by destroying its membrane (neshani et al., ) . the peptide jellein derived from bee royal jelly which has introduced above and -amino acid amp kdel (lysine, aspartic acid, glutamic acid, and leucine) has shown a significant effect on the leishmania parasite (cao et al., ; zahedifard et al., ) . however, it should be noted that their mechanisms are not the same. cyanobacterial peptides differ from higher-eukaryote amps because their antiparasitic action depends on specific protein targets. thus, these target parasites can be distinguished accurately even though they belong to the same family or genus (rivas and rojas, ) . the acps show anticancer mechanisms by ( ) recruiting immune cells (such as dendritic cells) to kill tumor cells, ( ) inducing the necrosis or apoptosis of cancer cells, ( ) inhibiting angiogenesis to eliminate tumor nutrition and prevent metastasis, and ( ) activating certain regulatory functional proteins to interfere with the gene transcription and translation of tumor cells (wu d. et al., ; ma et al., ) . tritrpticin and its analogs induce considerable toxicity toward jurkat cells in vitro, whereas indolicidin and puroindoline a can also act as acps (arias et al., ) . it should be noted that both net charge and hydrophobicity play important roles in optimizing the anticancer activity of acps and they can constrain and influence each other. thus, achieving a balance between net charge and hydrophobicity is important for better anticancer activity. besides the peptide mentioned above, anti-inflammatory, anti-diabetic peptides, spermicidal peptides etc. have been noticed, but they are not the same as antimicrobial peptides. simply put, anti-inflammatory peptides decrease the release of inflammatory mediators and inflammatory cytokines (nitric oxide, interleukin- , and interleukin- β) and some of them also inhibit inflammatory signals like nf-κb, mapk, and jak-stat pathways (meram and wu, ; gao et al., ) . anti-diabetic peptides play their function by modulating the g proteincoupled receptor kinase (grk / ) or activating glucagonlike peptide- (glp- ), glucagon receptors (marya et al., ; graham et al., ) . however, it is not accurate to classify these types of peptides as amps and bioactive peptides may be more convincing. proline is a typical non-polar amino acid. pramps behave differently from other amps, that is, they enter bacterial cytoplasm by the inner membrane transporter sbma instead of killing bacteria through membrane destruction (mattiuzzo et al., ) . once in the cytoplasm, pramps target ribosomes and block the binding of aminoacyl-trna to peptidyltransferase center or trap decoding release factors on the ribosome during the termination of translation to interfere with protein synthesis (seefeldt et al., ) . for instance, tur a, which is an orthologous amp of bovine pramp bac discovered from tursiops truncatus, interferes with the transition from the initial phase to the extension phase of protein synthesis by binding to ribosomes. in addition, different pramps lack a high sequence similarity but have short motifs containing repeating proline and arginine (arg) residues (e.g., -ppxr-in bac and -prpxin bac ) (mardirossian et al., (mardirossian et al., , . although pramps mainly kill gram-positive bacteria, ppr-amp , a proline-rich amp identified from crab (scylla paramamosain), exhibits antimicrobial activity against gram-positive and gram-negative bacteria (imjongjirak et al., ) . besides, pieces of research have shown that pramps have immunostimulation activity (li w. et al., ) . tryptophan (trp), as a non-polar amino acid, has a remarkable effect on the interface region of the lipid bilayer, whereas arg, as a basic amino acid, confers peptide charge and hydrogen bond interactions, which are essential properties to combine with the bacterial membrane's abundant anionic component. and it seems that trp residues play the role of natural aromatic activators of arg-rich amps by ion-pair-π interactions (walrant et al., ) , thereby promoting enhanced peptide-membrane interactions (chan et al., ) . in addition to indolicidin and triptrpticin which both are famous amps that rich in arg and trp residues. octa (rrwwrwwr) is also a typical trp-and arg-rich amp that inhibits gram-negative e. coli and pseudomonas aeruginosa and gram-positive s. aureus. and short trp-and arg-rich amps designed based on bovine and murine lactoferricin have also shown strong inhibitory action against bacteria (strøm et al., ; bacalum et al., ) . histidine is a common basic amino acid, and histidine-rich amps show good membrane permeation activity. hv is a histidinerich amp designed based on rr(xh) xdpgx(yh) rr-nh (where x represents i, w, v, and f). this peptide increases the permeability of bacterial cell membranes to cause cell membrane rupture and death. in addition, hv inhibits bacterial movement in a concentration-dependent manner and shows a strong anti-inflammatory effect by inhibiting the production of tumor necrosis factor α (tnf-α) ). an amp designed based on octa has shown good therapeutic potential by replacing its arg residues with histidine (bacalum et al., ) . furthermore, l h , which is designed based on the linear cationic amphiphilic peptide magainin, also shows good antibacterial activity and cell penetration properties by inserting four histidine sequences in leucine and alanine (lointier et al., ) . the r group of glycine is generally classified as a non-polar amino acid in biology. glycine-rich amps, such as attacins and diptericins, widely exist in nature (lee et al., ; kwon et al., ) . these peptides contain % to % glycine residues, which have an important effect on the tertiary structure of the peptide chain. a glycine-rich amp derived from salmonid cathelicidins activates phagocyte-mediated microbicidal mechanisms, which differ from the mechanism of conventional amps (d'este et al., ) . furthermore, the glycine-rich central-symmetrical gg is an ideal commercial drug candidate against clinical gramnegative bacteria . antimicrobial peptides can be divided into four categories based on their structures including linear α-helical peptides, β-sheet peptides, linear extension structure, and both α-helix and β-sheet peptides ( figure ) (lei et al., ) . moreover, progressively cyclic peptides and amps with more complex topologies (including lasso peptides and thioether bridged structures) are reported (koehbach and craik, ) . the membrane-targeting mechanisms of amps can be described through models, including the pole and carpet models and the pole model can be further divided into the toroidal pore and barrel-stave models (figure ). the toroidal pore model is also known as the wormhole model. in this model, amps vertically embedded in the cell membrane accumulate and then bend to form a ring hole with a diameter of - nm (matsuzaki et al., (matsuzaki et al., , . the typical examples of this model are magainin , lacticin q, and arenicin. furthermore, cationic peptides, including tc , tc , and bp , compromise the membrane barrier by creating fluid domains (omardien et al., ) . antimicrobial peptides aggregate with each other, penetrate the bilayer of the cell membrane in the form of multimers, and form channels that result in the cytoplasmic outflow. in severe cases, amps can induce cell membrane collapse and lead to cell death (lohner and prossnigg, ). for instance, alamethicin performs its pore-forming activity by using this model. besides, hairpin amp protegrin- can form stable octameric β-barrels and tetrameric arcs (half barrels) in implicit and explicit membranes by simulations (lipkin and lazaridis, ) . antimicrobial peptides are arranged parallel to the cell membrane. their hydrophilic end faces the solution, and their hydrophobic end faces the phospholipid bilayer. amps will cover the membrane surface that similar to a carpet and destroy the cell membrane in a 'detergent'-like manner (oren and shai, ) . however, this pore-forming mechanism requires a certain concentration threshold and the required concentration of amps is high. human cathelicidin ll- exhibits its activity through this mechanism, and amps with β-sheet structure also play a role in this model (shenkarev et al., ; corrêa et al., ) . polarized light-attenuated total reflection fourier transform infrared spectroscopy (atr-ftir) was used to study the effect of amp cecropin p on the bacterial cell membrane and found that it was an applied flat on the surface of the pathogen's cell membrane to destabilize and eventually destroy the cell membrane . membrane targeting mechanisms (the cell membrane composition differences of bacteria and fungi shown in figure ) can be further refined to address the large differences in the lipid composition of the cell membranes of bacteria, fungi, and mammals. the main lipids in cell membranes include glycerophospholipids (gpls), lysolipids, sphingolipids, and sterols. phosphatidylethanolamine (pe), phosphatidylglycerol (pg), and cardiolipin (cl) are the most common anionic lipids in bacteria, whereas phosphatidylcholine (pc), phosphatidylinositol (pi), pe, and phosphatidic acid (pa) are the main gpls in fungal cell membranes (ejsing et al., ; singh and prasad, ; li et al., ) . furthermore, fungal cell membranes are more anionic than mammalian cell membranes and have higher pc content. meanwhile, ergosterol is the sterol found in the plasma membrane of lower eukaryotes, such as fungi, whereas that of animals contains cholesterol (faruck et al., ) . many amps take advantage of differences in membrane components to exert their effects. antimicrobial peptides are promising to be anti-biofilm agents but it should be noticed that they are different from the cell penetrating peptides (cpps) which typically comprise - amino acids and can translocate across the cell membrane. cpps could be categorized according to physicochemical properties into three classes: cationic, amphipathic, and hydrophobic, but anti-biofilm peptides have stricter requirements for these physicochemical properties. anti-biofilm peptides target the biofilms by different mechanisms including ( ) degradation of signals within biofilms; ( ) permeabilize within cytoplasmic membrane/eps; ( ) modulating eps production etc. and then can address chronic multi-resistant bacterial infections (pletzer et al., ; ribeiro et al., ; guidotti et al., ; derakhshankhah and jafari, ; rajput and kumar, ) . for instance, saap- , synthesized based on ll- , showed activity to prevent biofilm formation by s. aureus and a. baumannii (crunkhorn, ) . the way of amps entering cells is direct penetration or endocytosis. after entering the cytoplasm, amps will identify and act on the target. depending on the target, amps can be divided into the following categories. antimicrobial peptides affect transcription, translation, and assembly into functional peptides through molecular chaperone folding by interfering with related enzymes and effector molecules. for example, bac - targets ribosomes to inhibit protein translation (mardirossian et al., ) , whereas tur a inhibits protein synthesis in e. coli and thermus thermophilus by inhibiting the transition from the initial phase to the extension phase. however, the differences between tur a and bac also lead to various ways of binding to ribosomes and interacting with the ribosomal peptide exit tunnel (mardirossian et al., ) . but some amps' have different targets. for instance, genome-wide transcription shows that the amp dm can affect many important intracellular pathways of protein biosynthesis . chaperones are key proteins for correctly folding and assembling newly synthesized proteins and make them have stereoisomerism, which makes amps have cell selectivity and can prevent cytotoxicity. according to a previous review: both pyrhocoricin and drosocin can prevent dnak from refolding misfolded proteins by inducing a permanent closure of the dnak peptide-binding cavity (kragol et al., ; le et al., ; wrońska and boguś, ) . antimicrobial peptides can affect key enzymes or induce the degradation of nucleic acid molecules to inhibit nucleic acid biosynthesis. indolicidin, a c-terminal-amidated cationic trprich amp with amino acids, specifically targets the abasic site of dna to crosslink single-or double-stranded dna and it can also inhibit dna topoisomerase i (subbalakshmi and sitaram, ) . tfp (tissue factor pathway inhibitor) - tc , which is an amp from tongues, enters the cytoplasm of target cells after the rupture of cell membrane and then degrades dna and rna (he et al., ) . many amps can inhibit various metabolic activities by inhibiting protease activity. for example, histatin has a strong inhibitory effect on the proteases secreted by the host and bacteria. amps enap- and indolicidin inhibit microbial serine proteases, elastase, and chymotrypsin (le et al., ) . cathelicidin-bf is a peptide isolated from the venom of bungarus fasciatus, it can effectively inhibit thrombin-induced platelet aggregation and further block protease-activated receptor (shu et al., ) . antimicrobial peptides inhibit cell division by inhibiting dna replication and dna damage response (sos response), blocking the cell cycle or causing the failure of chromosome separation (lutkenhaus, ) . for instance, app (glaraltrllrqltrqltra), which is an amp with amino acid residues, can efficiently kill c. albicans because of its cell-penetrating efficiency, strong dna-binding affinity, and ability to induce s-phase arrest in intracellular environment . mciz, which has amino acid residues, is an effective inhibitor of bacterial cell division, z-ring formation, and localization (cruz et al., ) . moreover, it has been reported that several afps have damaging effects on the organelles of fungi. for example, histintin can interact with mitochondria, causing the production of ros, and inducing cell death (helmerhorst et al., ) . in addition to intracellular targets, differences in cell wall composition, such as lipopolysaccharide (lps), lipid a and mannoproteins, are potential targets for amps. specifically, gram-positive and gram-negative bacteria are classified based on their bacterial cell wall structure. gram-positive bacteria have a layer of cross-linked peptidoglycan, whereas gram-negative bacteria have an additional outer membrane with an inner leaflet containing only phosphatidic acid and an outer leaflet made of lps. lps has numerous negatively charged phosphate groups, which combine with a salt bridge with a divalent cation (e.g., ca + and mg + ) to form an electrostatic network (nikaido, ) . this electrostatic zone is the main barrier against hydrophobic antibiotics and causes the low permeability of gram-negative bacteria. the main components of the fungal cell wall are mannoprotein, β-glucans and chitin (polymers of , -β-n-acetylglucosamine) and the mutations in the relevant genes of the lps pathway and phospholipid trafficking provide resistance to the amps (cabib, ; spohn et al., ) . mannoproteins in fungal cell walls include a variety of proteins, including structural proteins, cell adhesion proteins (floccrin and lectin) and enzymes involved in cell wall synthesis and remodeling (hydrolytic enzymes and transglycosylase). these proteins differ from human cell membrane proteins and are potential targets of afps (rautenbach et al., ) . furthermore, teichoic acid and lipoteichoic acid in the cell wall are also potential targets of amps and these theories could support the design of amps with low cytotoxicity. antimicrobial peptides have good application prospects. however, amps have the following problems. ( ) amps damage the cell membrane of eukaryotes and cause hemolytic side effects; ( ) rising production costs and technical problems limit their manufacture; ( ) their stability is limited at certain ph; ( ) amps have reduced activity under the presence of iron and certain serum; ( ) amps are easily hydrolyzed by proteases. therefore, the ideal amp should meet the following characteristics: (i) high antimicrobial activity; (ii) low toxicity to mammalian membranes; (iii) high protease and environment stability; (iv) low serum binding capacity and (v) ease of access and low cost production . therefore, designing amps to achieve the desired effect has attracted increasing attention. the rational design of antibacterial peptides should focus on the following five aspects: chain length, secondary structure, net charge, hydrophobicity, and amphiphilicity and these have been mentioned in many studies and this review will focus more on several specific methods of antimicrobial peptide design. site-directed mutation refers to the redesign of natural antimicrobial peptides by adding, deleting or replacing one, or several amino acid residues (torres et al., ) . the de novo design of peptides attaches importance to the design of amphiphilic amps (guha et al., ) . for example, gala is a well-known de novo-designed amp. amphipathic α-helical peptide gala is created by placing protonatable glutamic acid residues in most positions with the spacing of i to i + (goormaghtigh et al., ) . the repeated sequence (xxyy)n, where x and x are hydrophobic amino acids, y and y are cationic amino acids, and n is the number of repeat units, is designed based on the hydrophobicity cycle that mimics natural α-helical amps and successfully designs broadspectrum α-helical amps. sequences (lkkl) and (wkkw) . have the highest selectivity (khara et al., ) . moreover, l l k m w model peptides are also de novo-designed peptides. amphipathic helical properties were conferred by using leucines and lysines, and two tryptophan residues were positioned at the amphipathic interface between the hydrophilic ending side and the hydrophobic starting side. among the model peptides, l k w has good anti-mrsa activity (lee et al., ) . sequence templates can be obtained by comparing a large number of structurally homologous fragments of natural amps (such as hdps) and extracting conservative patterns based on the type of residue (such as charged, polar, hydrophobic, etc.) (zelezetsky and tossi, ) . based on the modification, the parameters, such as helix formation tendency, cationic, amphiphilicity and overall hydrophobicity, can be systematically changed. for instance, cecropin, magainin, protegrin, and lactoferrin have all been used as amp templates (fjell et al., ) . peptides can form nanostructures, such as micelles, vesicles, nanotubes, nanoparticle nanobelt, and nanofibre nanotube, and can increase or impart antibacterial activity to amps during the self-assembly of peptides. for example, kld- (kld) is a self-assembling peptide with amino acid residues that can adopt nanostructures and are known for their tissue engineering properties. the addition of arg residues in kld shows no remarkable change in its β-sheet secondary structure and the self-assembly characteristics of the forming nanostructures (tripathi et al., ) . dimer structure can also be used to enhance the antimicrobial activity of amps and reduce toxicity, but membrane-destabilizing effects are reduced after dimer formation (malekkhaiat häffner and malmsten, ). various chemical modifications of amps, including residue phosphorylation, the addition of d-amino acids or unnatural amino acids (homoarginine), cyclization, halogenation, acetylation, and peptidomimetics, have been used to improve the stability of peptides against proteases. given that the enzyme is stereospecific, the incorporation of unnatural d-amino acids into the amp sequence can reverse the stereochemistry and prevent protease degradation (zhong et al., ) . the so-called peptidomimetics, whose main elements mimic the structure of peptides, are usually produced by modifications, such as chain extension or heteroatom incorporation of existing peptides (patch and barron, ) . ornine, which is an unnatural residue with a positive charge and has a high resistance to protease activity, is also used in non-chemical modification. replacing trp residues with family residues, such as β-dihydrophenylalanine, can stabilize secondary structures and improve antibacterial properties (maurya et al., ) . halogenation is highly related to the activity, specificity, and stability of amps. in the latest report, halogen is introduced into jelleine-i which is a short peptide isolated from the royal jelly of honeybees (apis mellifera) by replacing phenylalanine with a halogenated phenylalanine analog, increasing the antibacterial activity in vitro and anti-biofilm activity. in addition, the proteolytic stability of jelleine- is increased by - times by halogenation (jia et al., ) . the halogenated peptidomimetic α,α-disubstituted β-amino amides are also promising bacteriostatic drugs that have inhibitory effects on more than multi-resistant clinical isolates of gram-positive and gram-negative bacteria (paulsen et al., ) . halogenation is also related to the specificity of amps. the o-fluorine substitution in phenylalanine residues maintains the activity of temporin l on e. coli but leads to the loss of activity on s. aureus and p. aeruginosa (setty et al., ) . three modes of cyclisation, including cyclisation via disulfide bonds, head-to-tail cyclisation and internal bonding between side chains, have been found in natural amps. the synthesis of disulfide bonds often complicates the development of synthetic peptides. the circularisation of the main chain of arenicin- molecule resulted in increased activity against drug-resistant clinical isolates but caused no substantial effect on cytotoxicity (orlov et al., ) . the hdps tachyplesins i, ii, and iii and their cyclic analogs cti, ctii, and ctiii, respectively, have similar structures and activities and can resist bacterial and cancer cells. the cyclisation of the backbone reduces the hemolytic activity and improves the stability of the peptides whilst maintaining effective anticancer and antibacterial activities (vernen et al., ) . capping refers to the addition of specific motifs or modifications, such as amidation at the c-terminus and acetylation at the n-terminus, rendering amps with more natural peptide characteristics. post-translational modifications play an important role in the function of amps and are the most commonly used in peptide design. the c-terminal rana box (consisting of a c-terminal cyclic heptapeptide with a conservative disulfide bond) and amide group are important c-terminal capping methods. for example, the c-terminal amide group of maximin h can enhance antibacterial efficacy without increasing lytic ability (dennison et al., ) . the n-terminal lipidated analog c vg krkp shows enhanced antibacterial activity against various gram-negative bacteria. the functions of n-terminal lipidation include (i) increasing lps neutralization, (ii) increasing stability to proteases and peptidases, and (iii) reducing cytotoxicity (datta et al., ) . furthermore, hydrophobic end labeling is a commonly used method to increase the activity of antimicrobial peptides. acyl lipid peptides have a linear or cyclic structure in which one or more hydrocarbon tails are connected to the n-terminus of a short oligopeptide (chu-kung et al., ) . lipopeptides have covalently attached hydrophobic moieties, such as sterols or fatty acids. aromatic amino acid terminal labeling is also the main hydrophobic terminal labeling method. tryptophan (w) and phenylalanine (f) are the commonly used aromatic amino acids. their large and polarisable residues have an affinity for the interface, and the w/f tag is also sensitive to the differences between ergosterol and cholesterol and can prevent self-assembly. this condition results in low aggregation numbers and high critical aggregation concentrations (schmidtchen et al., ) . peptide conjugation has been the goal of most research in recent years to produce active and stable amps with high selectivity. different side chains or amp fragments can be used aside from the repetition of the same amino acid motifs. for example, conjugating fatty acids with a length of - carbon atoms to the th or th side chain of the d-amino acids of ano-d , improves antibacterial selectivity and anti-biofilm activity. in addition, the new peptide exhibits high stability against trypsin, serum, salt, and different ph environments (zhong et al., ) . the conjugation of different amps can also be performed. for example, the hybrid peptide (pa -gnu ) constructed by the addition of pa to gnu has a high activity and specificity to p. aeruginosa . smamps include a broad family of molecular entities based on the structure and function of amps. however, their backbones are not entirely based on α-amino acids, including β-amino acid oligomers, arylamide oligomers, and phenylene ethynylenes (michael henderson and lee, ) . for instance, smamp , which is a potential drug for intravenous treatment, causes no drug resistance and has a strong inhibitory effect on mrsa and vancomycin-resistant enterococcus faecium (tew et al., ) . peptoids are peptide isomers, in which the side chain is bonded to the main chain nitrogen instead of α-carbon or poly-nsubstituted glycine in which the side chain is connected to amide nitrogen instead of the α-carbon on the main chain (andreev et al., ) . for example, the cationic peptide sa (iowagolfolfo-nh ) and its poly-n-substituted glycine homolog spo (ninonwnangnonlnfnonlnfno-nh ) inhibit the planktonic and biofilm formation of a. baumannii strains, which are susceptible to multi-drug resistance (sharma et al., ) . motifs with specific functions have been reported increasingly. these motifs can be repeated units for combining into new antimicrobial peptides, or specific amino acid combination units appearing at the end (such as capping) of or even in the peptide chain. this motif includes two tripeptide structures, including gly-gly-his or val-ile-his, which are added at the end of the peptide chain. atcun-containing amps in the presence of hydrogen peroxide and ascorbic acid combine with cu + to induce the valence of copper ions between + and + oxidation states and form an atcun-cu (ii) complex, generating ros by fenton-like reactions. extracellular polymeric substances (eps) are important for biofilms and can enhance the resistance of cells to antibacterial agents (flemming, ) . atcun-amps have been used to degrade environmental dna, which is one of the major components of eps. several related practical applications have been reported. for example, the biological activity against carbapenem-resistant enterobacteriaceae is increased by adding this motif to the n-terminus of an alpha-helical amp (such as cm ). besides, the cu-atcun derivative of ov- containing a c-terminal ggc sequence showed high levels of membrane permeation and lipid peroxidation. the concept of catalytic metal drugs has attracted widespread attention although the concept is still in its infancy because of the role of metal ions (alexander et al., ; agbale et al., ) . rana box: rana box is a heptapeptide motif (cglxglc) from the nigrocin family. rana box consists of two cysteine residues that are separated by four or five other residues on the side and can form a cyclic disulfide bond. rana box peptide has shown structural analogies with polymyxin (colistin), and the primary structure of the rana box motif is important in determining bacteriostatic activity (kozić et al., ) . the deletion of the 'rana box' motif will cause the amp antibacterial effect to disappear, but replacing the natural 'rana box' sequence of amps with amidated phenylalanine can expand its efficacy against antibiotic-resistant microorganisms, including mrsa and p. aeruginosa, and reduce cytotoxicity. this phenomenon also shows that the effect of the motif on amps needs to be determined based on the specific situation and is not completely beneficial (bao et al., ) . the lps binding motif (g-wkrkrf-g) can produce a broad spectrum of antibacterial activity when introduced into the c-terminus of temporin- ta and temporin- tb (close isoforms of temporin) (mohanram and bhattacharjya, ) . antifungal peptides have a conserved gxc(x − ) c γ-core motif (residues - , gkcykkdnic; d-isomer) at its n-terminus, which is a cation part of the ring. this conserved motif interferes with the integrity of the plasma membrane of the cell (yount and yeaman, ) . conserved γ-core motifs are directly involved in protein-membrane interactions and strongly contribute to membrane binding (utesch et al., ) . if replace d-phe -pro sequence in peptide chain with d-phe- -abz turn motif ( -abz is an abbreviation of -aminobenzoic acid d-amino acid) in amp tyrc a, and nuclear magnetic resonance shows that this change retains the β-hairpin structure. unlike the traditional β-turn motif, the d-phe- -abz motif can be used as a tool for β-hairpin libraries. the hydrophobic peptide can be formed into the nucleated β-hairpin formation by adding the d-phe- -abz motif. moreover, the inclusion of this part in two designed cationic amphiphilic peptides can produce broadspectrum antibacterial activity and low hemolysis rate (cameron et al., ; cameron et al., ) . the ngr motif is composed of asn-gly-arg, and amps with this structure have strong cytotoxicity ( table ). the data indicate that the new amps containing ngr may bind to cd + or αvβ + tumor cells by binding to cd or αvβ , respectively, to exert anti-tumor activity, especially on cd + tumor cells . the central gxxxg motif can induce strong self-assembly and have been already used in the design of amps (brosig and langosch, ; krauson et al., ) . bovine lactoferrin b is an amp composed of amino acid residues and has antibacterial, antifungal, and antiparasitic activities. the multivalent molecules lfcinb ( - ) and lfcinb ( - ) contain the lfcinb ( - ) motif (rrwqwr) and show inhibition activity against e. coli, p. aeruginosa, and s. aureus. chimeric peptide chimera containing two motifs, namely, the rrwqwr of lfcinb ( - ) and the rllr of bfii computer design includes simple statistical modeling, structureactivity relationships study (abdel monaim et al., ) , neural networks (müller et al., ) , deep learning (veltri et al., ) , word embedding (hamid and friedberg, ) and machine learning. for example, a machine learning method by matlab is proposed based on the concept of scoring the contribution of each amino acid's antibacterial activity (wu x. et al., ) . the genetic algorithm was used to design the amphiphilic α-helical peptide guavalin , which has an uncommon amino acid composition (three tyrosine and three glutamine residues) and interestingly causes membrane hyperpolarization, which is a different mechanism from those of other amps (porto et al., ) . two research methods have been developed based on the research background of quantitative structure-activity relationships: prediction method based on amp therapeutic index and the identification of novel potential amps from the expressed sequence tag database based on the principles of the highly conserved signal peptide subclasses related to amps (juretić et al., ) . in this way, a variety of amp variants can be obtained. if combined with high-throughput screening, it can effectively obtain the desired amp. for instance, some new amps are designed by the combinatorial peptide library of melittin and show higher activity and lower cytotoxicity (krauson et al., ) . cations, such as na + and mg + , may affect amp activity . however, the different valences of metal ions have varied effects on amps. for example, divalent cations show stronger antagonism to bacteria than monovalent cations with thanatin and s-thanatin, which are insect amps (wu et al., ) . in the presence of nacl, the signal response during the association phase remarkably decreased in single-cycle and multi-cycle kinetic experiments, resulting in a decreased association rate. this occurrence may be caused by the shielding effect of nacl between the cationic peptide and the zwitterionic membrane. another possible reason is that na + can bind to the phospholipid bilayer, where the ions interact with the phosphate and the carbonyl oxygen of lipid head groups (sabapathy et al., ) . the reduced activity of synthetic peptide [rllr] under high salt concentration is possibly caused by the destruction of its α-helix structure. table shows that several amps, including histatin, myxinidin, and hepcidin, contain atcun motifs (amino terminal copper and nickel with xxh sequence). iron is the most abundant metal ion in human saliva, but the combination with this metal ion results in the loss of the α-helix of histatin and greatly reduces its antifungal activity (puri et al., ) . however, the coordination of copper (ii) and nickel (ii) ions can induce the formation of ros, which is essential for bactericidal activity (jeżowska-bojczuk and stokowa-sołtys, ). anionic amps have a large number of negatively charged aspartic and glutamic acid residues (lakshmaiah narayana and chen, ) . they require zinc as a functional cofactor and the zinc complex shows stronger antibacterial activity (jiang et al., ) . several of these amps use metal ions to form cationic salt bridges with the negatively charged components of the microbial membrane to penetrate the membrane. anionic amps may attach to ribosomes or inhibit ribonuclease activity when in the cytoplasm (jeżowska-bojczuk and stokowa-sołtys, ). metal ions also affect the self-assembly of peptides. these ions can recognize specific amino acids, such as lysine and glutamic acid, and may form salt bridges between peptide molecules to induce peptide self-assembly. for example, zn + can stabilize the aggregation of peptides on the cell membrane, which results in the enhanced antibacterial effect of dcd- l in the presence of zn + (tian et al., ) . ph many amps are stable and retain their antimicrobial activity in a wide ph range. amps have enhanced activity at low ph because of their basic properties. this condition is related to the protonation of histidine at acidic ph, which promotes electrostatic interactions with anionic surfaces, including lps and the anions of phospholipids, and subsequently enhances antibacterial properties. the effect of ph on the antibacterial activity of amps varies. for example, thanatin's activity at neutral ph is slightly higher than that under acidic conditions. by contrast, the activity of xylan on e. coli, listeria, and c. albicans is remarkably higher at ph . than at ph . (holdbrook et al., ) . the inactivation of the histidinecontaining amp c g-his under low ph conditions involves ph-dependent changes in the state of the aggregates in the solution, because the aggregates, which are sensitive to ph and lipid composition, may be affected by binding and conformation. peptides can also enhance bacterial membrane permeability at low ph (hitchner et al., ) . thrombin-derived c-terminal peptides (tcps) will also change the mode of cd (a protein that is abundant in human plasma) from anti-inflammatory mode to bacterial elimination mode from ph . to ph . (holdbrook et al., ) . a dimer (e.g., p- ) can be created to provide amps with resistance to a higher ph range. the sensitivity of this ph-sensitive amp can be used to achieve a certain targeting effect in practical applications. in addition, charge interaction is one of the most important factors in peptide self-assembly. ph affects the charge state of amino acid and substituent functional groups. therefore, adjusting the ph is the most common method for controlling peptide assembly and disassembly (tian et al., ) . proteases have a strong destructive effect on amps. for instance, ll- , which has the strongest inhibitory effect on chlamydial infection, is inhibited by the protease chlamydial protease-like activity factor (cpaf) secreted by chlamydia (tang et al., ) . studies have been focused on the design of amp carriers to solve this problem (lewies et al., ; nordström et al., ) . the presence of chitosan-silica solid support of kr- peptide can protect it be hydrolyzed by α-trypsin, and the degree of protection is increased by % compared with the free kr- (diosa et al., ) . however, several enzymes, such as protease , esterase and phosphatase , cut the blocking group of the peptide and trigger the self-assembly of the peptide, which positively affects amps (tian et al., ) . antimicrobial peptides can regulate pro-inflammatory reactions, recruit cells, stimulate the proliferation of cells, promote wound healing, modify gene expression and kill cancer cells to participate in the immune regulation of human skin, respiratory infections, and inflammatory diseases (de la fuente-núñez et al., ) . for example, α-defensins hnp- , hnp- , and hnp- showed effective antibacterial activity against adenovirus, human papilloma virus, herpes virus, influenza virus and cytomegalovirus. pulmonary diseases, such as idiopathic pulmonary fibrosis, alveolar proteinosis, and acute respiratory distress syndrome, show elevated levels of amps (guaní-guerra et al., ) . likewise, amps secreted by the paneth cells in the mammalian gut are important to shape the gut microbiota (bevins and salzman, ) . the application of amps in medicine, such as dental, surgical infection, wound healing and ophthalmology is developing now. but there are only three amps that have been approved by fda including gramicidin, daptomycin, and colistin. dental caries, endodontic infections, candidiasis, and periodontal disease are common diseases in the human oral cavity. dental caries is a prevalent oral disease and some acidogenic bacteria like streptococcus sp. are the main cariesassociated pathogens (izadi et al., ) . several amps have good application potential. for instance, peptide zxr- (fkiggfikklwrslla) has shown potent activities against pathogenic bacteria of dental caries, streptococcus mutans, streptococcus sobrinus, and porphyromonas gingivalis and peptide pac- (clinical trial identifier: nct ) that has been sold over the counter in taiwan for treating oral candidiasis (chen l. et al., ) . in surgical infection and wound healing: surgical infection occurs after surgery, burns, accidental injury, skin disease, and chronic wound infections have a serious hazard to human life (thapa et al., ) . several amps have shown the therapeutic potential of these diseases. for example, amp pxl shows pronounced efficacy as an anti-infective agent in burn wounds in mice and amp d a has been in the third phase of clinical trials for treating burn wound infections (björn et al., ) . in ophthalmology: human eyes are prone to be infected by several organisms including bacteria and fungi in which s. aureus, streptococcus pneumoniae, p. aeruginosa, aspergillus spp., and c. albicans are the most relevant pathogens (silva et al., ) . although amps such as lactoferricin b, protegrin- exhibited antimicrobial activity against these pathogenic bacteria, their application in the field of ophthalmology is only at the theoretical stage. with the popularity of contact lenses and the increase in cases of related eye infections, antimicrobial peptides have shown good application prospects in ophthalmology (khan and lee, ) . additional methods need to be performed for the application of amps as drugs in medicine. the main strategies include ( ) constructing precursors to reduce cytotoxicity and improve protease stability, ( ) using amps in combination with existing antibacterial agents, ( ) inducing the correct expression of amps with appropriate drugs and using engineering probiotics as vectors to express amps. for example, in the field of wound repair, different formulation strategies, such as loading amps in nanoparticles, hydrogels, creams, gels, ointments, or glutinous rice paper capsules, have been developed to effectively deliver amps to the wound (borro et al., ; thapa et al., ) . in recent research, the sponges developed from modified starch and hs-peg-sh are covalently immobilized with amp showed effective antibacterial activity (yang et al., ) . more technical means, including pheromone-labeled amps, local environment-triggered amps (enzyme precursor drug release system, ph-activated amps, etc.), have been developed to improve the targeting mechanism of amps. furthermore, nanotubes, quantum dots, graphene, and metal nanoparticles have been proposed to be a potential method to enhance drug delivery of amps (magana et al., ) . hybrid peptides have also been used to build targeting peptides. for example, pa , which is a p. aeruginosa-targeting peptide, was combined with gnu (a broad-spectrum amp) to construct a hybrid peptide (pa -gnu ) that targets oprf protein and has good bactericidal activity and specificity . furthermore, some antibiotics, for instance, daptomycin (a lipopeptide), lugdunin which is a -membered cyclic peptide consists of amino acid residues plus a thiazolidine moiety and telavancin (a glycopeptide) have been widely used for the clinic (durand et al., ; lampejo, ) . although they are antibiotics, they have provided broader ideas for the design of amps. food preservatives have potential harm to the human body. therefore, natural preservatives are being advocated by more people. amps have a good inhibitory effect on common bacteria and fungi in food, and many amps are resistant to acids, alkalis, and high temperatures are easily hydrolyzed by proteases in the human body. thus, amps are a promising alternative to preservatives. nisin is a bacteriocin produced by l. lactis subspecies. lactic acid bacteria have been widely used as food preservatives. nisin is categorized as generally recognized as safe (gras) by the us food and drug administration (fda) and is used as a food preservative in other countries (khan and oh, ) . however, only nisin and polylysine are currently approved by the fda as food additives (santos et al., ) . pedocin pa- , a bacteriocin consisting of amino acids produced by a diplococcus, is also used as a food preservative and is sold on the market under the trade name alta . pedocin pa- is used as a food additive to inhibit the growth of l. monocytogenes, which can cause meat deterioration (settanni and corsetti, ) . enterocin as- is an amp used to preserve cider, fruit and vegetable juices, and enterocin ccm is used to preserve soy milk (rai et al., ; santos et al., ) . encapsulating bacteriocins into liposomes is a new method used to overcome the problems of amps in food applications (such as proteolytic degradation or interaction with food ingredients) (da silva malheiros et al., ) . moreover, active packaging by adding amps is a novel packaging method that has great potential in the food industry. for instance, ε-poly-l-lysine is used in conjunction with starch biofilms to show good inhibitory effects on aspergillus parasiticus (aflatoxin producer) and penicillium expansum and nisin have the potential to be dairy preservative because it is a highly surface-active molecule (luz et al., ) . the european union banned the use of animal growth promoters in animal feed in . thus, a new antibacterial strategy is needed. many amps are the potential to be used in poultry, swine, and ruminants breeding and aquaculture because they can improve production performance (liu et al., ; bao et al., ) , immunity and promote intestinal health and some of them have a stronger inhibitory effect on bacterial inflammation if used with antibiotics cote et al., ) . for example, siamp has a good effect on the treatment of ibv in chicken (sun et al., ) . by adding swine gut intestinal antimicrobial peptides (sgamp), broilers showed higher average daily gain and feed efficiency under chronic heat stress conditions (hu et al., ) . frog caerin . , european sea bass dicentracin and nk-lysine peptides (nklps) have good inhibitory effects on nodavirus, septicaemia haemorrhagic virus, infectious pancreatic necrosis virus and spring viremia carp virus, which are devastating to fish farming (león et al., ) . the amp in soybean meal fermented by b. subtilis e effectively inhibits v. parahaemolyticus and vibrio alginolyticus and enhances the resistance level of litopenaeus vannamei against v. parahaemolyticus when added to feeds (cheng et al., ) . for agriculture, the plant pathogenic infection of bacteria and fungi causes the loss of economy, for instance, aspergillus flavus infection of corn and peanuts, citrus green mold caused by penicillium digitatum, gray mold disease caused by botrytis cinerea on strawberries and geotrichum citriaurantii infection of citrus fruit all cause great harm to the growth and post-harvest of agricultural products (liu et al., ; liu et al., ) . several afps have shown prospect to control these problems. however, the practical application of antimicrobial peptides in the transportation and preservation of agricultural products is still lacking, because the use of antimicrobial peptides will greatly increase the cost in the transportation of fruits and vegetables (application examples of amps in these four fields are shown in table ). antimicrobial peptides constitute a global research hotspot, but many key issues in design and application need to be solved urgently. several restrictive factors hinder the application of amps. the interaction of multidisciplinary subjects, such as biology, materials science, chemistry, bioinformatics, molecular informatics and pharmacy can further develop prospective amps. computer molecular dynamics simulation, cell membrane simulation, and more methods are being applied to study the mechanism of amps. how to further understand the correlation between amps and various targets instead of conducting one-sided experimental research might improve experimental designs to obtain stronger systemic and scientific demonstrations. on this basis, further animal experiments are required instead of simple cell-level experiments to test the effect of amps under complex physiological conditions. several complicated methods, such as the chemical method of peptidomimetics and non-natural amino acid modifications, have been applied in designing amps to solve the problem of protease hydrolysis. most methods use chemical substrates, but the cost of these methods cannot be ignored in practice. in addition, chemical synthesis and the use of engineered bacteria are currently the mainstream for such procedures. finding a better biological preparation method, reducing the cost and increasing the yield is important problems in practical application. furthermore, studying the amp expression of the organism itself and finding a better expression vector are necessary for mass production in the future as more amps in nature are discovered. further research is needed on the reported amps to solve the problem on structure-function relationship. as a branch of peptide drugs, amps need to progress with the advancement of medical science against the background of the current low success rate of the clinical application of amps. more attention can be 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promises antimicrobial potency against multidrug-resistant bacteria isolation and structure of corticostatin peptides from rabbit fetal and adult lung characterization of antimicrobial activity and mechanisms of low amphipathic peptides with different α-helical propensity we thank the national key r&d program of china ( yfd ). key: cord- -xqmir ca authors: olaimat, amin n.; shahbaz, hafiz m.; fatima, nayab; munir, sadia; holley, richard a. title: food safety during and after the era of covid- pandemic date: - - journal: front microbiol doi: . /fmicb. . sha: doc_id: cord_uid: xqmir ca the coronavirus disease (covid- ) is a clinical syndrome caused by severe acute respiratory syndrome corona virus- (sars-cov- ). covid- was declared a pandemic by the world health organization (who) on march , due to its rapid and extensive spread among many countries through its very contagious nature and its high mortality among the elderly and infirm. recently, data on the survival of sars-cov- on contact surfaces has been reported, but there is none on the survival of covid- on food surfaces and packages. the potential survival and transmission of sars-cov- on/via food and packages are discussed based on data available for other respiratory viruses such as sars-cov and mers-cov. however, studies are needed to explore its transmission via food and survival on food packaging materials. the implementation of food safety management systems such as hazard analysis and critical control points (haccp), and good manufacturing practices (gmp) are important to reduce the risk of covid- infection. cleaning, sanitation, good hygienic practices, and active packaging are also needed from farm to fork. in december , sars-cov- was initially detected in patients who suffered from unusual viral pneumonia in wuhan, hubei, china (kaul, ; naserghandi et al., ; petrosillo et al., ) . the virus was first named novel coronavirus ( -ncov) by the who and later, when it was found that . % of the novel virus genome was similar to the sars-cov genome, the virus was renamed sars-cov- (chang et al., ; the lancet infectious diseases, ) . covid- is the clinical syndrome caused by sars-cov- infection which is characterized by a respiratory disease with symptoms ranging from mild influenza (flu-like) to severe pneumonia and acute respiratory distress syndrome (petrosillo et al., ) . the clinical manifestations of covid- are non-specific and variable among patients, and between countries. generally, covid- symptoms involve fever, sore throat, runny or stuffy nose, dry cough, headache, myalgia or fatigue, sputum production, dyspnea, chest pain or pressure, joint pain, chills, loss of taste or smell, and a rash on the skin or discoloration of toes or fingers. abdominal pain, dizziness, diarrhea, nausea, and vomiting are less common symptoms (kaul, ; naserghandi et al., ; petrosillo et al., ) . borges do nascimento et al. ( ) found that covid- -related symptoms among , patients in studies were: fever ( %), cough ( %), muscle aches and/or fatigue ( %), dyspnea ( %), headache ( %), sore throat ( %), and gastrointestinal symptoms ( %). on average, the incubation period takes - days for a patient to show the symptoms after infection, however, it may reach up to days (world health organization [who] , ). covid- infection is highly contagious among the population and now almost all countries have reported cases and deaths. on march , , covid- was characterized as a pandemic by the who. as of early july, over million confirmed cases and , deaths of covid- have been reported worldwide (world health organization [who], ). at the beginning of the pandemic, two-third or out of reported cases had previously visited the huanan seafood wholesale market where live animals were sold close to seafood and meat products, suggesting that the virus was transmitted from animals to humans (guan et al., ; harapan et al., ; naserghandi et al., ) . the genome of sars-cov- is closely related to the genomes of the sars-cov that caused the sars epidemic during and the sars-related-covs (sarsr-covs) that was isolated from horseshoe bats. this suggested that the primary host of sars-cov- was a bat and other animals including pigs, or pangolins are potential secondary hosts to the virus (lai et al., ; sun et al., ) . to date, the impact of the seafood market in spreading covid- is not fully understood (harapan et al., ) . it has been proposed that sars-cov- was introduced to the seafood market in wuhan, hubei, china and then the disease spread more rapidly through human-to-human interactions, which has been confirmed by the occurrence of infection among the family members and medical workers attending the victims (chan et al., ; yu et al., ) . the most plausible transmission routes are respiratory droplets dispersed via talking, sneezing and coughing or direct contact with infected persons. other suggested routes are fecal-oral transmission, contaminated fomite transmission and mother-fetal vertical transmission (naserghandi et al., ; wang et al., ) . it has been demonstrated that covid- can also be transmitted by asymptomatic patients who are harboring the virus during its early incubation period before symptoms appear ye et al., ; zhang et al., ) . additionally, sars-cov- has reappeared in recovered patients leading to a reoccurrence of illness. this has been confirmed through the detection of viral nucleic acid using a real-time rt-pcr after patient recovery and discharge from the hospital lan et al., ; qiao et al., ) . however, the exact reason for the reappearance of sars-cov- is not well understood. foodservice operators were among the first workers in frontline employment sectors experiencing the impact of the covid- pandemic. however, there is no study to date which reports that covid- spreads via food products. further, no evidence is available showing that viruses which infect the respiratory tract can be transmitted via food or food packaging (food and agriculture organization of the united nations [fao] and world health organization [who] , ). the transmission of sars-cov and mers-cov through the consumption of foods does not appear to have occurred yet (european food safety authority [efsa], ). however, it has been reported that human coronavirus e (hucov- e) survived for at least days on the surfaces of polyvinyl chloride (pvc), polyfluorotetraethylene (teflon, ptfe), glass, ceramic tiles, and stainless steel and for days on silicon rubber surfaces at • c with a relative humidity of - % (warnes et al., ) . similarly, sars-cov- survived on stainless steel and plastic up to and days, respectively, at - • c and a relative humidity of %; however, the virus was not detected on copper and cardboard, after and h, respectively (van doremalen et al., ). these results indicated that sars-cov- can be transmitted via contact surfaces because of the ability of the virus to survive on the surfaces for several days. coronaviruses can persist for long periods in environmental samples which may enhance the probability of transmission via package contact surfaces (geller et al., ) . it has been confirmed that virulence of variola (smallpox) virus and influenza virus were positively correlated with survival time in the external environment, which explained their high mortality rate compared to other viruses with low survival rates in environmental samples such as the virus causing parainfluenza and rhinovirus (walther and ewald, ) . food and agriculture organization of the united nations [fao] and world health organization [who] ( ) proposed that touching food packages or containers contaminated with sars-cov- could transmit the virus to the mouth, nose, or eyes. however, this is not considered the main route for disease spread because the virus shows poor survival on these surfaces. a previous study reported that food products were a plausible transmission route for respiratory viruses including sars-cov- and influenza (klein, ) . in another study, the risk of ebola infection to individual humans in the united states resulting from contaminated cocoa beans, palm oil, or cashews imported from south africa was considered negligible to low (bergeron et al., ) . in addition, several studies showed that transmission of avian influenza through poultry products (golden et al., ; bauer et al., ; sánchez-vizcaíno et al., ) or water consumption was a remote possibility, but possible (schijven et al., ) . similarly, the probability that consumers from the united kingdom might get infected with covid- via the consumption of food or the handling of material contacting food or packaging was considered negligible to very low. as mentioned, the genome of sars-cov- is closely related to sars-cov for which the transmission via foods has not been confirmed. it has been suggested that the potential foodborne transmission of sars-cov- may occur due to the consumption of foods originating from infected animals or the consumption of cross-contaminated foods (oakenfull and wilson, ) . more effort is needed to address the transmission of sars-cov- from the respiratory tract to food package surfaces or through food consumption. the food and drug administration [fda] ( b) proposed guidelines for consumers during food shopping, food handling and food preparation. it is worth mentioning that food handlers including food establishment employees and consumers should adhere to good sanitation and hygienic practice guidelines to avoid sars-cov- transmission and comply with at least the minimum requirements of the food safety system. it is widely known that viruses cannot multiply in food products because they need an animal or human host to grow. however, to date, no study has investigated the survival of sars-cov- in foods. to the best of our knowledge, only two studies reported the survival of infectious respiratory viruses in food products. adenovirus survived on both lettuce and strawberries at • c for up to days. in contrast, coronavirus survived only days on lettuce, and it was not recovered from the surface of strawberries after inoculation (yépiz-gómez et al., ) . these results indicated that respiratory viruses may transfer from food surfaces to the hands and subsequently to the mouth, nose or eyes. the survival of mers-cov in different types of milk (camel, goat and cow milk) at or • c has been investigated. mers-cov titers were decreased by less than log in all types of milk after h at • c. higher log reductions were observed when milk was stored at • c since the virus titers decreased by ≤ . log with h of storage. low temperature, long time pasteurization ( • c/ min) of raw milk completely eliminated the virus from the milk of the three different animals (van doremalen et al., ) . the infectious dose of most respiratory viruses is low; thus, the handling or consumption of food products could represent a risk for infection. as a result, preventive measures such as washing and sanitizing of fresh produce surfaces as well as the implementation of good personnel hygiene and practices among workers would seem reasonable ways to reduce the risk of virus transmission. another issue is that several viruses that cause respiratory infections have been found in the human gastrointestinal tract and were capable of proliferation there. these include enterovirus (coxsackie a, b virus), parechovirus, orthomyxovirus (avian influenza virus), henipavirus (nipahand hendra viruses), mastadenovirus (adenovirus), alphatorquevirus (torque teno virus), and coronavirus (bosch et al., ) . in the later case, lymphocytes and mucosal epithelial cells of the patients' intestine were positive for sars-cov (shi et al., ) . this may mean that the viruses can be acquired by humans via the consumption of contaminated foods. however, these results may not apply to sars-cov- , which points out the need for studies to investigate the survival of sars-cov- in different foods and on food packages. as mentioned, covid- virus has an ability to stay alive for up to h as a virion on inanimate objects after completing its life cycle in the body of an infected person (van doremalen et al., ) . therefore, if the respiratory discharges of the covid- patient come in contact with food, the food items can become a fomite (carrier), and if these items are contacted by other individuals, the virus is more likely to gain entry to their respiratory epithelium when unsanitized hands touch the nose, eyes, and mouth (bundesinstitut für risikobewertung [bfr], ; centers for disease control and prevention [cdc], ). the surfaces of utensils, packaging material, counters, conveyor belts, interiors of transport vehicles, and all other food work stations where there might be human contact with food should remain a focus of attention where food handlers can act to impede the spread of covid- . therefore, the proper use of personal protective equipment and adherence to the guidelines issued by public health authorities that include regular hand washing when exchanging goods, plus the use of hand sanitizers, wearing masks and gloves, and the maintenance of at least feet between personnel are most important. a range of disinfectants and sanitizers are available in the marketplace. if disinfectant labels suggest that they are effective against coronaviruses or norovirus, then they should be effective against sars-cov- as well. additionally, complete instructions are given on epa disinfectant labels regarding the contact time, concentration, and appropriate surfaces for application (environmental protection agency, ). all food industry organizations should strictly follow the protocols of food safety management systems (fsms) given by authorities based on haccp principles and should be kept updated in response to new pieces of evidence for viruses when required. in food companies where haccp protocols are not being implemented, an expert should be appointed who will remain in contact with public health authorities to seek advice during the pandemic situation. hand washing stations should be maintained for the workforce with the provision of normal soap, warm running water, hand sanitizers, and posters designed for displaying information regarding effective hand washing and sanitization. the physical distancing of feet should be implemented among workers as infected people may remain asymptomatic or be pre-symptomatic during the course of the disease and may spread the infection when close to others (kimball et al., ; pan et al., ; tong et al., ; wei et al., ; yu et al., ) . the introduction of staggered workstations is an effective method to overcome the challenge of physical distancing in food industry facilities. it is advised to minimize the contact between people during the outbreak; therefore, online food deliveries are more desirable. these allow physical distancing between customers and sales personnel. at this stage, proper dissemination of information on food handling practices is also required. since food packages and paper currency are exchanged between consumers and retailers, proper precautions are needed to minimize the potential for virus transfer during the transaction. some third-party delivery companies have also introduced contact-free delivery to homes. the packaging can be discarded after keeping track of important information mentioned on it. the proper use of gloves, sanitizers, and disinfectants can minimize the risk of virus spread and disease transmission (food and agriculture organization of the united nations [fao] and world health organization [who], ; food and drug administration [fda], a). maintaining the movement of food along the food chain is an important function that requires all involved to contribute and stay vigilant. it is necessary to maintain the confidence and trust of consumers regarding food safety and food availability. in case of limitations on the foodservice industry, home deliveries can be promoted, however, a safe and secure environment for food retail shops and canteens must be ensured at both the consumer's and retailer's end. retailers can play their roles by ensuring the provision of sanitary facilities including wipes, disinfectants, sanitizers, and display of sanitary practices through visual aids. physical distancing can be maintained by having the floors marked as a reference for maintaining minimum distance required. plexiglass can be installed to avoid contact at cashier counters, and food tasting for promotional campaigns should be avoided ( at the consumer end, people should make sure that family members belonging to vulnerable groups (immunecompromised, elderly, children, should stay at home (centers for disease control and prevention [cdc] , ). besides, the use of masks, wearing gloves, using hand sanitizers, using wipes before handling food carts, avoiding reusable shopping bags and opting for acceptable respiratory etiquette should be prioritized. if reusable bags are deemed regionally acceptable, their disinfection should be done instantly after use. it is advised to stock food items according to their perishability in order to minimize the number of visits to markets. after safe purchasing with all protocols, the most neglected thing is the safe handling of food. with respect to food handling practices in our kitchens, evidence that has been obtained with coronaviruses indicated that they cannot survive cooking, but if food remains unwashed and is frozen afterward, the virus can survive up to years during frozen storage (bundesinstitut für risikobewertung [bfr], ). therefore, scrubbing of food items like fruits and vegetables is essential if they are not to be cooked for longer periods or to be eaten uncooked. for canned foods, the lids should be wiped before opening. ensure the disinfection of utensils, pots, countertops, and fridge at every use and minimize the risks of cross-contamination among food items during storage. in addition, respect cooking protocols to ascertain food safety and avoid a false sense of security by following proper cooking time, temperature, and thawing protocols. consumers' concern regarding the ability of sars-cov- to survive on the surface of packages has led to an increasing interest in the development of polymers and biopolymers with antiviral properties. the applications of polymers and biopolymers have shown high efficacy against hepatitis a virus (hav) and human norovirus (hunov) (randazzo et al., ) . a previous study showed that the release of copper ions can help in the inactivation of hucov- e on copper or copper alloy surfaces (warnes et al., ) . these findings are a source of validation for the recent findings by van doremalen et al. ( ) regarding the decreased viability of sars-cov- on copper surfaces and inactivation within h. the lack of trials on food matrices and related food regulatory requirements remain the hurdles to the adoption of novel food packaging. the development of biopolymers with antiviral properties and their applications in the food area remains an open field of research. for example, it has been recently reported that the use of nanomaterial coatings or films containing copper, silver, and zinc nanoparticles has a potential against sars-cov- to prevent contamination of food packaging surfaces and thus reduce its transmission (sportelli et al., ) . in conclusion, there are only shreds of evidence regarding the duration of coronavirus survival on different contact surfaces and in foods under certain conditions which suggests the need for advanced studies in understanding the risk of covid- spread associated with food and food packages. research trials are required to find a link between the ingestion of food contaminated with sars-cov- and the probability of infections as well as the development of antiviral active packaging using nano-based biopolymer materials. the current guidelines issued by public health authorities are based on the disease patterns of previously encountered coronaviruses and they need to be updated according to the novel coronavirus sars-cov- as this virus is likely to persist and people will have to modify their "normal behavior" to a "new normal." all datasets generated for this study are included in the article/supplementary material. ao, hs, and nf wrote the draft of the manuscript. ao, hs, nf, and sm reviewed and edited the final manuscript version. rh critically revised the final manuscript version. all authors contributed to the article and approved the submitted version. interagency risk assessment for the public health impact of highly pathogenic avian influenza virus in poultry, shell eggs, and egg products. food safety and inspection 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presymptomatic transmission of sars-cov- stability of middle east respiratory syndrome coronavirus in milk aerosol and surface stability of sars-cov- as compared with sars-cov- pathogen survival in the external environment and the evolution of virulence unique epidemiological and clinical features of the emerging novel coronavirus pneumonia (covid- ) implicate special control measures human coronavirus e remains infectious on common touch surface materials presymptomatic transmission of sars-cov- -singapore coronavirus disease (covid- ) pandemic delivery of infection from asymptomatic carriers of covid- in a familial cluster survival of respiratory viruses on fresh produce a familial cluster of infection associated with the novel coronavirus indicating potential person-to-person transmission during the incubation period covid- transmission within a family cluster in yancheng the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © olaimat, shahbaz, fatima, munir and holley. this is an openaccess article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- - drr p authors: massey, bill w.; jayathilake, karuna; meltzer, herbert y. title: respiratory microbial co-infection with sars-cov- date: - - journal: front microbiol doi: . /fmicb. . sha: doc_id: cord_uid: drr p co-infection with additional pathogens is a well-known feature of pandemics. we determined the prevalence and type of a wide variety of respiratory pathogens in , united states subjects tested for sars-cov- infection in march and april . infections with other respiratory pathogens, which on their own produce at least some sars-cov- symptoms including mortality, were present in both sars-cov- + and sars-cov- - subjects. non-sars-cov- infection rates were significantly higher in sars-cov- + ( %) patients than sars-cov- – patients ( %) (p < . ). among the co-pathogens present in both subject groups were k. pneumoniae and m. catarrhalis which can produce serious respiratory illness on their own, advanced age and nursing home status were associated with higher sars-cov- + and co-infection rates. testing for the presence of co-pathogens going forward will assist in the diagnosis and optimal treatment of suspected sars-cov- respiratory infections in the current pandemic. introduction sars-cov- infected individuals present with fever, aches, headache, cough, runny nose, nasal congestion, coagulation-induced vascular pathology, and fatigue (world health organization, ). an estimated to % of infected individuals are asymptomatic, while to % will die despite current approaches to treatment (nishiura et al., ) . these symptoms are also present in other, sometimes fatal respiratory infections, e.g., k. pneumonia, and m. catarrhalis (cox et al., ) . patients with primary sars-cov- or other respiratory infections may have increased susceptibility to a secondary respiratory infection, with the probable result of exacerbations of disease severity should both be present (cox et al., ) . it is likely that co-infection with some pathogens will lead to greater severity of illness and complicate treatments which address only sars-cov- or the co-pathogen (cox et al., ) . there is limited information about respiratory co-infection in sars-cov- subjects. a few studies have reported co-infection rates in sars-cov- infected patients. a united states study of sars-cov- + subjects found that % were co-infected with other pathogens (kim et al., ) . a recent online study in a metropolitan new york population, sampled in march , examined a limited number of relatively benign pathogens in , sars-cov- + cases and found coinfection in only sars-cov- + patients ( %), while / ( . %) of sars-cov- -patients were co-infected (nowak et al., ) . the study tested for influenza a and b, respiratory syncytial virus, other coronaviruses, rhinovirus, human metapneumovirus, adenovirus and parainfluenza viruses. bacterial pathogens were not tested in this study. the most common detected pathogen in sars-cov- -patients was rhinovirus ( . %), whereas in sars-cov- + patients the most common co-pathogen were non-sars-cov- coronaviridae viruses (nowak et al., ) . of note, only . % of the , sars-cov- + and . % of the , sars-cov- -patients were tested for co-pathogens. the basis for sample selection was not disclosed and may not be representative of the population at risk. studies examining coinfection rate in sars-cov- infected patients have revealed a wide variance in coinfection rates, ranging from . to % (lai et al., ) . the observed coinfected pathogens have been viral (e.g., influenza, rhinovirus, parainfluenza, metapneumovirus, hiv), bacterial (e.g., staphylococcus aureus, streptococcus pneumoniae, klebsiella pneumoniae, mycoplasma pneumoniae, chlamydia pneumoniae, legionella pneumophila), and fungal (e.g., candida species, aspergillus flavus) (lai et al., ) . the most common coinfected pathogens varied from study to study, and this variability may be due to geographic factors, differences in the breadth of organisms tested, or disparate patient demographic factors (lai et al., ) . in addition, several case studies of coinfection in sars-cov- patients have been reported and include coinfection with influenza a (d' ardes et al., ; konda et al., ; wu et al., ) . as an indication of the importance of this issue, bacterial co-infection was reported in half of the deceased patients in a retrospective study of sars-cov- from wuhan, china (zhou et al., ) . this may be due to increased risk of pneumonia, the primary cause of sars-cov- deaths in coinfected individuals (gao et al., ) . the fatal pneumonia cases may result from an excessive immune reaction to sars-cov- called "cytokine storm, " elicited by the response to multiple pathogens (zhou et al., ) . the extent to which one type of infection affects the susceptibility to the other in sars-cov- is unknown. respiratory co-infections were reported to be major contributors to morbidity and mortality in previous viral respiratory epidemics, such as the influenza pandemics of and (morens et al., ; macintyre et al., ) . during the influenza pandemic, bacterial infections were found in almost % of patients and were associated with higher rates of intensive care unit admissions and death (macintyre et al., ) . in the present study, we present data on the prevalence of sars-cov- and other bacterial, viral and fungal respiratory pathogens in samples taken from over , united states symptomatic subjects tested for the presence of sars-cov- in a manner which permitted assessment of the presence of co-pathogens as well as sars-cov- . the data were obtained from , subjects from whom nasopharyngeal swabs were collected with the principal aim to detect presence or absence of sars-cov- infection. subjects consent to use de-identified data for research purposes was obtained with the test requisition form. these samples were collected and submitted to vikor scientific, a clia-certified and cap-accredited clinical laboratory located in charleston, south carolina between march , and april , . the samples were collected from multiple types of health facilities throughout the united states and shipped by overnight mail for analysis. pcr analyses were conducted the day of arrival at the lab to identify sars-cov- and a panel of other bacterial, fungal, and viral respiratory pathogens. pathogen testing was performed by real time pcr amplification to detect presence of tested pathogens by amplifying genomic dna/rna. the microorganisms tested are listed in table . amplification and detection were performed using an applied biosystems tm quantstudio tm , k flex real-time pcr system (catalog # a ; thermo fisher scientific, waltham, ma, united states). real-time pcr amplification was performed using taqman tm assays from thermo fisher scientific consisting of two pcr primers and one fluorescently labeled probe which hybridizes to the target organism's genomic nucleic acid. semiquantitative determination of pathogen load for co-infected pathogens was derived from c t values and presented as number of cells per ml for bacterial pathogens (e.g., × cells/ml) and as number of viral copies per ml for viral pathogens (e.g., × viral copies/ml). sars-cov- testing was performed using the taqpath tm covid- combo kit (catalog # ccu ; thermo fisher). the assay detected three sars-cov- -specific gene targets: orf ab; s protein; and n protein. full validation studies established a limit of detection % (lod ) of . copies/ul. linear dynamic range sensitivity over a positive control range of - , copies/ul was demonstrated at r > %. no cross-reactivity was seen in the multi-target plasmid control containing common respiratory pathogens nor to any respiratory pathogens tested using thermo fisher taqman tm assays. detection of the sars-cov- -specific gene targets were each considered positive at c t values < , inconclusive at c t values to , and negative at c t values > . patients were considered positive for sars-cov- if two or more of the sars-cov- genes were detected. analytical accuracy was evaluated using the control material contained in the thermo fisher taqpath tm covid- combo kit (catalog # a ) for each of the target sequences for gene assays orf ab, s protein, and n protein. controls with concentrations of x, x, and x lod were used and run in triplicate. a positive/negative amplification status was used to satisfy the c t criteria and considered successful when the "actual" call concurred with the "expected" call. analytical accuracy was evaluated based on the comparison of "expected" call and "actual" call for rna spiked samples from x- x lod and the non-template control (water without nucleic acid). results based upon concordance between "actual" and "expected" calls were % concordant for all gene targets of sars-cov- . co-infected pathogens were tested using respira-id tm (vikor scientific, charleston, sc, united states), a custom respiratory pathogen panel consisting of taqman tm assays on a preloaded taqman tm array card. the list of tested respiratory pathogens is shown in table . full validation studies established a lod of copies/preampreaction of input, with only the herpesvirus assay showing an lod at copies/preampreaction. linear dynamic range sensitivity over a positive control range of logs was demonstrated with r values > . . potential cross reactivity was evaluated using well-characterized, normalized genomic dna pools, with specificity across all assays shown to be . %. analytical accuracy was evaluated based on comparison to well-characterized, normalized genomic dna and multi-target plasmid controls and found to be %. a data query engine was developed and used to mine data from the laboratory information management system (ovdx, v . ; ovation.io, inc., cambridge, ma, united states). the data query engine extracted all available information on all tested patients, including all test results and all patient information provided on the test requisition submitted by the ordering physician. these data were organized in a spreadsheet format for importation into the sas tm statistical software used for analyses. the sars-cov- and co-infection data were then analyzed for the frequency and pattern of co-detection of other pathogens. patients were grouped by health facility type, age and gender for additional analysis. patients were grouped into the following age categories: > years of age (elderly), - years of age (adult), and < years of age (young). the source of the samples, age, and gender of the samples are provided in table . no samples were collected from incarcerated individuals. the t-test for independent group comparison was used to compare age of the study subjects across gender, sars-cov- ± patients and residential versus outpatients. the interaction effect of sars-cov- ± patients and gender on age was tested using an anova model. the effects of gender, age group, residential status, and presence or absence of coinfections on sars-cov- ± rates were analyzed by chi-square. prediction of sars-cov- + rates by age group, gender, number of co-infections, quantitative load of each co-infection and residential status was done by logistic regression. all analyses were performed using sas tm (sas institute, inc., cary, nc, united states) statistical software. in sars-cov- + patients, the target were detected in the following frequency: orf ab, . %, n protein, . %; and s protein, . %. there was % concordance between all three targets in all sars-cov- -patients. the samples were collected from , subjects from facilities throughout the united states. these consisted of doctor's offices, medical clinics and hospitals, community health care centers, urgent care centers, assisted living facilities, retirement community centers and nursing homes. the average age of the population was . ± . years; and males ( . ± . years) and females ( . ± . years) were similar in age. sars-cov- + patients ( . ± . ) were . years older than sars-cov- -patients ( . ± . ) and this was statistically significant with p < . . patients from nursing homes, hospice facilities, senior living facilities, long-term care facilities and rehab facilities were grouped together as residential (n = , . %). healthcare or medical centers, urgent care facilities, community health centers, assisted living retirement communities were categorized as nonresidential or outpatient (n = , . %). residential patients were, on average, . years older than outpatients. the subject's race was recorded only for % (n = , ) of subjects and the racial breakdown of the patients was . % (n = , ) caucasian, . % (n = ) african american, and . % (n = ) other races (asian, hawaiian and american indians). fourteen percent (n = ) of the samples were found to be sars-cov- +. of the of the subjects who reported their gender information, . % of the males and . % of the females tested as sars-cov- +. racial data was available only for % of the subjects. no differences in sars-cov- infection rates were observed between the different races, with . % of caucasian subjects and . % of the african american subjects found to be sars-cov- +. of the subjects from other races, % were found to be sars-cov- +. using chi-square tests it was found that gender and race had no effect on the sars-cov- infection rate. however, there were differences in sars-cov- infection rates between patients from residential facilities and outpatients, with . % of residential subjects being sars-cov- + compared to % of the outpatient subjects (p < . ). also, the elderly group (age ≥ years) had a sars-cov- infection rate of % compared to . % in the younger patients (age < years; p < . ). thus, race and gender had no effect on sars-cov- infection rate, while both residential status and age group had a significant effect on sars-cov- infection rate. the results are displayed in the table . in the total population, patients also had other non-sars-cov- infections such as s. aureus ( . %), epstein-barr virus (hhv ) ( . %), human herpes virus (hhv ) ( . %), m. catarrhalis ( . %), k. pneumoniae ( . %), human metapneumovirus (hmpv) ( . %), and human adenovirus (adv) ( . %). these data and the prevalence rates of co-infections in sars-cov- patients are listed below in table . it was noted that % more patients had s. aureus in sars-cov- + group as compared to the sars-cov- -group. similarly, hhv , hhv , m. catarrhalis and k. pneumoniae were found in , , , and % more patients in the sars-cov- + group compared to the sars-cov- -group, respectively. in contrast, significantly increased infection rates of hmpv and adv were seen in sars-cov- patients as compared to sars-cov- + patients. the data are presented in table . some of the subjects tested positive for more than one copathogen, with co-infection rates being greater in sars-cov- patients. in sars-cov- + patients, . % had at least one co-infection compared to . % in the sars-cov- -group (p < . ). the degree of disparity between sars-cov- + and sars-cov- -groups increased with increasing number of coinfected pathogens. in patients who tested sars-cov- +, % had two or more co-infections compared to . % of the sars-cov- -subjects (p < . ). the percentage of subjects with three or more co-infections who were sars-cov- + ( . %) was significantly greater than the percentage of sars-cov- subjects with three or more co-infections ( . %; p < . ). likewise, the percentage of sars-cov- + subjects with four or more co-infections ( . %) was significantly greater than the percentage of sars-cov- -subjects with four or more coinfections ( . %; p < . ). this data is presented in table . in further analysis of grouping of co-infections it was also noted that patients with hhv and hhv co-infections were twice as likely to be sars-cov- + ( . %) than sars-cov- -( . %; p < . ). sars-cov- + patients were more likely than sars-cov- -patients to have co-infections with hhv + s. aureus, hhv + k. pneumoniae, hhv + s. aureus, hhv + k. pneumoniae, or s. aureus + k. pneumoniae (p < . ). co-infection with s. aureus + m. pneumoniae was more prevalent in sars-cov- + versus sars-cov- -patients (p = . ). analysis also revealed that sars-cov- + patients are more likely to have multiple co-infections, whereas the percentage of patients with a single co-infected pathogen is higher in the sars-cov- -( . %) versus sars-cov- + group ( . %), and this difference was statistically significant (p < . ). a logistic regression model was used to predict sars-cov- positivity using other associate variables in the study. in the model, age group, gender and resident status were used as categorical variables, while log-transformed quantitative loads of hhv , hhv , s. aureus, k. pneumoniae, m. catarrhalis, and m. pneumoniae were used as continuous variables. the loads of the co-pathogen ranged from to . the logistic model found that m. pneumoniae was not a predictor for sars-cov- + status, so it was removed from the model. it was found that all variables except gender are significantly associated with predicting sars-cov- + rates. the data is presented similarly, residential patients (or = . , p < . ), higher hhv viral load (or = . , p < . ), higher hhv viral load (or = . , p < . ), and higher bacterial loads of s. aureus (or = . , p < . ), k. pneumoniae (or = . , p = . ) and m. catarrhalis (or = . , p < . ) were significant predictors of sars-cov- + status. the lower confidence intervals for all variables were above . the use of logistic regression models to predict sars-cov- + status from the above-listed independent variables was supported by the higher model fit criteria of percent concordant statistic ( . ) and c statistic ( . ). the logistic regression models were repeated adding race into the model and found that race was not significantly associated with predicting sars-cov- + rate. initially, caucasian versus african american patients were examined, followed by caucasian versus everyone else. neither of the models displayed any significant race effect (results not presented). this is the first large-scale assessment of sars-cov- and co-infection rates with other respiratory pathogens in a consecutive series of people presenting for sars-cov- testing during the covid- pandemic in the united states. the major findings were that almost all patients have one or more co-pathogens, and that non-sars-cov- co-infection rates were significantly higher in sars-cov- + ( %) patients than sars-cov- -patients ( %) (p < . ). s. aureus, epstein-barr virus (hhv ), human herpes virus (hhv ), m. catarrhalis, and k. pneumoniae were the most common copathogens in sars-cov- + patients. in sars-cov- -patients, the most common pathogens detected, in order of frequency, were s. aureus, human herpes virus (hhv ), epstein-barr virus (hhv ), m. catarrhalis, hmpv, k. pneumoniae and adv. the respiratory symptoms produced by these non-sars-cov- pathogens in % of sars-cov- -patients may have been the reason for sars-cov- testing, as the testing guidelines in place at the time were heavily weighted toward symptomatic patients. the pcr test used here, which tested for three different sars-cov- associated proteins, showed high reliability of the data that were used in this analysis. some of the bacterial pathogens that were significantly increased in sars-cov- + compared to sars-cov- -patients (s. aureus, k. pneumoniae, and m. catarrhalis) can themselves compromise pulmonary function and cause pneumonia, especially in immunocompromised individuals (henig and kaye, ; martin and bachman, ) . previous studies have shown that respiratory viral infections predispose patients to bacterial infection with a more severe clinical course with greater morbidity and mortality (prasso and deng, ) . post-viral bacterial pneumonia produces complex interactions between the bacteria and viruses, normal nasopharyngeal bacterial flora, and the host immune system (lee et al., ) . for example, viral infection can damage the respiratory epithelium, enabling infiltration and infection by bacterial flora and subsequent pneumonia, and can also upregulate bacterial binding sites, facilitating infection (lee et al., ) . additionally, viral infection can impair activity of monophages, a key participant in immune function (lee et al., ) . a characteristic of sars-cov- infections is significantly reduced lymphocyte counts (li et al., ) . lymphocytes are essential for adaptive immunity. the very high levels of pro-inflammatory cytokines in sars-cov- "cytokine storm, " can cause lymphocyte apoptosis and resultant lymphocytopenia (jensen et al., ) . this lymphocytopenia constitutes an immunodeficient state, which could make these patients more susceptible to coinfection with other pathogens (li et al., ) . in the present study, the observed rate of co-infection in sars-cov- + patients ( %) and sars-cov- -patients ( %) was significantly higher than the rates observed in previous studies ( . to %). the higher coinfection rate observed in the present study may be due to the criteria applied to sars-cov- testing eligibility at the time, which was heavily weighted toward symptomatic patients, and whom would be more likely to have a current infection. the samples were consecutive samples from a -week period beginning in the last week of march. since that time, great differences in the rates of sars-cov- infection in different parts of the united states have evolved, and continue to evolve, most likely based on rates of opening of businesses and social venues, social distancing, use of masks, hand washing, etc. a repeat analysis of this kind, with the opportunity to recontact those who test positive for sars-cov- with and without infection with other pathogens known to produce serious respiratory symptoms, is under consideration. such follow up data would be useful to determine the contribution of the coinfection to outcome. since the subjects in this study came from many parts of the united states, the findings reported here may be representative of the united states as whole at the time of sampling. nevertheless, this was not an epidemiologic selected sample and may not reflect the true prevalence of the other pathogens. thus, data from an epidemiologic sample is needed. the large number and diversity of subjects allowed for examination of sars-cov- and other pathogen infection rates, and co-infection by age group, residential facilities (i.e., nursing homes and assisted living facilities), race, and gender. as expected, co-infection rates were higher in elderly patients, however, young adults and some children were also co-infected. rates of co-infection were similar in both caucasians and african americans, and males and females. as with sars-cov- , infection rates for common respiratory pathogens increased with age. the rate of sars-cov- infection in the > years age group was more than double that observed in younger patients. co-pathogen rates were also greater in the elderly age group as a whole and even higher than those for sars-cov- . infection with non-sars-cov- pathogens was also greater in the sars-cov- group in the elderly, indicating age-related vulnerability is a general phenomenon. it suggests that a negative test for sars-cov- in a symptomatic subject, particularly an elderly one, should be followed by testing for non-sars-cov- pathogens if such testing is not done during the testing for sars-cov- . given how much more prevalent these non-sars-cov- respiratory infections are than sars-cov- itself, it may be worthwhile to also test for them in all elderly patients with moderate-severe respiratory symptoms. absence of association with others infected with sars-cov- or a lack of travel to places with high rates of infected individuals supports testing for co-pathogens in the initial testing effort. for the % of sars-cov- -patients co-infected with one or more respiratory pathogens, it is likely that respiratory symptoms in at least some are due, in part, to the coinfection, and the patient would benefit from treatment for that pathogen, e.g., k. pneumoniae. co-pathogen testing can include testing for antibiotic resistance genes to aid in the selection of effective treatments for infections due to s. aureus, k. pneumoniae and m. catarrhalis, the most common serious co-pathogens detected in sars-cov- + patients in the present study. in conclusion, we have utilized a highly reliable pcrbased test for sars-cov- and other respiratory pathogens, to provide the first countrywide data on the rate of infection with respiratory pathogens other than sars-cov- . the infection rate for these other respiratory pathogens throughout the united states is much greater than that of sars-cov- itself in subjects seeking sars-cov- testing. infection with these other respiratory pathogens as well as co-occurrence of infection with sars-cov- was increased in the elderly and those who reside in nursing homes. gender and race did not impact rates of infection with other respiratory pathogens. these results suggest that patients with pre-existing respiratory infections are at increased risk to develop a sars-cov- infection, or conversely, that once infected with sars-cov- , vulnerability to other respiratory pathogens is increased. identifying and treating other respiratory pathogens along with sars-cov- testing should facilitate better outcomes in the current pandemic. the raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. ethical review and approval was not required for the study on human participants in accordance with the local legislation and institutional requirements. written informed consent to participate in this study was provided by the participants' legal guardian/next of kin. bm conceived and designed the study and was primary author the manuscript. kj designed and conducted the statistical analyses. hm provided guidance and was secondary author of the manuscript. all authors contributed to the article and approved the submitted version. this study was supported, in part, by a donation from the weisman family. co-infections: potentially lethal and unexplored in covid- a case of coinfection with sars-cov- and cytomegalovirus in the era of covid- breakthrough: chloroquine phosphate has shown apparent efficacy in treatment of covid- associated pneumonia in clinical studies bacterial pneumonia in older adults sepsisinduced t cell immunoparalysis: the ins and outs of impaired t cell immunity rates of coinfection between sars-cov- and other respiratory pathogens co-infection with sars-cov- and influenza a virus co-infections among patients with covid- : the need for combination therapy with non-anti-sars-cov- agents the role of respiratory viruses in the etiology of bacterial pneumonia: an ecological perspective coinfection with sars-cov- and other respiratory pathogens in covid- patients in guangzhou the role of pneumonia and secondary bacterial infection in fatal and serious outcomes of pandemic influenza a(h n )pdm colonization, infection, and the accessory genome of klebsiella pneumoniae predominant role of bacterial pneumonia as a cause of death in pandemic influenza: implications for pandemic influenza preparedness estimation of the asymptomatic ratio of novel coronavirus infections (covid- ) co-infection in sars-cov- infected patients: where are influenza virus and rhinovirus/enterovirus? postviral complications bacterial pneumonia available online at:˜www.who.int/news-room/q-a-detail/q-a-coronaviruses#:˜:text=symptoms co-infection with sars-cov- and influenza a virus in patient with pneumonia. china emerg clinical course and risk factors for mortality of adult inpatients with covid- in wuhan, china: a retrospective cohort study the authors would like to acknowledge vikor scientific llc for the use of their database. the authors would also like to acknowledge tony donofrio and kevin yee of ovation.io inc. for creation of the data query engine and data acquisition, and robin prince for antimicrobial treatment recommendations. the supplementary material for this article can be found online at: https://www.frontiersin.org/articles/ . /fmicb. . /full#supplementary-material key: cord- - ea gz s authors: li, guiping; zhou, lijuan; zhang, can; shi, yun; dong, derong; bai, miao; wang, rong; zhang, chuanfu title: insulin-like growth factor regulates acute inflammatory lung injury mediated by influenza virus infection date: - - journal: front microbiol doi: . /fmicb. . sha: doc_id: cord_uid: ea gz s the acute inflammatory lung injury is an important cause of death due to influenza a virus (iav) infection. insulin-like growth factor (igf ) played an important role in the regulation of inflammation in the immune system. to investigate the role of igf in iav-mediated acute inflammatory lung injury, the expression of igf and inflammatory cytokines was tested after iav a/puerto rico/ / (h n ; abbreviated as pr ) infection in a cells. then, a balb/c mouse model of pr infection was established. on days , , , and post-infection, the mice lung tissue was collected to detect the expression changes in igf mrna and protein. the mice were divided into four groups: ( ) pbs (abbreviation of phosphate buffered saline); ( ) pr + pbs; ( ) pr + igf ; and ( ) pr + ppp (abbreviation of picropodophyllin, the igf receptor inhibitor). the body weight and survival rate of the mice were monitored daily, and the clinical symptoms of the mice were recorded. on day post-infection, the mice were sacrificed to obtain the serum and lung tissues. the expression of inflammatory cytokines in the serum was detected by enzyme linked immunosorbent assay; lung injury was observed by hematoxylin-eosin staining; the viral proliferation in the lung was detected by real-time quantitative pcr; and the protein expression of the main molecules in the phosphatidylinositol- -kinases/protein kinase b (pi k/akt) and mitogen-activated protein kinase (mapk) signaling pathways was detected by western blot. it was found that igf expression is upregulated in a cells and balb/c mice infected with pr , whereas igf regulated the expression of inflammatory cytokines induced by pr infection. overexpression of igf aggravated the iav-mediated inflammatory response, whereas the inhibition of igf receptor reduced such inflammatory response. the phosphorylation of igf receptor triggered the pi k/akt and mapk signaling pathways to induce an inflammatory response after iav infection. therefore, igf plays an important immune function in iav-mediated acute inflammatory lung injury. igf may provide a therapeutic target for humans in response to an influenza outbreak, and inhibition of igf or igf receptor may represent a novel approach to influenza treatment. the acute inflammatory lung injury is an important cause of death due to influenza a virus (iav) infection. insulin-like growth factor (igf ) played an important role in the regulation of inflammation in the immune system. to investigate the role of igf in iav-mediated acute inflammatory lung injury, the expression of igf and inflammatory cytokines was tested after iav a/puerto rico/ / (h n ; abbreviated as pr ) infection in a cells. then, a balb/c mouse model of pr infection was established. on days , , , and post-infection, the mice lung tissue was collected to detect the expression changes in igf mrna and protein. the mice were divided into four groups: ( ) pbs (abbreviation of phosphate buffered saline); ( ) pr + pbs; ( ) pr + igf ; and ( ) pr + ppp (abbreviation of picropodophyllin, the igf receptor inhibitor). the body weight and survival rate of the mice were monitored daily, and the clinical symptoms of the mice were recorded. on day post-infection, the mice were sacrificed to obtain the serum and lung tissues. the expression of inflammatory cytokines in the serum was detected by enzyme linked immunosorbent assay; lung injury was observed by hematoxylin-eosin staining; the viral proliferation in the lung was detected by real-time quantitative pcr; and the protein expression of the main molecules in the phosphatidylinositol- -kinases/protein kinase b (pi k/akt) and mitogen-activated protein kinase (mapk) signaling pathways was detected by western blot. it was found that igf expression is upregulated in a cells and balb/c mice infected with pr , whereas igf regulated the expression of inflammatory cytokines induced by pr infection. overexpression of igf aggravated the iav-mediated inflammatory response, whereas the inhibition of igf receptor reduced such inflammatory response. the phosphorylation of igf receptor triggered the pi k/akt and mapk signaling pathways to induce an inflammatory response after iav infection. therefore, igf plays an important immune function in iav-mediated acute inflammatory lung injury. igf may provide a therapeutic target for humans in response to an influenza outbreak, and inhibition of igf or igf receptor may represent a novel approach to influenza treatment. keywords: influenza virus, igf , acute inflammatory lung injury, pi k/akt, mapk introduction influenza is an acute infectious disease caused by influenza virus infection, which is primarily characterized by respiratory damage. moreover, it is associated with serious features (e.g., acute onset, wide spread, strong contagiousness, and great harm), which seriously threatens human health. studies have shown that a series of symptoms and consequences caused by the influenza virus are not caused by the direct action of the influenza virus itself but are rather due to the inflammatory injury caused by the excessive activation of the immune response induced by an influenza virus infection (oda et al., ; taubenberger and morens, ; basler and aguilar, ; li and cao, ) . during the process of an iav infection, there are different cytokine waves from the initial early responses of monocyte chemotactic protein- (mcp- ), interleukin- (il- ), and interferon-alpha/beta/kappa (ifn-α/β/κ) to the later responses of il- α/β, il- , il- , tumor necrosis factor-alpha (tnf-α), ifn type i, macrophage inflammatory protein- alpha/beta (mip- α/β), mip- α, mcp- , and interferon-inducible protein- (ip- ), which are mainly secreted by infected macrophages in the lower respiratory tract (julkunen et al., ; xagorari and chlichlia, ; jewell et al., ; gu et al., ) . the modest cytokine response contributes to the induction of antiviral and th -type immune responses in the body; however, excessive inflammatory responses can be harmful. for example, the main reason for the high pathogenicity of the h n avian influenza virus is the exaggerated generation and secretion of excessively high levels of pro-inflammatory cytokines, known as a "cytokine storm" (kobasa et al., ; allen et al., ) . in , the "spanish flu" also caused fatal inflammatory damage to the lung tissue by triggering an overreaction of the human immune response (ocana-macchi et al., ). this inflammatory injury to the lung tissue is both an important cause of death due to influenza virus infection and a major cause of lung infections caused by severe acute respiratory syndrome (sars), sepsis, and aspiration pneumonia (imai et al., ; nicholls and peiris, ) . currently, immunosuppressive agents (e.g., glucocorticoids) are often used in clinical practice to inhibit inflammatory cytokine responses, thereby blocking disease progression and improving the clinical therapeutic effect. the literature reports that - % of patients with severe h n influenza are treated with glucocorticoids, which have a specific therapeutic effect (aikawa et al., ) ; however, glucocorticoids systemically inhibit the immune function of the infected individual. moreover, the long-term treatment with such medication can induce or aggravate the infection, causing a variety of complications and serious side effects (e.g., femoral head necrosis). therefore, it is imperative to develop a deep understanding of the pathogenesis of influenza virus and to identify new strategies of treating influenza more safely and effectively. insulin-like growth factor (igf ) belongs to the insulinlike growth factor family, which also includes growth hormone (gh), insulin-like growth factor ii (igf ), insulin-like growth factor receptor (igf r), insulin-like growth factor ii receptor (igf r), and insulin-like growth factor binding protein - (igfbp - ; jogie-brahim et al., ) . this family plays an extremely important role in the process of cell growth, differentiation, and apoptosis (stewart and rotwein, ; granata et al., ) . igf mainly functions by binding to igf r, a transmembrane protein composed of two α domains and two β domains (siddle et al., ) . the α domain binds to igf to activate the β domain (siddle et al., ) . since the β domain has tyrosine kinase activity, it can promote the phosphorylation of the substrate hepatocyte growth factor (hgf), docking protein insulin receptor substrate (irs), vascular endothelial growth factor (vegf), and growth factor receptor binding protein (grb ; wilden et al., ; hubbard, ) . some phosphorylated substrates activate the downstream phosphatidylinositol- -kinases/protein kinase b (pi k/akt) and mitogen-activated protein kinase (mapk) signaling pathways to regulate a range of biological responses (myers et al., ; egan et al., ) . recent studies have found that igf plays an important role in the regulation of inflammation in the immune system. igf mrna expression in the bronchial cells of asthmatic patients was significantly higher than that of normal people and was significantly associated with fibrosis in epithelial cells (hoshino et al., ) . the associated mechanism is that igf binds to the receptor and activates the pi k/akt signaling pathway and induces akt activation, which further activates the downstream il- -mediated inflammatory pathway (lee et al., ) . in addition, the levels of serum igf protein in patients with type diabetes are significantly higher than those in healthy people. obesity is closely related to the pathogenesis of type diabetes, as long-term obesity will lead to il- and il- -mediated chronic inflammation (cohen and leroith, ; ge et al., ) . in non-autoimmune inflammatory contact dermatitis, igf relieves the inflammatory response by recruiting regulatory t cells to release the anti-inflammatory cytokine, il- (johannesson et al., ) . in the nervous system, igf recruits anti-inflammatory proteins to protect nerve cells from degeneration (arroba et al., ) . other studies have shown that igf plays an important role in regulating the function of lactating buffalo oocytes and preventing inflammation induced by post-partum genital tract infections. a concentration of ng/ml igf acts on lipopolysaccharides (lps; μg/ml)-infected buffalo granulosa cells, reducing the expression of the inflammatory cytokines, il- , tnf-α, and il- β, as well as decreasing akt phosphorylation in pi k/akt signaling pathway and extracellular-regulated kinase / (erk / ) phosphorylation in the mapk signaling pathway (onnureddy et al., ) . however, whether igf plays a significant role in mediating inflammation and pathology during influenza infection and its associated mechanism remains unknown. in this study, we found that igf mrna and protein increased after influenza virus infection. overexpression of igf aggravated cytokine expression during infection by influenza, while blocking of igf production in mice treated with igf r inhibitor, decreased immunopathology. the phosphorylation level of igf r was elevated after influenza virus infection, triggering the pi k/ akt and mapk signaling pathways to induce an inflammatory response. thus, igf could regulate influenza virus-mediated acute inflammatory lung injury, which may provide a therapeutic target for humans in response to an influenza outbreak. the human alveolar epithelial cell line a is particularly sensitive to influenza virus infection and has been widely used as a good in vitro model to study influenza virus for nearly years. the cell line a was purchased from the american type culture collection (atcc, usa) and propagated in dulbecco's modified eagle's medium (dmem; life technologies, usa) supplemented with % fetal bovine serum (fbs; hyclone, usa) at °c in a % co incubator. the mouse adapted influenza a virus (iav) a/puerto rico/ / (h n ; abbreviated as pr ) was kindly provided by prof. shihui sun (beijing institute of microbiology and epidemiology) and propagated in -to -day-old spf chicken embryos. the allantoic fluid was collected and titrated to determine the % tissue culture infection dose (tcid ) in a cells and the median lethal dose (ld ) in mice following the reed-muench method (reed and muench, ) . specific pathogen free (spf) grade female balb/c mice aged - weeks (body weight: - g) were purchased from the experimental animal center of the military medical research institute. amplification of human igf open reading frame (orf; guangzhou genecopoeia biotechnology co., ltd.) using primers containing xba i and xho i restriction sites (forward: ′-tgctctagaatgggaaaaatcagcagtct- ′; reverse: ′-ccgctcgagctacatcctgtagttcttgt- ′) ligated into a pcdna . expression vector, constructing pcdna . -igf . the pcdna . -igf vector was transfected into a cells with lipo . the cell line overexpressing igf was screened with g ( μg/ml). the human igf shrna lentiviral particles (sc- -v) were purchased from santa cruz company. the total cellular rna was extracted using trizol (invitrogen, cat: - ). the cdna was synthesized by reverse transcription using a tianscript rt kit (tiangen, cat: kr ), followed by quantitative pcr (qpcr) using sybr premix ex taq ii (takara, cat: rr a). the primer sequences that were used are presented in table . when detecting the viral proliferation in the lungs of mice, a realtime fluorescent quantitative pcr probe method was used, and the probe sequence was fam-tgcagtcctcgctcac tgggcacg-bhq . the primer sequence of matrix protein (m ) was forward: ′-gaccratcctgtcacctctgac- ′; reverse: ′-gggcattytggacaaakcgtctacg- ′. gapdh was selected as the internal reference, and the results were analyzed using the −△△ct method. the reaction conditions were set as follows: step : °c for s; step : °c for s, °c for s, cycles; and step : dissolution curve analysis. for the cultured cells, the total cellular protein was extracted using the whole cell lysate (beijing comwin, cat: cw ) containing a protease inhibitor cocktail (roche, germany). the protein was quantified using a bicinchoninic acid (bca) protein assay kit (beijing comwin, cat: cw ), and a μg protein sample was obtained for polyacrylamide gel electrophoresis (page). the lung tissues from six mice of each group were mixed and ground in liquid nitrogen, and then the total cellular protein was extracted using the whole cell lysate (beijing comwin, cat: cw ) containing a protease inhibitor cocktail (roche, germany). protein was quantified using a bca protein assay kit (beijing comwin, cat: cw ). a μg protein sample was obtained for page and subsequently transferred to a polyvinylidene fluoride (pvdf) membrane. the primary antibodies of rabbit anti-glyceraldehyde phosphate dehydrogenase (gapdh) and goat anti-igf (abcam) were diluted to : , . the secondary antibodies of horseradish peroxidase (hrp)-goat anti-rabbit immunoglobulin g (igg) and hrp-rabbit anti-goat igg (zsgb-bio) were diluted to : , . the signals were detected using a western hpr substrate peroxide solution (millipore). the quantification of western blot analysis was performed by using image j software, and the protein expression levels were normalized to gapdh levels. the level of cytokine expression was detected using mouse igf elisa kit, il- elisa kit, tnf-α elisa kit, ifn-γ elisa kit, and il- β elisa kit (all purchased from r&d systems). for viral infection, a cells were washed with phosphate buffered saline (pbs) and subsequently infected with pr at the multiplicity of infection (moi) of . or . in infection medium, which was dmem supplemented . mg/ml n-p-tosyl-phenylalanine primer ′- ′ chloromethyl ketone (tpck)-treated trypsin (sigma, usa) and . % bovine serum albumin (bsa; sangon, china). after h, cells were incubated with fresh infection medium at °c for the indicated times. balb/c mice were intraperitoneally injected with sodium pentobarbital ( mg/kg), and the mice were induced into a deep anesthetic state and administered a nasal inhalation of phosphate buffered saline (pbs) or pr (diluted -fold, μl/mouse, ld ). in the pr + igf group, igf ( μg/ kg, peprotech) was intraperitoneally injected h before infection, and after infection, igf was intraperitoneally injected every h. the pr + ppp group was intraperitoneally injected with the igf receptor inhibitor, and picropodophyllin (ppp, mg/kg, selleck) h before infection and after infection ppp was intraperitoneally injected every h. after the continuous administration for days, changes in the clinical symptoms of mice were observed daily, and changes in the body weight and survival were recorded. on days , , , and after pr infection, six mice of each group were sacrificed every time. the blood was taken by excising the eyeballs, and the lung tissues were collected. the blood was kept at room temperature for h and centrifuged at , g for min. the serum was collected and aliquoted and stored at − °c for later use. all animal experimental procedures were approved by the animal care and use committee of the academy of military medical sciences (amms; id: syxk - ) and were carried out in strict accordance with the guidelines. all experiments involving the live virus were performed in an approved biosafety level facility. after removing the whole lung tissue of the mice, damage to the lung tissue was observed. the degree of lung injury visible to the naked eye was dark red due to edema. the area ratio of lung injury to the total lung tissue was estimated. each sample was estimated by at least three different individuals, and the average was obtained. finally, the lung injury area of six mice in each group was counted. the wet weight of the lung tissue was weighed. lung index = lung wet weight/ body mass. all experiments were repeated at least three times. data were expressed as means ± standard deviation (sd). the graphical representation of the data was performed using graphpad prism software. the grayscale analysis of the western blot images was performed using image j software by comparing the integrated density of the igf band with the control gapdh. the statistical analyses were performed using spss . statistical software with two-tailed student's t test for two comparisons, *p < . ; **p < . ; and ***p < . . a p below . was considered statistically significant. to detect the regulation of igf by iav, a cells were infected with pr ( . tcid /ml) at different multiplicity of infection (moi) of . , . , or . , and the level of igf mrna and protein expression was detected at , , , and h post-infection. as shown in figure a , compared with the mock-infected controls, the level of igf mrna expression in a cells increased gradually at , , and h after pr infection at a moi of . . the level of igf mrna expression peaked at h post-infection, which was . ± . times higher than that of the mock controls. the level of igf mrna expression decreased slightly at h post-infection but still reached . ± . times that of the mock group. as shown in figure b , in a cells infected with different mois of pr , the level of igf mrna expression increased following the increasing of moi at h post-infection. at a moi of . , the level of igf mrna increased to . ± . times that of the mock group. the level of igf protein expression was also upregulated at , , , and h post-infection of pr ( figure c ) and following the increasing of moi at h post-infection in a cells ( figure d) . to compare the trend in igf variability more intuitively, the grayscale analysis of figures c,d was performed and plotted according to the ratio of the integrated density of igf /gapdh. figures e,f show that the level of igf protein expression in the a cells following pr infection was consistent with the level of igf mrna; however, such upregulation times were lower than the level of mrna. compared with the mock group, the level of igf protein expression peaked . times at h after pr infection at a moi of . ( figure e) . the level of igf protein expression was also increased following the increasing of moi. at a moi of . , the level of igf protein expression reached . times that of the mock group at h post-infection ( figure f) . the above results indicate that igf expression is upregulated in pr -infected a cells. to investigate the role of igf in the inflammatory response to influenza virus infection, we established two stable a cell lines, in which igf is overexpressed or inhibited. using pcdna . -igf to overexpress igf , figure a showed that the level of igf mrna was increased . ± . times that of the control group. the expression of igf in the cells was inhibited by lentiviral packaged shrna (lenti + shigf ), after which the level of igf mrna was reduced to . ± . times that of the control group (figure a) . as shown in figure b , the level of igf protein was also increased in stable a cell line with pcdna . -igf and inhibited in stable a cell line with lenti + shigf . the stably transfected a cell lines were infected with pr at a moi of . . after h, real-time quantitative pcr (qpcr) was used to detect the changes in cytokine expressions [il- , il- , il- , monocyte chemotactic protein- (mcp- , or ccl ), tnf-α, and il- β], which were related to the inflammatory response. the results in figure c showed that compared with the mock controls, the cytokine levels were significantly increased following pr infection in control cells (pr + pcdna . -con group and pr + lenti + con group). igf overexpression further increased the level of cytokine mrna expression, whereas the levels of cytokine expression in the pr -infected cells with igf knocked down (pr + lenti + shigf ) were significantly decreased far lower than the pr -infected control cell (pr + lenti + con group), as well as the uninfected mock group. especially the lowest ccl and tnf-α mrna of pr + lenti + shigf group were downregulated to less than half that of the mock group. these results indicate that the overexpression of igf aggravated the inflammatory response induced by influenza virus infection, whereas the inhibition of igf expression can alleviate this inflammatory response. to detect whether influenza virus can also regulate igf expression in vivo, iav pr (diluted -fold, μl/mouse, ld ) was intranasally inoculated into balb/c mice. mice were sacrificed on days , , , and post-infection to obtain mice lung tissue and serum, and the samples of six mice in every group were mixed. the level of igf mrna expression in the lung tissue of mice was detected by qpcr. as shown in figure a , the level of igf mrna expression in the lung tissue gradually increased at days , , and after pr infection. on day post-infection, igf mrna increased to . ± . times that of the control group and decreased slightly on day post-infection. a western blot was used to detect changes in igf protein expression in the lung tissue following pr infection ( figure b ). the grayscale analysis by comparing the integrated density of the igf band with the control gapdh showed that the trend in the level of igf protein expression was figure c , and plotted as the ratio of the integrated density of igf / gapdh; (f) grayscale analysis of figure d , and plotted as the ratio of the integrated density of igf /gapdh. data were the means ± sd from three independent experiments. **p < . consistent with that of the mrna data ( figure c) . the content of igf in the serum of mice was detected by enzyme linked immunosorbent assay (elisa). as shown in figure d , compared with the pbs control group, the content of igf protein in serum increased significantly on day post-infection of pr and decreased on day . these results were also consistent with the qpcr and western blot findings. to detect whether igf regulates acute inflammatory lung injury in pr infection, balb/c mice were tested as follow. balb/c mice were randomly divided into four groups: ( ) pbs; ( ) pr + igf ; ( ) pr + ppp; and ( ) pr + pbs. the pbs group was intranasally inoculated with μl pbs, whereas the other three groups were intranasally inoculated with μl influenza virus pr ( ld ). from h before the inoculation to day post-infection, the mice in the pr + igf group were intraperitoneally injected with igf protein ( μg/kg/ h); pr + ppp mice were injected intraperitoneally with an igf receptor inhibitor, picropodophyllin (ppp, mg/kg/ h); and the pr + pbs group was injected with pbs ( μl/ h). the clinical symptoms of the mice were observed daily for days, and changes in body weight and survival were recorded. six mice of each group were sacrificed at day post infection of pr , and the serum and lung tissues of each group were collected and mixed. the extent of lung injury and inflammatory cell infiltration of the mice in each group was observed by histologically. changes in the level of serum cytokines were detected by elisa. a western blot was used to detect the changes in the expression of major proteins in the igf /igf r-related signaling pathways. there was no change in the general appearance of the pbs control group mice. in the pr + pbs group, flu-like symptoms began to appear on day after pr inoculation, such as reduced activity, ruffled fur, and slight weight loss. on day postinfection, these conditions worsened with % weight loss, canthus secretion, hunched back. the symptoms of the pr + igf group were more severe than those of the pr + pbs group, whereas the pr + ppp group displayed milder clinical symptoms than that of the pr + pbs group (figure a) . figure b showed that the body weight of the mice decreased significantly on day post-infection of pr , and the body weight of the mice on day decreased to the lowest. although the changes in body weight were similar between the pr + ppp group and pr + pbs group, the survival rate of the pr + ppp group was dramatically increased up to % compared with the survival rate of the pr + pbs group was only %, with the first death not occurring until the day post-infection ( figure c ). the first death in the pr + igf group was accelerated to day post-infection, and all mice have succumbed to the infection by day . these findings indicated that the intraperitoneal injection of igf promoted the death of pr -infected mice, whereas the inhibition of igf increased the survival rate of pr -infected mice. figure a showed that in iav pr -infected mice, the lungs exhibited differential degrees of damage, and the color of the damaged parts changed from pink to dark red, with the presence of edema. the extent of lung injury in the pr + igf group was significantly more severe than that of the pr + pbs group, as the lung color was darker, and the lesion area was larger. the degree of lung injury in the pr + ppp group was significantly less severe than that of the pr + pbs group. figures b,c showed that the lung index of uninfected mice was approximately %. compared with the pr + pbs group, the lung index of the pr + igf group increased significantly from . ± . to . ± . %, and the area of lung injury also increased from . ± . to . ± . , approximately twice that of the control group. in the pr + ppp group, the lung index decreased to . ± . %, and the lung injury area also decreased to . ± . , which were both lower than that of the pr + pbs group. as shown in figure d , the lung structure of the mice in the pbs group was clear, with intact pulmonary alveoli, clear alveolar septum, and without hemorrhaging and inflammatory cell infiltration. the pulmonary alveoli of the pr + pbs, pr + igf , and pr + ppp groups exhibited varying degrees of damage. the level of inflammatory cell infiltration in the lung tissue of the pr + igf group was greater than that of the pr + pbs group, and there was lower inflammatory infiltration in the pr + ppp group than the pr + pbs group. whether igf regulates the viral replication of iav, qpcr was used to detect changes of the viral matrix protein (m ) expression in the lungs of pr -infected mice, which could indirectly reflect the viral load. the viral load in the lungs of the pr + igf group was significantly increased to nearly two-fold of the pr + pbs group ( figure e) . however, the viral load of the pr + ppp group and the pr + pbs group was similar, indicating that igf responded to influenza infection via the host immune response rather than targeting viral replication. the cytokine content in the serum of pr -infected mice was detected. figures a-d showed that there was a significant increase in the serum inflammatory following pr infection. figure | lung injury following pr infection in balb/c mice. groups of balb/c mice (n = , females, - weeks) were intranasally infected with pr ( ld ), treated with igf or ppp, and at day post-infection, the mice lungs were examined for changes in (a) morphology, (b) lung injury, (c) injury areas, (d) histopathology, and (e) viral load. data were the means ± sd from three independent experiments. **p < . vs. pr + pbs group by t test. moreover, the inflammatory cytokine levels in the intraperitoneal injected igf protein group were further increased, in which il- β was highly variable, and there was no statistical difference with the pr + pbs group. the levels of inflammatory factors ifn-γ, tnf-α, il- , and il- β in the serum of the pr + ppp group were significantly decreased, and il- β was lower than that of the uninfected mice. the results further confirm that igf induced an inflammatory response following influenza virus infection, which can be alleviated by inhibiting igf . to explore the mechanism by which igf regulates acute inflammatory lung injury induced by iav infection, a western blot was used to detect the expression of molecules in key signaling pathways in the lung tissue of pr -infected mice ( ld pr ). each sample was from six mice in a group and was tested for three times. figures were representative of three independent experiments. figure a showed that the phosphorylation level of igf r increased gradually following pr infection, peaked at day post-infection, and then decreased slightly on day , which was consistent with the changes in the trend of igf protein expression ( figure b ). figures b-d showed that the pi k/akt and mapk signaling pathways were activated following pr infection; p-akt expression was upregulated in the pi k/akt signaling pathway; and p-p and p-jnk expression were upregulated in the mapk signaling pathway. the levels of p-akt and p-p expression were further increased in the pr + igf group. the expression level in the pr + ppp group was between that of the pbs and pr + pbs groups. there was no significant difference in the level of p-jnk expression between the groups infected with influenza virus. the results indicated that the administration of igf protein following influenza virus infection promoted the activation of the pi k/akt and mapk signaling pathway. intervention with the igf r inhibitor ppp inhibited influenza virus mediated pi k/akt and mapk signaling pathways. these results suggest that igf affected the expression of key proteins associated with mapk and the pi k/akt signaling pathway in response to iav-mediated inflammation. in this study, we investigated that igf played a significant role in mediating inflammation and pathology during iav infection. igf expression was upregulated in a cells and balb/c mice infected with pr , which modulated inflammatory cytokine expression. igf overexpression aggravated influenzamediated inflammatory responses, whereas the inhibition of igf expression reduced such inflammatory responses. the phosphorylation of igf r triggered the pi k/akt and mapk signaling pathways to induce inflammation. thus, inhibiting igf or igf r expression to block downstream signaling may be a novel treatment strategy for influenza infection. following an influenza virus infection, a series of immunological responses are initiated. first, the host's innate immune system, consisting of interferon, cytokines, macrophages, neutrophils, nk cells, and dendritic cells (dcs), all rapidly respond to the viral infection and activate adaptive immunity (gu et al., ) . although the innate immune response is important, the host typically eliminates the viral infection with the aid of the adaptive response (bonilla and oettgen, ) . in humoral immunity, neutralizing antibodies can prevent the adsorption of the virus, via opsonization, and primarily interact with extracellular free viruses (murasko et al., ) . activated th cells release various cytokines (i.e., ifn-γ and tnf), activate macrophages and nk cells to induce further inflammatory reactions, and promote the proliferation and differentiation of cytotoxic t lymphocytes (ctls), which play an important role in antiviral infection (julkunen et al., ; farsakoglu et al., ) . however, studies have shown that while an inflammatory response helps to clear pathogens, excessive inflammatory reactions do not only protect the host organism but also cause the additional damage. many studies have shown that the cause of death from an influenza virus infection is the pulmonary respiratory syndrome induced by inflammation caused by an excessive reaction of the body's immune system (blach-olszewska and leszek, ) . in the present study, changes in the level of igf mrna and protein expression in a cells were detected. the results showed that igf mrna and protein expression were significantly altered following iav infection. to further confirm the important role of igf in iav-mediated inflammation and the associated signaling mechanism, igf protein was administered as an intervention to pr -infected mice. compared with the untreated infected mice, the infected mice administered igf displayed more severe clinical symptoms; in particular, the serum levels of ifn-γ, tnf-α, and il- β were significantly increased, and lung damage was more serious. igf is known to function by binding to the receptor, igf r. in this study, the igf r inhibitor ppp was used to inhibit igf r function, thereby disrupting its downstream signaling capacity. by monitoring the clinical symptoms and inflammation indicators in mice, it was found that during the same period of viral infection, when ppp was used to inhibit igf , the clinical symptoms of mice were alleviated; the lung injury area of the mice was reduced; the degree of lung injury was reduced; the lung index was significantly decreased; the serum levels of inflammatory cytokines ifn-γ, tnf-α, il- β, and il- were significantly decreased; and the survival rate increased from to % compared with the untreated group. the viral proliferation in the lungs of mice treated with igf was significantly increased; however, the viral proliferation of the mice treated with ppp was similar to that of the pr + pbs group. thus, the inhibition of igf only affects the immune response but has no significant effect on iav replication. thus, the inhibition of igf , rather than antiviral pathways that affect viral replication, may provide a new therapeutic avenue for influenza that limits detrimental inflammatory responses to infection. this is consistent with the previously reported effective treatments for influenza-mediated inflammation, which prolong the survival of older influenza-infected individuals, compared to those treated with antivirals (pillai et al., ) . the inflammation-associated pi k/akt and mapk signaling pathways are known to be activated in response to influenza virus infection (dong et al., ; hirata et al., ) . although igf primarily regulates cell growth and apoptosis through the pi k/akt and mapk signaling pathways, whether igf plays a role in these pathways in the context of influenza virus-mediated inflammation remains unknown. in this study, a western blot was used to detect the expression of key proteins in the pi k/akt and mapk signaling pathways. the results showed that p-igf r was upregulated following pr infection; p-akt expression was upregulated in the pi k/akt signaling pathway; and p-p and p-jnk expression were upregulated in the mapk signaling pathway compared with the pbs group. the expression of p-akt and p-p in the pr + igf group was higher than that of the pr + pbs group, and the expression in the pr + ppp group was between that of the pbs and pr + pbs group. there was no significant difference in the level of p-jnk expression between the groups infected with influenza virus. this suggested that igf affected the expression of key proteins associated with p in the mapk and the pi k/akt signaling pathways in the context of influenza virusmediated inflammation, which confirmed our hypothesis. therefore, we conclude that igf expression is upregulated following influenza virus infection, and igf receptor phosphorylation is elevated, triggering two signaling pathways downstream of pi k/akt and mapk to induce inflammation. thus, the inhibition of igf or igf r expression to block such downstream signaling pathways may represent a novel approach for the treatment of influenza virus infection. the data used to support the findings of this study are available from the corresponding author upon request. the animal study was reviewed and approved by academy of military medical sciences (amms; id: syxk - ). chz and gl contributed to conceptualization. chz, gl, and lz contributed to methodology. lz, caz, and ys contributed to investigation. rw and chz contributed to writing the original draft. rw and chz contributed to writing, reviewing, and editing. chz contributed to funding acquisition. lz, dd, and mb contributed to resources. rw, gl, and chz helped in supervision. glucocorticoid: major factor for reduced immunogenicity of influenza a (h n ) vaccine in patients with juvenile autoimmune rheumatic disease the nlrp inflammasome mediates in vivo innate immunity to influenza a virus through recognition of viral rna autophagy resolves early retinal inflammation in igf -deficient mice progress in identifying virulence 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differentiation, and survival: multiple physiological functions for insulin-like growth factors influenza: the mother of all pandemics insulin receptor kinase domain autophosphorylation regulates receptor enzymatic function toll-like receptors and viruses: induction of innate antiviral immune responses the authors thank prof. shihui sun (beijing institute of microbiology and epidemiology) for kindly providing the iav a/puerto rico/ / (h n ). key: cord- -ilir i authors: wu, zhiqiang; liu, bo; du, jiang; zhang, junpeng; lu, liang; zhu, guangjian; han, yelin; su, haoxiang; yang, li; zhang, shuyi; liu, qiyong; jin, qi title: discovery of diverse rodent and bat pestiviruses with distinct genomic and phylogenetic characteristics in several chinese provinces date: - - journal: front microbiol doi: . /fmicb. . sha: doc_id: cord_uid: ilir i bats and rodents are widely distributed worldwide and can be native or intermediate reservoirs of many important zoonotic viruses. pestiviruses are a group of virus species of the genus pestivirus under the family flaviviridae that can infect a wide variety of artiodactylous hosts, including swine and ruminants. two classic types of pestiviruses, bovine viral diarrhea virus and classical swine fever virus, are important causative agents of mild-to-severe disease in bovine and swine hosts, respectively, and cause tremendous economic losses in these industries. recent reports revealed that bats and rodents could also act as natural hosts of pestiviruses and an atypical porcine pestivirus, which cause disease in piglets, showed a close genetic relationship with a specific bat pestivirus, rapestv- . this study aimed to describe the detection and characterization of novel pestiviruses from bats and rodents in different locations by analyzing the available bat and rodent virome data from throughout china. two bat pestivirus species and four rodent pestivirus species that are distinct from other known viruses were identified and sequenced. these viruses were identified from two bat species and four rodent species in different chinese provinces. there were two distinct lineages present in these viruses, that differ from artiodactylous pestivirus. these findings expand our understanding of the genetic diversity of pestiviruses in bats and rodents and suggest the presence of a diverse set of pestiviruses in non-artiodactylous hosts. this study may provide new insight for the prevention of future viral disease outbreaks originating from bats and rodents. the genus pestivirus is contained within the family flaviviridae, which includes positive, single-stranded rna viruses with ∼ kb genomes and a single open reading frame that encodes a polyprotein flanked by -and -untranslated regions (utrs). the polyprotein is translated from genomic rna and translation is initiated by a cap-independent mechanism through a type iv internal ribosomal entry site within the -utr. the single polyprotein is then cleaved into four structural and eight non-structural proteins by cellular and viral proteases. pestiviruses were previously divided into four species, including bovine viral diarrhea viruses and (bvdv- and bvdv- ), classical swine fever virus (csfv), and border disease virus (bdv) of sheep or goats (becher et al., ; simmonds et al., ) . more recently, the original four species were re-classified as pestivirus a, pestivirus b, pestivirus c, and pestivirus d. since several new pestiviruses have been characterized in other hosts, seven new species of pestivirus were identified: pestivirus e (pronghorn antelope pestivirus), pestivirus f (porcine bungowannah virus), pestivirus g (giraffe pestivirus), pestivirus h (hobi-like bovine pestivirus), pestivirus i (sheep aydin-like pestivirus), pestivirus j (rat pestivirus), and pestivirus k (atypical porcine pestivirus, appv) (avalos-ramirez et al., ; kirkland et al., ; becher et al., ; liu et al., ; firth et al., ; hause et al., ; de groof et al., ; smith et al., ) . pestiviruses can infect a variety of artiodactylous hosts, including swine and ruminants (tautz et al., ) . diseases associated with pestiviruses include hemorrhagic symptoms, abortion, respiratory disease, and fatal mucosal disease. bvdv and csfv are severely pathogenic in cattle and swine, respectively, and are endemic in many countries (liu et al., ; chander et al., ) . recently, the divergent pestiviruses, bungowannah virus and appv, began to be considered a threat to pig health (kirkland et al., ; hause et al., ) . in previous work from our lab, we found partial rapestv- genome sequence in rhinolophus bats from hainan province in china. as such, we were the first to describe the identification of pestivirus in species outside the order artiodactyla ). an rapestv- related pestivirus, appv, was recently discovered in north america and europe, and was highly suspected to be the causative agent of a type a-ii congenital tremor (ct a-ii) in piglets (hause et al., ; de groof et al., ; postel et al., ; beer et al., ; lamp et al., ) . recently, the identification of a divergent pestivirus (norway rat pestivirus, nrpv) in new york city suggested that norwegian rats (rattus norvegicus) can also serve as natural hosts for pestivirus (firth et al., ) . these findings suggested a wider pestivirus host range than previously known and highlighted the need for an improved understanding of the biodiversity and evolution of pestivirus in bats and rodents. bats (order chiroptera) and rodents (order rodentina) are two of the most widely geographically distributed mammals and display the most extensive species diversity (wilson and reeder, ) . they are also considered to be major natural hosts of a large variety of viruses, including many important zoonotic viruses that can cause severe diseases in humans and domestic animals, including henipaviruses, severe acute respiratory syndrome coronavirus, hantaviruses, and arenaviruses (wang et al., ; meerburg et al., ; smith and wang, ; yang et al., ; han et al., ; gao et al., ; wu et al., a) . in the present study, we use virome analysis by using sequence-independent pcr amplification, next-generation sequencing, and sequence similarity comparisons to classify identified pestiviruses. novel sequencing reads related to pestivirus were found in samples collected from different bat and rodent species. genomic and phylogenetic analyses of these viruses revealed the presences of six novel pestivirus species in bat and rodent hosts. bats and rodents were treated in accordance with the guidelines outlined in the regulations for the administration of laboratory animals ( a total of , individuals bats across species and , rodents covering species were sampled by obtaining both pharyngeal and anal swabs between october and july throughout china (wu et al., (wu et al., , b . all bats and rodents collected in this study were considered to be apparently healthy and showed no overt signs of disease. pharyngeal and anal swab samples from captured bats and rodents were immersed in virus sampling tubes (yocon, china) containing maintenance medium and temporarily stored at − • c. the samples were then transported to the laboratory and stored at − • c. the samples from each species were pooled by adding ml from each maintenance media sample into a fresh sample tube. the pooled samples were classified by species, and then processed using a viral particle-protected, nucleic acid purification method as described in our previous studies (wu et al., , . the extracted rna and dna were amplified using sequence-independent pcr. amplified viral nucleic acid libraries were then sequenced using an illumina hiseq sequencer with single, - bp reads. the raw sequence reads were then filtered using previously described criteria to obtain valid sequences (wu et al., , li et al., a) . sequence similarity-based taxonomic assignments were conducted as described in our previous study (wu et al., ) . valid sequence reads were aligned to sequences obtained from the ncbi non-redundant nucleotide database (nt) and non-redundant protein database (nr) using blastn and blastx, respectively. the taxonomies of the aligned reads with the best blast scores (e score < − ) were parsed using the megan -metagenome analyzer (huson et al., ) . sequence reads classified into the same virus family or genus with megan were extracted. the accurate loci of each reads and the relative distances between reads of the same virus were determined based on the alignment results obtained with megan . we designed specific nested primers based on alignment results for rt-pcr to screen individual samples for the presence and prevalence of pestivirus. the located reads and contigs were used for reads-based pcr to identify partial genomes in positive individual samples (the primer sequences are available in supplementary table ) . based on partial genomic sequences of each virus, the remaining genomic sequences were determined with genome walking, and and rapid amplification of cdna ends (race) by using genome walking kit (takara), race kit (invitrogen), and full race core set, version . (takara). the polyproteins were deduced by comparing them with the sequences in other pestiviruses. the conserved protein families and domains were predicted using either pfam and interproscan or the conserved domain database of ncbi. routine sequence alignments were performed using clustal omega, needle , megalign (lasergene), and t-coffee with manual curation. mega . was used to align nucleotide sequences and deduced amino acid sequences using the muscle package with the default parameters (tamura et al., ) . the best substitution model (lg + g for polyprotein, lg + g for npro, jtt + g for erns, and lg + g + i for ns ) was then evaluated using the model selection package. finally, a maximum-likelihood method with an appropriate model was used to conduct phylogenetic analyses with , bootstrap replicates (tamura et al., ) . all genome sequences have been submitted to genbank. the accession numbers for the four bat pestiviruses are mh -mh . the accession numbers for the five rodent pestiviruses are ky -ky . the ga ii sequence data have been deposited into the ncbi sequence reads archive (sra) under accession numbers sra and prjna . a systematic survey of pestiviruses was performed using small mammal virome data obtained throughout china from october to may and indicated the presence of diverse pestiviruses in the mammals tested (wu et al., (wu et al., , b . a total of , sequence reads of - bp were classified into the genus pestivirus based on the nr alignment results generated with megan (wu et al., ) . these reads showed - % amino acid (aa) identity with known pestiviruses in the genbank database. two bat species identified from two provinces and four rodent species from four provinces were found to be pestivirus-positive (figure and table ). besides the previously reported rapestv- identified from samples of rhinolophus affinis bats in haikou city of hainan province, all other bat-derived reads related to pestivirus were only found in samples from scotophilus kuhlii bats in the cities of nanning and beihai in guangxi province. however, reads related to pestivirus were found in samples collected from multiple rodent species, including those in the subfamily murinae taken from four different locations, including niviventer excelsior in ganzi tibetan autonomous region of sichuan, apodemus peninsulae in the korean autonomous prefecture of yanbian of jilin, apodemus draco in ankang, shaanxi, and niviventer confucianus in wuhan, hubei as well as in hanzhong, shaanxi. the prevalence of specific pestiviruses were then re-evaluated by single-strain screening with specific reads-based nested-pcr, as shown in table . by this method, five bat anal swab samples, twelve rodent anal swab samples, and two rodent pharyngeal swab samples were confirmed to be pestivirus-positive. two rodent individuals were pestivirus-positive in both pharyngeal and anal swab samples. the genome sequences of four viral strains identified from pestivirus-positive s. kuhlii bats in sichuan were characterized (figure ) . the bpv strains were described as follows: the full-length genome of strain btsk-pv- /gx was sequenced and yielded an , nt genome that encoded a , aa polyprotein. strains btsk-pv- /gx , btsk-pv- /gx , and btsk-pv- /gx were partially sequenced, obtaining partial genome lengths of , , and nt, respectively, that shared - % nt identity between one another and with btsk-pv- /gx . the polyprotein of these four pestiviruses shared % aa identity with rapestv- , % aa identity with appv from pestivirus k, and < % aa identity with pestiviruses of rodents and other hosts. according to the general rules of taxonomy proposal accepted by ictv (smith et al., ) , the four identified bpvs could be defined as bpv species , and the previously partially sequenced strain, rapestv- , was defined as bpv species . the -utr, mature pestivirus peptides, and -utr of bpvs were predicted by sequence alignment with available pestivirus reference genomes and known cleavage sites, which have been determined previously in appv and other artiodactylous pestiviruses (figure ) . for bpv species , we used strain btsk-pv- /gx as a reference, for which the -utr was nt and the -utr was nt. each of these are consistent with the lengths of other pestivirus and -utr's. each peptide of bpv species and aligned best with those of appv, even when the overall identities figure | occurrence of pestivirus-related reads in bats and rodents from different locations. provinces with pestivirus-related reads are labeled in green and aqua and the animal species from each province is noted in red to the right of the map. between bpv species and appv were relatively low when compared with the identities between bpv species and appv. each mature peptide had very low aa sequence identity when compared with those of other pestiviruses. the non-structural autoprotease (npro) protein of bpv species was predicted to be self-cleaved from the polyprotein between cys and ser , and the conserved catalytic residues were identified as glu , his , and cys . a slightly larger core protein (c) was located between npro and the three envelope (e) glycoproteins (erns, e , and e ). an rnase t superfamily domain with uridylate specificity was identified in erns between aa and of the polyprotein. a peptidase s domain between aa and , and a helicase associated domain between aa and were identified in the ns protease. due to the lack of significant similarity to other pestiviruses, some domains that were conserved in previously reported artiodactylous pestiviruses were not found in the polypeptides of bpv species and . the genome sequences of five virus strains from representative positive samples were characterized (figure ) . the strains were described as follows: strain rtap-pv/jl , identified in a. peninsulae from jilin had a , nt genome, encoding a , aa polyprotein. rtnc-pv/hub , which was found in n. confucianus from hubei had a , nt genome, encoding a , aa polyprotein. rtad-pv/sax in a. draco from shaanxi had a partially sequenced genome, of , nt, which encoded a , aa polyprotein, denoted as a partial polyprotein. rtne-pv/sc in n. excelsior from sichuan had an , nt partially sequenced genome, encoding a , aa partial polyprotein, and lastly, we identified rtnc-pv/sax in n. confucianus from shaanxi with an , nt partially sequenced genome with a , aa partial polyprotein. we propose the name rodent pestivirus (rpv) for these five pestivirus strains. the five rpvs shared similar genome sizes with one other and were each larger than all known pvs from artiodactylous hosts (figure ) . the polyproteins encoded by three of the five rpvs (rtap-pv/jl , rtad-pv/sax , and rtne-pv/sc ) had , , and % aa identities to each other, respectively, while they had only % aa identity to that of nrpv, and < % aa identity to those of bpvs and other pestiviruses. the polyproteins of the remaining two rpvs (rtnc-pv/hub and rtnc-pv/sax ) showed % aa identity to each other, % aa identity to that of nrpv, and < % aa identity to bpvs and other pestiviruses. according to the ictv criteria, the five identified rpvs could be further classified into two four species, named as rpv species (rtnc-pv/hub and rtnc-pv/sax ), species (rtap-pv/jl ), species (rtad-pv/sax ), and species (rtne-pv/sc ), with only - % intergroup aa identities. the -utr, mature pestivirus peptides, and -utr of rpvs were predicted by aa alignment to pestivirus reference genomes and known cleavage sites, as determined in nrpv and other artiodactylous pestiviruses. the aa identity of mature peptides between rpvs and other pestiviruses were then calculated from pairwise alignments (figure ) . despite the extensive sequence diversity, each peptide of rpv species matched best with that of nrpv, the identities between rpv species and nrpv were higher than between rpv species - and nrpv. the p peptides were the most divergent regions of rpvs, as they showed only - % aa identity with p of nrpv, respectively, and lacked significant similarity to any other proteins in genbank. the npro proteins were - aa, which is longer than those found in other pestiviruses. the conserved npro catalytic residues were identified in both rpvs (rtnc-pv/hub : glu , his , and cys ; rtap-pv/jl : glu , his , and cys ; rtne-pv/sc : glu , his , and cys ), but the self-cleavage site for npro of rpv species - is between cys and asn (rtap-pv/jl and rtne-pv/sc ) or between cys and glu (rtad-pv/sax ), which differs from those of rpv species and other pestiviruses. the rnase t superfamily domains in erns and peptidase s domains and helicase associated domains in the ns protease were also identified in rpvs. phylogenetic trees based on the aa sequences of each strain's partial polyprotein, erns, and ns were used to describe the evolutionary relationships between pestiviruses. consistent with the phylogeny of their hosts in order level, all pestiviruses were divided into three main lineages; the artiodactylous lineage, bat-swine lineage, and rodent lineage (figure ) . most pestiviruses that are associated with animals of the families suidae, bovidae, cervidae, antilocapridae, and giraffidae under the order artiodactyla clustered together under the artiodactylous lineage. however, viruses under the artiodactylous lineage tend to be inconsistent with host phylogenies, and multiple incongruous relationships between the phylogenies of hosts and viruses were identified. for example, diverse bovine and swine pestiviruses were scattered in the genus pestivirus with much higher genetic diversity than pestiviruses of other host species. in accordance with pair-wise similarity analysis, bpvs identified from animals of the families vespertilionidae and rhinolophidae were classified into bpv species and in the bat lineage, respectively, while rpvs identified from animals of the subfamily murinae were classified into rpv species - in the rodent lineage. the only exception was appv. this viral species was identified in pigs but formed a distinct lineage located outside the artiodactylous lineage, but instead, was associated within the bat-swine lineage. bats and rodents are the most abundant and diverse mammals in the world. they often live in close contact with human populations and their domestic animals, and act as a bridge between humans, domestic animals, and other wildlife, thus exposing humans to some zoonoses in these natural ecosystems (meerburg et al., ; smith and wang, ; chen et al., chen et al., , han et al., ; olival et al., ) . many viral causative agents of human and domestic animal diseases, including severe respiratory syndrome coronavirus, henipaviruses, hantaviruses, and arenaviruses, are widely recognized as having originated or been transmitted from wild bat or rodent hosts (wang et al., ; guo et al., ; wu et al., ; li et al., b; blasdell et al., ) . recently, a novel causative agent of fatal disease in pigs known as swine acute diarrhea syndrome coronavirus, was confirmed as being an hku -related alphacoronavirus that originated in horseshoe bats (rhinolophus spp.) (zhou et al., ) . furthermore, an atypical porcine pestivirus, appv, which caused piglet ct a-ii in europe and north america showed close resemblance to a horseshoe bat pestivirus, rapestv- (hause et al., ; de groof et al., ; postel et al., ; beer et al., ) . the identification of diverse rodent arteriviruses in our recent report also revealed a much closer common ancestor of porcine reproductive and respiratory syndrome virus in the rodents pestiviruses (wu et al., b) . these new findings further emphasize the importance of bats and rodents as natural reservoirs of viruses that threaten the health of pigs and by extension, cause tremendous economic losses to the swine industry. though a large volume of wildlife virome data has been obtained in the past decade, a predictive analysis revealed that the viral richness in wildlife is somewhat limited, and there is still a large number of "missing potentially zoonotic viruses." this missing data merits further systematic global surveillance, especially in bats, rodents, and primates (olival et al., ) . this study described the identification of novel bpvs and rpvs in different bat and rodent species across several chinese regions, which revealed that these two mammals served as natural hosts for pestiviruses. though the virome analyses were conducted on highly diverse samples of bat and rodent species throughout the country, only two bat species in two provinces and four rodent species from four provinces were found to be figure | phylogenetic analysis. phylogenetic trees based on pestivirus (a) partial polyprotein, (b) erns, and (c) ns were constructed (left). all bpvs and rpvs are labeled in red font. the evolutionary lineages of involved artiodactylous hosts (right) were drawn from genus to family according to previous reports (jansa and weksler, ; meredith et al., ) , viruses of different hosts are labeled in different colors. pestivirus positive. this suggested that rpvs and bpvs are not as broadly distributed across hosts and geographical distance as other bat-or rodent-borne rna viruses such as coronaviruses, picornaviruses, and hantaviruses (guo et al., ; du et al., ; wu et al., ) . the hosts for bpvs can only be found in south china, while rpvs showed much broader host and geographical distributions then rpvs, with rodents in north, central, and west china serving as natural hosts of rpvs. the newly identified bpvs and rpvs potentially represented six new species under the genus pestivirus. considering their divergent phylogenetic positions, different genome sizes and structures, and the minimal sequence similarities of bpvs and rpvs when compared to other pestiviruses, we propose classifying members of the genus pestivirus into three main lineages; bat-swine, rodent, and artiodactylous lineages, based on the order level of their hosts. bpv species and , and appv represent the preliminary species under the bat-swine lineage, and rpv species - represent the preliminary species under the rodent lineage. furthermore, the close relationship between appv and bpvs indicates the presence of a much closer common ancestor of appv in bats than in other swine pestiviruses. these viruses may have evolved independently in pigs and bats, eventually forming at least three sub-lineages under bat-swine lineage. given that pestiviruses can cross species barriers to infect a wide range of artiodactylous animals (becher et al., ) , further work should be conducted to investigate the prevalence of bpvs and rpvs worldwide and to evaluate their virulence in animal experiments. evidence for the presence of two novel pestivirus species genetic and antigenic characterization of novel pestivirus genotypes: implications for classification phylogenetic analysis of pestiviruses from domestic and wild ruminants complete genome sequence of a novel pestivirus from sheep high prevalence 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rodents: relationships within and among major lineages as determined by irbp gene sequences identification of a novel virus in pigs-bungowannah virus: a possible new species of pestivirus novel pestivirus species in pigs unprecedented genomic diversity of rna viruses in arthropods reveals the ancestry of negative-sense rna viruses isolation and characterization of a novel arenavirus harbored by rodents and shrews in zhejiang province atypical pestivirus and severe respiratory disease in calves rodent-borne diseases and their risks for public health impacts of the cretaceous terrestrial revolution and kpg extinction on mammal diversification host and viral traits predict zoonotic spillover from mammals presence of atypical porcine pestivirus (appv) genomes in newborn piglets correlates with congenital tremor ictv virus taxonomy profile: flaviviridae proposed revision to the taxonomy of the genus pestivirus, family flaviviridae bats and their virome: an important source of emerging viruses capable of infecting humans mega : molecular evolutionary genetics analysis version . the molecular biology of pestiviruses review of bats and sars mammal species of the world: a taxonomic and geographic reference detection of hantaviruses and arenaviruzses in three-toed jerboas from the inner mongolia autonomous region comparative analysis of rodent and small mammal viromes to better understand the wildlife origin of emerging infectious diseases virome analysis for identification of novel mammalian viruses in bat species from chinese provinces deciphering the bat virome catalog to better understand the ecological diversity of bat viruses and the bat origin of emerging infectious diseases novel henipalike virus, mojiang paramyxovirus, in rats novel sarslike betacoronaviruses in bats, china fatal swine acute diarrhoea syndrome caused by an hku -related coronavirus of bat origin zw and qj conceived the experiments, analyzed the results, and wrote the manuscript. bl, jd, yh, hs, ly, sz, and ql conducted the experiments and analyzed the results. jz, ll, and gz collected the specimens. all the authors reviewed the manuscript. the supplementary material for this article can be found online at: https://www.frontiersin.org/articles/ . /fmicb. . /full#supplementary-material key: cord- - pjg kn authors: chen, shilong; wang, long; chen, jieying; zhang, lanlan; wang, song; goraya, mohsan u.; chi, xiaojuan; na, yang; shao, wenhan; yang, zhou; zeng, xiancheng; chen, shaoying; chen, ji-long title: avian interferon-inducible transmembrane protein family effectively restricts avian tembusu virus infection date: - - journal: front microbiol doi: . /fmicb. . sha: doc_id: cord_uid: pjg kn avian tembusu virus (atmuv) is a highly pathogenic flavivirus that causes significant economic losses to the chinese poultry industry. our previous experiments demonstrated that atmuv infection effectively triggered host innate immune response through mda and tlr -dependent signaling pathways. however, little information is available on the role of interferon-stimulated genes (isgs) in defending against atmuv infection. in this study, we found that atmuv infection induced robust expression of type i and type iii interferon (ifns) in duck tissues. furthermore, we observed that expression of interferon-inducible transmembrane proteins (ifitms) was significantly upregulated in def and df- cells after infection with atmuv. similar results were obtained from in vivo studies using atmuv-infected ducklings. importantly, we showed that knockdown of endogenous ifitm or ifitm by specific shrna markedly enhanced atmuv replication in df- cells. however, disruption of ifitm expression had no obvious effect on the atmuv replication. in addition, overexpression of chicken or duck ifitm and ifitm in df- cells impaired the replication of atmuv. taken together, these results reveal that induced expression of avian ifitm and ifitm in response to atmuv infection can effectively restrict the virus replication, and suggest that increasing ifitm proteins in host may be a useful strategy for control of atmuv infection. tembusu virus (tmuv) is a member of the ntaya virus group within the genus flavivirus of family flaviviridae (yan et al., ) . tmuv strains were firstly isolated from mosquitoes in malaysia and thailand, but their pathogenicity is not fully understood pandey et al., ) . sitiawan virus, a broiler-origin tmuv strain, was the first pathogenic virus causing encephalitis and retarded growth in broiler chicks (kono et al., ) . since , chinese domestic poultry including ducks, chickens, and geese have been manifesting a new epidemic disease caused by a tmuv-related flavivirus, named as avian tembusu virus (atmuv). this outbreak was quickly spread to many provinces of china and several south-eastern asian countries (homonnay et al., ; thontiravong et al., ) . atmuv genome consists of a single strand positive-sense rna and encodes three structural proteins [capsid (c), premembrane (prm/m), and envelope (e)] and seven non-structural proteins (ns , ns a, ns b, ns , ns a, ns b, and ns ) homonnay et al., ) . atmuv-infected adult animals showed symptoms of hemorrhagic ovaritis and acute egg drop syndrome, high fever, anorexia, diarrhea, ataxia, weight loss and paralysis, with a high morbidity ( - %), and mortality ranged from to % depending on different management and weather conditions, leading to enormous economic losses to poultry industry in china (cao et al., ; su et al., ; yan et al., ; liu et al., liu et al., , chen et al., ) . the young flocks are more vulnerable to atmuv infection, characterized by similar clinical symptoms including anorexia, diarrhea, high fever and severe neurologic dysfunction, with a higher mortality than adult birds (vaidya et al., ; ti et al., ) . atmuv was easily detected in ovaries and theca folliculi of infected animals, suggesting that the reproductive tissues were the major targets for the viral infection and replication . viral rna was also detected in spleen, trachea, kidney, brain, and blood of infected host (yan et al., ; liu et al., ) . in addition, it was observed that atmuv could infect various cell lines including def, cef, df- , bhk- , vero, a , t, and hela, resulting in a noticeable cytopathic effect (cpe) characterized by cell shrinkage, rounding and detachment (chen et al., a; . it has been reported that atmuv rna and neutralizing antibodies was detected in duck farm workers in shandong province of china (tang et al., ) , suggesting that this virus could infect human. but a disease associated with atmuv infection has not been found in human. moreover, it was shown that atmuv failed to cause any clinical manifestation or viremia in non-human primates, indicating that atmuv is unlikely to emerge as a human pathogen for the time being . however, due to zoonotic nature of its genus flavivirus relatives, atmuv might be a potential threat to human health in the future liu et al., ) . host innate immune response serves as the first line of defense against the infection of pathogens at the early stages. innate immune system recognizes viruses invasion via specific pattern recognition receptors (prrs) to sense pathogen associated molecular patterns (pamps) expressed by viruses. activated prrs then interact with adaptor proteins such as interferonβ promoter stimulator- (ips- ), myd , and trif (takeuchi and akira, ). the ligations of prrs with adaptor proteins result in the activation of the transcription factors, including nf-κb and interferon regulatory factors (irf and irf ) (takeuchi and akira, ) . irfs and nf-κb translocate to the nucleus where they stimulate the expression of type i and type iii interferons (ifns). then interaction between the ifns and their receptors causes activation of jak-stat signaling pathway. phosphorylated stat proteins translocate to the nucleus and combine with interferon regulatory proteins to promote an abundant expression of a wide array of genes, including ifnstimulated genes (isgs) (takeuchi and akira, ; tan et al., ; bailey et al., ) . these isgs encode distinct antiviral proteins with diverse biological effects that block multiple stages of the viral lifecycle including viral entry, translation, replication, assembly, and spread (diamond and farzan, ) . the interferon-inducible transmembrane proteins (ifitms) are a family of small transmembrane proteins belonging to isg superfamily and strongly induced by ifns (perreira et al., ) . the ifitm proteins are the distinct restriction factors known to block viral entry, including restriction of virus fusion with the late endosomal or lysosomal compartments and penetration into the cytoplasm (diamond and farzan, ; . it has been shown that gene cluster iftim , , and , the immunerelated genes, are critically involved in immune defense against a broad variety of viruses, including influenza virus, dengue virus, filoviruses, coronavirus, hepatitis c virus, lyssaviruses, and west nile virus (brass et al., ; huang et al., ; lu et al., ; smith et al., ; wilkins et al., ) . conversely, ifitms have little or no effect on several other viruses, including human papillomavirus, human cytomegalovirus and adenovirus type , arenavirus, murine leukemia virus, and foot-and-mouth disease virus (warren et al., ; bailey et al., ; zhang et al., ) . despite the progresses in understanding of ifitmmediated antiviral ability, host ifitm expression profiles in response to atmuv infection are still not clear. little is known about this immune-related isg family in restricting atmuv pathogenesis. in the present study, we examined the expression of key ifns and ifitms in host cells after atmuv infection in vitro and in vivo. interestingly, we observed that atmuv infection could trigger duck innate immune response including robust expression of particular type i and type iii ifns and ifitm family proteins. using df- cell system, we found that knockdown of endogenous ifitm and ifitm by short hairpin rna (shrna) markedly enhanced atmuv infection in host. however, silencing ifitm had no significant effect on atmuv replication. furthermore, overexpression of chicken or duck ifitm and ifitm could strongly inhibit the replication of atmuv. these results reveal that avian ifitm and ifitm but not ifitm serve as the critical components of host innate immune defense against atmuv infection. the antibodies used in this study are described as follows: mouse anti-β-actin (ab , abcam, cambridge, uk), mouse anti-flag (ht , transgen, beijing, china), hrp goat anti-mouse igg (lp a, abgent, usa). a monoclonal antibody against e protein of atmuv was prepared in our lab using the method described previously (chen et al., ) . fitc (fluorescein isothiocyanate) conjugated goat anti-mouse igg was purchased from boster (wuhan, china). chicken type i interferon was obtained from dalian sanyi animal medicine co. ltd. (dalian, china). lipofectamine was obtained from invitrogen (carlsbad, ca, usa). cell lines, birds, virus, and infection atmuv cjd strain was isolated from a chicken farm with acute egg-drop syndrome in fujian, china . duck embryo fibroblasts (defs) were prepared from -dayold mule-duck embryo as previously described (shahsavandi et al., ) . df- (immortalized chicken embryo fibroblast cell line) and t cells were obtained from american type culture collection (manassas, va). def, df- , and t cells were cultured at • c with % co in dmem (sigma, usa) supplemented with % fetal bovine serum (fbs, hyclone, utah, usa), units of penicillin g and µg of streptomycin. df- and def were infected with atmuv cjd as previously described (chen et al., a) at the multiplicity of infection (moi) of . and harvested at the indicated times after infection. twenty five -day-old mule healthy ducklings were challenged with . ml of cjd (the th passage allantoic fluid virus, eld = − . /ml) per duckling by intramuscular injection. each group of three randomly selected ducklings was sacrificed at , , , , and h post-infection (hpi), and their spleen, kidney, bursa of fabricius, pancreas, and brain were harvested for detection of viral infection by indirect immunofluorescence assay. these tissues were also used for total rna extraction to examine the mrna expression of ifns/ifitms by quantitative real-time rt-pcr (qrt-pcr). the sera and spleen homogenates ( %w/v) of the ducklings were prepared for detecting of viral titers by % tissue culture infectious dose (tcid ) assay during the infection. other infected ducklings and control ducklings (inoculated with . ml sterile pbs per duckling) were used to monitor clinical signs and rectal temperature daily for days. quantitative real-time rt-pcr and rt-pcr total rna was extracted from the cultured cells and duckling spleen tissues using trizol reagent (transgen biotech, beijing, china) according to the manufacturer's instructions. equal amount of rna ( µg) was reverse transcribed into cdna utilizing m-mlv reverse transcriptase (promega, usa). the cdna was analyzed by qrt-pcr using transstart green qpcr supermix (transgen) and pcr using rtaq dna polymerase (takara bio). the primers specific for chicken β-actin, ifna, ifn-β, ifn-λ, and atmuv e gene have been previously described (chen et al., a) . the primers specific for atmuv ns , duck β-actin, ifn-a, ifn-β, ifn-λ, ifitm , , , and chicken ifitm , , were designed using the primer software ( table ) . the relative mrna abundances were analyzed using the ′ ct method with housekeeping gene (β-actin) as an internal normalization and plotted as fold changes compared with the mock-infected samples. the specific short hairpin rnas (shrna) were designed for knockdown of chicken ifitm , , and . all the shrna sequences are shown in table . luciferase control shrna was described previously (wang et al., ) . df- cell lines stably expressing shrna targeting chicken ifitms (chifitms) the italic underlined nucleotides are restriction enzyme sites. were generated using lentiviral vectors as previously described . briefly, t cells were cotransfected with shrna construct and hiv-based packaging constructs (plp, plp , and plr ). supernatant of the cultured cells containing pseudotyped lentiviruses with indicated shrnas were collected at h post-transfection and filtered through the . µm syringe-driven filter. df- single-cell suspension was incubated with the supernatant and µg/ml of polybrene (sigma) and centrifuged at , rpm, • c for min. df- cells were then cultured in dmem supplemented with % fbs for further studies. qrt-pcr was performed to determine the interference efficiency and mrna expression of viral genes in df- cell lines at indicated times after infection. the viral titers of supernatants were examined by tcid assay in def cells. full-length cdna encoding chicken or duck species ifitm and ifitm was subcloned into pflag-cmv- a vector with a flag tag in the cooh terminus to create dna constructs chifitm , chifitm , duck ifitm (difitm ), and difitm , respectively. the specific primers with restriction enzyme sites are shown in table . df- cells were seeded onto -well plates and cultured in dmem with % fbs. when the cells reached - % confluence, they were transfected with µg of plasmid dna/well using lipofectamine (invitrogen). twenty four hours later, transfected df- cells were infected with atmuv cjd and harvested at indicated times ( , , and hpi) for further qrt-pcr or western blotting analysis. supernatants were collected for detection of viral titers by tcid assay. indirect immunofluorescence assay was performed as previously described (chen et al., b) . briefly, the parenchymal organs sections and def cells infected with atmuv were incubated with mouse anti-atmuv e protein monoclonal antibody for min at • c and then washed with pbs, followed by incubation with fitc conjugated goat anti-mouse igg at • c for min before imaging with fluorescence microscope (nikon, japan). df- cells were lysed with lysis buffer containing × complete protease inhibitor cocktail for min on ice according to our previous method (wang et al., ) . cell lysates were separated on % sds-page gels, transferred to pvdf membranes and blocked with % (w/v) milkpowder in tris-buffered saline (ph . , tbs) for h at room temperature. the membranes were incubated with indicated primary antibodies for . h at room temperature and washed with tbs, followed by incubation with appropriate secondary antibodies at room temperature before imaging with the proteinsimple fluorchem m system (bio-techne, usa). data represented the mean ± sd. statistical significance was determined by one tail student's t-test analysis. differences were considered statistically significant with p < . . the animal protocol used in this study was approved by the research ethics committee of college of animal science, fujian agriculture and forestry university (permit number pzcasfafu ). the procedures were carried out in accordance with the approved guidelines. the inoculated ducklings began to display clinical symptoms on day post-infection (dpi) characterized by anorexia and weight loss. two infected ducklings got slight legs paralysis and were reluctant to move on - dpi. infected ducklings developed a fever, showing that rectal temperatures increased from day to day post-inoculation and then gradually returned to normal from to dpi (figure d ). at necropsy, no lesions were observed except swollen spleens on - dpi. no duckling was died throughout the monitoring period. later on ifa test was carried out to detect viral antigens. we observed that viral antigens were detectable in the spleen, kidney, and bursa of fabricius tissues, with a higher viral burden in the spleens (figures a,b) . surprisingly, viremia ( . tcid / . ml) was observed in serum samples from to dpi ( figure c) . no virus was detected in other tissues including brain, pancreas, and liver. all the ducklings in control group were healthy and no viral antigen was detected. these findings indicate that atmuv exhibits mild pathogenicity in young ducklings following intramuscular injection. our previous studies had shown that atmuv infection effectively triggers the host innate immune response, including robust upregulation of type i and type iii ifns and some critical isgs in chicken and cef (chen et al., a) . however, innate immune response against atmuv infection in duck still remains to be determined. importantly, the role of ifitms in defending against atmuv infection has not been well-defined in any avian species. here, we examined the expression profile of ifns and ifitms in parenchymal organs of ducks infected with atmuv using qrt-pcr and rt-pcr analysis. the results showed that atmuv has higher replication in duckling spleen tissues, as indicated by obvious expression of viral e gene ( figure a ; supplementary figure ). remarkably, the mrna levels of type i and type iii ifns were gradually elevated and reached their maximum value on hpi and then declined gradually. in particular, ifn-α and ifn-β were greatly upregulated by -and , -fold at hpi, respectively (figures a,b) . the expression of ifn-λ was modestly upregulated by . -fold at hpi ( figure c) . the expression of duck ifitm , , and were gradually induced and reached their maximum value at hpi. strikingly, expression of duck ifitm was markedly increased by over -folds at hpi (figures d-f) . these observations were further confirmed by rt-pcr analysis (supplementary figure ) . moreover, we examined the mrna expression profile of ifns and ifitms in kidney and bursa of fabricius tissues. we found that in kidneys, the mrna levels of ifns and ifitms were modestly upregulated until hpi and then declined (supplementary figure ) . ifns and ifitms expressions in bursa frontiers in microbiology | www.frontiersin.org figure | expression of ifns and ifitm family is strongly upregulated in duck in response to atmuv infection. twenty five -day-old healthy mule ducklings were challenged by intramuscular injection with . × eld of atmuv in a volume of . ml per duckling. three randomly selected ducklings were sacrificed at , , , , and hpi, respectively. their spleen tissues were collected for examination of ifn-α (a), ifn-β (b), ifn-λ (c), and ifitm , , (d-f) mrna expression using qrt-pcr. the mrna levels were normalized to the endogenous β-actin level and the expression at hpi was set to . . expression at - hpi was compared to its expression at hpi. each sample was analyzed in triplicate and the results are depicted as means ± sd (n = ). statistical significance was determined by one tail student's t-test analysis. *p < . , **p < . . of fabricius were slightly increased with the highest levels at hpi and then declined (supplementary figure ) . taken together, these data indicate that atmuv infection can trigger innate immune response in ducklings, including upregulation of ifns and ifitms. ifitms are significantly induced in def and df- cells after atmuv infection or treatment with ifn to confirm duck innate immune response induced by atmuv, we further determined the expression of type i and type iii ifns and ifitms following atmuv infection in vitro. for this, defs were prepared and infected with atmuv, and the mrna expression of particular ifns and ifitms were then analyzed using qrt-pcr. atmuv replicated well in defs with an obvious cytopathic effect (cpe) characterized by cell shrinking, rounding and detachment at hpi ( figure a) . analysis of ifa showed that viral antigens were detected in def cells at - hpi, indicating that def cells could be infected by the virus (figure b) . the mrna levels of ifn-α, ifn-β, and ifn-λ were significantly elevated in atmuv infected defs during atmuv infection as compared to mock-treated control. expression of ifn-α and ifn-β was increased by about -and -fold at hpi, respectively (figures c-e) . ifn-λ expression was also significantly induced with a highest . -fold at hpi. qrt-pcr analysis was performed to examine the mrna expression of duck type i and type iii ifns (c-e) and ifitm , , (f-h). the mrna levels were normalized to the endogenous β-actin level and the expression at hpi was set to . . expression at - hpi was compared to its expression at hpi. plotted are the average levels from three independent experiments with three replicates per experiment (means ± sd). statistical significance was determined by one tail student's t-test analysis. *p < . , **p < . . frontiers in microbiology | www.frontiersin.org these data were consistent with the in vivo studies presented above. strikingly, duck ifitm was greatly induced and reached their maximum value ( -fold) at hpi and then declined gradually ( figure f) . expression of duck ifitm and ifitm were enhanced modestly and reached their maximal levels by . and . -fold at hpi, respectively (figures g,h) . these results were further confirmed by rt-pcr analysis (supplementary figure ) . taken together, these data reveal that innate immune response is triggered in defs infected with atmuv, as indicated by significant upregulation of ifns and ifitms. because def cells are hard to survive after several passages, we employed chicken df- cell line as a model system to further investigate the interaction between host innate immune system and atmuv, and the role of ifitms in defense against viral infection. as expected, mrna levels of ifn-α, ifn-β, and ifnλ were greatly elevated by atmuv infection and reached the maximal levels at hpi and then declined gradually as compared to mock treatment (figures a-c) . similarly, expression of chicken ifitm (chifitm ) was highly upregulated by fold at hpi. expression of chicken ifitm (chifitm ) and ifitm (chifitm ) was also significantly increased in response to atmuv infection (figures d-f) . these observations were further confirmed by rt-pcr analysis (supplementary figure ) . since atmuv infection induced significant upregulation of ifns and ifitms in vivo and in vitro, we asked whether increased ifitm expression was stimulated by atmuv-induced ifns in df- cells. to this end, df- cells were treated with chicken ifns ( iu/ml) and mrna levels of ifitms were examined by qrt-pcr. indeed, we found that expression of ifitm , , was significantly upregulated in df- cells after treatment with chicken ifns (figure g ). results presented above revealed that atmuv infection effectively induced the expression of particular type i, type iii ifns, and ifitms in vivo and in vitro. furthermore, we tested that whether these ifitms functioned in defense against the virus infection. for this, we generated stable df- cell lines expressing specific shrnas targeting ifitm , ifitm , ifitm , or luciferase control, respectively. these cells were then infected with atmuv and harvested at indicated times ( , , and hpi) . interference efficiency of the shrnas was examined by qrt-pcr and viral load in cell culture supernatants was titrated via tcid assay. compared with the control shrna targeting luciferase, the specific shrnas caused decreased expression of ifitm , ifitm , or ifitm after atmuv infection, respectively (figures a-c) . interestingly, silencing endogenous ifitm or ifitm resulted in significant increase in viral load, as evidenced by higher viral titers in culture supernatants from the ifitm or ifitm knockdown cells than those in supernatants of luciferase control cells ( figure d) . however, knockdown of ifitm only slightly enhanced atmuv replication ( figure d ). to confirm these findings, df- cell lines expressing specific shrna targeting each of these ifitms were infected with atmuv and harvested at hpi, followed by qrt-pcr assay to detect mrna expression of viral genes. consistent with the results from tcid assay, data of qrt-pcr showed that mrna expression of viral envelope and ns genes was clearly increased in ifitm or ifitm knockdown cells as compared to the control cells. however, no significant difference was observed in viral load between ifitm knockdown and luciferase control cells after infection with atmuv ( figure e ). this data suggests that disrupting the endogenous expression of chicken ifitm and ifitm but not ifitm can strongly enhance the atmuv replication in df- cells. since silencing ifitm and ifitm could increase the susceptibility of df- cell to atmuv infection, we further investigated whether forced expression of ifitms could inhibit atmuv replication. thus, we cloned chicken and duck ifitm and ifitm , and their cdna sequences were analyzed and deposited in the genbank database. df- cells were then transiently transfected with constructs expressing chicken or duck ifitm and ifitm or empty vector, respectively and challenged with atmuv. virus titers in cell culture supernatants were determined by tcid assay at , , and hpi. the forced expression of ifitms in df- cells was examined by western blotting. as shown in figure a , flag-tagged ifitm proteins were expressed well in df- cells (figure a) . we observed that overexpression of chicken or duck ifitm and ifitm obviously suppressed atmuv replication in df- cells, as indicated by lower tcid titers in culture supernatants from the ifitm-overexpressing cells than those in control cells ( figure b) . to further confirm this observation, the df- cells were infected with atmuv and harvested at hpi, followed by qrt-pcr to examine the viral gene expression. as compared with control cells containing empty vector, overexpression of chicken or duck, ifitm or ifitm in df- cells reduced mrna expression of atmuv envelope gene by average of , , , %, respectively ( figure c) . these data suggest that avian ifitm or ifitm could strongly interfere with atmuv infection. our findings revealed that avian ifitm and ifitm were involved in cellular restriction of atmuv infection. next, we analyzed chicken (df- cell origin) and duck (duck embryo fibroblasts origin) ifitm and ifitm genes repertoire. the cdna sequences had been submitted in the genbank database (accession numbers: chifitm , kx ; chifitm , kx ; difitm , kx ; difitm , kx ). blastx search showed that the chifitm (kx ) shared , . , . , . , . , and . % amino acid identity to ifitm protein of chicken (kc . ), coturnix (xm_ . ), duck (kx ), human (ak . ), mouse (xm_ . ), and sus scrofa (xm_ . ), figure | ifitms are significantly induced in df- cells after atmuv infection or treatment with ifn. df- cells were infected with or without atmuv at a moi of . and harvested at , , , , and hpi, respectively. qrt-pcr was performed to determine the relative mrna expression of indicated ifns (a-c) and ifitm (d-f) genes compared with that at hpi. (g) df- cells were treated with chicken ifns ( iu/ml) for , , or h. qrt-pcr was performed to determine the relative mrna expression of ifitm genes compared with that without ifn treatment. the mrna levels were normalized to the endogenous β-actin level. plotted are the average levels from three independent experiments with three replicates per experiment (means ± sd). statistical significance was determined by one tail student's t-test analysis. *p < . , **p < . . respectively. chifitm (kx ) shared . , . , . , . , . , and . % amino acid identity to ifitm protein of chicken (xm . ), coturnix (xm_ . ), duck (kx ), human (bc . ), mouse (nm- . ), and sus scrofa (nm_ . ), respectively. two putative transmembrane domains were obtained by using transmembrane prediction programs tmhmm (http://www.cbs.dtu.dk/services/tmhmm/) and tmpred plotted are the average results from three independent experiments (means ± sd). statistical significance was determined by one tail student's t-test analysis. *p < . , **p < . . (http://www.ch.embnet.org/software/tmpred_form.html). chicken and duck ifitm and ifitm proteins could be divided into five domains reflecting their hydrophobicity and conservation as identified in other species (figure a) , including a variable, hydrophobic n-terminal domain (ntd), a conserved hydrophobic intramembrane domain (imd), a conserved intracellular loop (cil), a variable, hydrophobic transmembrane domain (tmd), and a short, highly variable c-terminal domain (ctd). although significant divergence is seen between avian and mammalian ifitm and ifitm sequences, several residues are conserved and important for antiviral function, including important amino acids (y , c , c , k , k , and k ) in ifitm (smith et al., ; blyth et al., ) . phylogenetic analysis exhibited that avian ifitm and ifitm are distinct from those of mammalian species, but avian ifitm is more conserved than avian ifitm ( figure b ). atmuv is a newly emerging member of the ntaya virus group within the genus of flavivirus, causing severe egg-drop syndrome and neurological disease in domestic poultry homonnay et al., ; thontiravong et al., ) . on the basis of typical clinical symptoms, atmuv was initially described as duck egg drop syndrome virus (dedsv). however, this virus exhibits a wide range of pathogenicity to avian species including ducks, chicken, geese, house sparrows, and racing pigeon, and its genome is closely related to tembusu virus strains. flavivirus isolates with a more than % nucleotide sequence homology in the ns region are considered to be the same species (kuno et al., ) . so we propose to name this novel flavivirus as "avian temubsu virus, atmuv." till now, there is no effective method for its prevention except vaccine. the previous studies have shown that atmuv infection induced an effective antiviral immunity through mda and tlr -dependent signaling pathways chen et al., a; fu et al., ) . however, our understanding of the molecular and cellular basis of interaction between the virus and host antiviral immune system is very limited. in this study, we examined the expression profile of key ifns and ifitms following atmuv infection in vivo and in vitro. we found type i, type iii ifns, and immune-related ifitms, including the viral titers in cell culture supernatants were determined by tcid assay in def cells. (c) the mrna expression of viral genes was examined by qrt-pcr at hpi. the mrna levels were normalized to the endogenous β-actin level and the expression in atmuv infected df- cells expressing empty vector was set to . . the mrna expression of viral genes in df- cells expressing exogenous ifitms was compared to that in control cells containing empty vector. plotted are the average results from three independent experiments (means ± sd). statistical significance was determined by one tail student's t-test analysis. *p < . , **p < . . ifitm , ifitm , and ifitm were significantly upregulated in response to atmuv infection. strikingly, the spleen tissue had a high viral load and showed strong innate immune response as compared to other organs. ifn-α/β expression levels in spleen were increased significantly at hpi and decreased after this time point, and the mrna levels of ifitm and ifitm were also upregulated. similar results were shown in kidney and bursa of fabricius tissues in infected animals. although ifn expression in defs was triggered and reached the maximum value at hpi, defs displayed an apparent cpe by atmuv infection at this time point. these data suggest that host innate immune response is triggered by atmuv, but the virus has developed multiple strategies to evade host antiviral immunity. in a recent study, wang and his co-workers found that atmuv ns could markedly suppress virus-induced ifn-β expression by inhibiting rlr receptor signaling . most flaviviruses are potent in blocking the jak-stat pathway to evade the antiviral effects of the host innate immune system (heim et al., ; basu et al., ; lin et al., ; green et al., ) . thus, further studies are needed to address the precise mechanisms by which atmuv circumvents the host innate immune response. type i and type iii ifns bind to their receptors, which stimulates the jak-stat pathway that triggers various isg expression. expression of ifitms is strongly induced by ifns (smith et al., ) . consistent with previous studies (smith et al., ) , our data showed that stimulation of df- cells with chicken ifns significantly enhanced the expression of ifitms. interestingly, we observed that ifitm in atmuv infected defs had a faster kinetic response than atmuv-induced ifns. thus, we assessed whether ifitms expression is totally ifn-dependent. for this, sirna specifically targeting chicken interferon alpha/beta receptor (chifnar -sirna) and negative control sirna (nc-sirna) were employed in this study. nm chifnar -sirna or nc-sirna was transfected into df- cell. twenty-four hours post-transfection, the cells were infected with atmuv and harvested at hpi. interestingly, disruption of endogenous ifnar by sirna greatly reduced ifitm mrna expression, but only caused modest downregulation of ifitm and ifitm (supplementary figure ) . the data suggest that chifitm may not share the same ifn-dependent pathways with chifitm and chifitm . previous study has found that murine ifitm expression was not only induced by increased expression of type i or ii ifns, but also upregulated by other cytokines such as il- (bailey et al., ) . other groups also found that expression of ifi- k, ifi- k, and isg was induced directly by virus and then triggered secondarily through virus-induced ifns (wathelet et al., ; holzinger et al., ) . these findings indicate that multiple signaling pathways might be involved in regulation of ifitm expression during the viral infection. ifitms serve as critical effector molecules in host innate immune system and effectively restrict a wide range of pathogenic viruses, such as dengue virus, influenza a virus, hepatitis c virus, west nile virus, and hiv- (brass et al., ; wilkins et al., ; yu et al., ) . in order to survive, some viruses such as arenavirus and foot-and-mouth disease virus have evolved multiple strategies to evade the antiviral effects of ifitms (bailey et al., ; zhang et al., ) . furthermore, human cytomegalovirus could exploit ifitms to facilitate morphogenesis of virion assembly compartment (xie et al., ) . to investigate the role of ifitm proteins in restricting atmuv replication, we generated df- cell lines stably disrupting chicken ifitm , ifitm , or ifitm expression. indeed, disruption of endogenous ifitm and ifitm by shrna greatly enhanced atmuv infection. furthermore, ectopic expression of chicken and duck ifitm or ifitm markedly inhibited atmuv replication. interestingly, chifitm was significantly upregulated after atmuv infection in vitro, but we found that altering ifitm expression had mild effect on atmuv infection. a previous report showed that duck ifitm was greatly upregulated after highly pathogenic iav infection but its antiviral activity was low in vitro (blyth et al., ) . the differences in viral entry mechanisms of iav and atmuv in the different types of host cells may account for the differential antiviral activity of ifitm proteins. five ifitm proteins have been identified in duck and chicken species including ifitm , ifitm , ifitm , ifitm , and ifitm . ifitm , ifitm , and ifitm belong to the immunerelated clade, whereas non-immune ifitm and ifitm make up the two remaining clades (blyth et al., ) . in this study, we observed that ifitm increase fold was much higher than those of ifitm and ifitm by in vivo and in vitro assays. this may be due to high basal expression of ifitm and ifitm in the cells tested. in addition, we cloned and characterized chicken and duck species of ifitm and ifitm genes. despite sharing low amino acid identity between chicken ifitm and duck ifitm ( . %) and high amino acid identity between chicken ifitm and duck ifitm ( . %), both chicken and duck origin ifitm and ifitm could effectively restrict atmuv replication. certain key amino acids in imd and cil domain are conserved in chicken and duck ifitms, suggesting their importance for functioning of the ifitms (smith et al., ) . our previous work demonstrated that pretreatment of cells with ifns could significantly impair atmuv replication (chen et al., a) . our present study further provides the evidence that host ifitm proteins have the ability to control the atmuv infection and likely restrict the viral reproduction as well. taken together, atmuv infection induces host's effective antiviral immune response involving several critical ifns and ifitms proteins, which can be useful for developing new antiviral drugs in future. however, further studies are required for better understanding of precise mechanisms underlying the antiviral activity of ifitm proteins. slc performed most of the experiments, collected and analyzed data and wrote the manuscript. lw, lz, ws, yn, and zy participated in preliminary data acquisition. jyc contributed to plasmids construction. xz helped with data analysis. sw and xc contributed to shrna design and revised the manuscript. jlc and mg revised the manuscript. syc and jlc conceived of the study, participated in study design and coordination. all authors 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caused by byd virus, a new tembusu-related flavivirus innate immunity to virus infection pattern recognition receptors and inflammation modeling and dynamical analysis of virus-triggered innate immune signaling pathways tembusu virus in human tembusu-related flavivirus in ducks effect of age and inoculation route on the infection of duck tembusu virus in goslings transmission dynamics of the recently-identified byd virus causing duck egg-drop syndrome the emerging duck flavivirus is not pathogenic for primates and is highly sensitive to mammal interferon signaling duck tembusu virus nonstructural protein antagonizes ifn-β signaling pathways by targeting visa influenza a virus-induced degradation of eukaryotic translation initiation factor b contributes to viral replication by suppressing ifitm protein expression transport of influenza virus neuraminidase (na) to host cell surface is regulated by arhgap and cdc proteins the antiviral restriction factors ifitm , and do not inhibit infection of human papillomavirus regulation of two interferon-inducible human genes by interferon, poly(ri).poly(rc) and viruses ifitm is a tight junction protein that inhibits hepatitis c virus entry human cytomegalovirus exploits interferon-induced transmembrane proteins to facilitate morphogenesis of the virion assembly compartment an infectious disease of ducks caused by a newly emerged tembusu virus strain in mainland china ifitm proteins restrict hiv- infection by antagonizing the envelope glycoprotein induction of systemic ifitm expression does not effectively control foot-andmouth disease viral infection in transgenic pigs key: cord- -fmb nyxu authors: liu, junli; wang, fangfang; du, liuyang; li, juan; yu, tianqi; jin, yulan; yan, yan; zhou, jiyong; gu, jinyan title: comprehensive genomic characterization analysis of lncrnas in cells with porcine delta coronavirus infection date: - - journal: front microbiol doi: . /fmicb. . sha: doc_id: cord_uid: fmb nyxu porcine delta coronavirus (pdcov) is a novel emerging enterocytetropic virus causing diarrhea, vomiting, dehydration, and mortality in suckling piglets. long non-coding rnas (lncrnas) are known to be important regulators during virus infection. here, we describe a comprehensive transcriptome profile of lncrna in pdcov-infected swine testicular (st) cells. in total, , annotated and , novel lncrna candidate sequences were identified. gene ontology (go) and kyoto encyclopedia of genes and genomes (kegg) analysis revealed that these lncrnas might be involved in numerous biological processes. clustering analysis of differentially expressed lncrnas showed that annotated and novel lncrnas were regulated after pdcov infection. furthermore, we constructed a lncrna-protein-coding gene co-expression interaction network. the kegg analysis of the co-expressed genes showed that these differentially expressed lncrnas were enriched in pathways related to metabolism and tnf signaling. our study provided comprehensive information about lncrnas that would be a useful resource for studying the pathogenesis of and designing antiviral therapy for pdcov infection. long non-coding rnas (lncrnas), which are transcripts larger than nt in length that lack protein-coding ability, have previously been described in mammalian cells (kapranov et al., ; mattick and rinn, ) . most of them have a structure similar to mrna; they have a methylguanosine cap and are usually spliced and polyadenylated at their termini. notably, lncrna expression shows significant cell and tissue specificity (mercer et al., ; derrien et al., ) . emerging evidence shows that non-coding rnas have a regulatory role in multiple cellular processes, such as genomic imprinting, chromatin modification, and alternative splicing of rna (mercer et al., ) . moreover, some diseases such as cancer and neurological disorders are also related to the dysregulated expression of lncrna (qureshi et al., ; tsai et al., ) . numerous studies have been conducted to ascertain their functional role during viral infection. for example, nrav can promote influenza virus replication and virulence through negatively regulating the initial transcription of varieties of interferonstimulated genes (isgs) (ouyang et al., ) . lncrna-acod , named by its neighboring coding gene aconitate decarboxylase , significantly reduces virus multiplication by directly interacting with the metabolic enzyme glutamic-oxaloacetic transaminase (wang et al., ) . neat , one of the lncrnas induced by hiv- infection, is retained in the nucleus and serves as a scaffold for the nuclear paraspeckle substructure. importantly, neat deficiency enhances hiv- replication (zhang et al., ) . although large amounts of data have proved that several lncrnas are involved in different kinds of virus infection, the mechanisms by which they act are still largely unknown. porcine delta coronavirus (pdcov), a novel emerging pathogenic enterocytetropic virus, was first discovered from the feces of pigs in hong kong in . it is an enveloped, single-stranded positive-sense rna virus. it belongs to the genus deltacoronavirus in the family coronaviridae of the order nidovirales (woo et al., ) . the genome length of pdcov is approximately . kb. it is similar in structure to other coronaviruses, with shorter non-coding regions ( -utr and -utr) at both terminals. the / genome from the end contains two overlapping open reading frames, orf a and orf b, encoding the pp a and pp b, respectively. the downstream of the genome encodes structural protein spike (s), envelope (e), membrane (m), accessory proteins ns , structural protein nucleocapsid (n), and accessory proteins ns and ns a. a total of non-structural proteins are encoded by the terminal of the genome (fang et al., ) . pdcov mainly causes acute, watery diarrhea, vomiting, dehydration, and mortality in suckling piglets, including lesions in the stomach and lungs (ma et al., ) . the first outbreak of pdcov infection was reported in the united states in early and, to date, it has been detected in canada, south korea, china, thailand, and vietnam, thus posing huge threat to the swine industry and attracting a great deal of attention (lee and lee, ; dong et al., ; lorsirigool et al., ; ajayi et al., ; saeng-chuto et al., ) . during infection, the accessory and non-structural proteins of pdcov usually perform multiple functions to promote replication in infected cells. a previous study showed that ns interaction with rig-i/mda attenuated the binding activity between rig-i/mda and double-stranded rna, resulting in a reduced level of ifn-β production (fang et al., ) . also, the non-structural protein nsp , a c-like protease, is an important molecule in suppressing type i ifn signaling (zhu et al., b) . in addition, nemo, an essential modulator of nf-κb, can also be cleaved by nsp , causing inhibition of ifn-β production (zhu et al., a) . though there are many reports of immune evasion by pdcov, the precise pathogenic mechanism of pdcov is largely unclear. based on the increasing number of reports on host lncrnas associated with virus infection, we are interested in investigating whether host lncrnas were involved in pdcov infection. in this study, genome-wide profiling of lncrnas in swine testicular (st) cells infected with pdcov was performed using rna-seq. we identified differentially expressed lncrnas from pdcovinfected cells. an integrative analysis of lncrna alterations suggested their putative role in regulating the expression of several key genes in metabolic and tnf signaling pathways during infection. in conclusion, this work supports the role of lncrnas as important regulators of pdcov infection. swine testicular cells and porcine jejunum intestinal epithelial cells (ipec-j ) were grown in dmem supplemented with % (vol/vol) fbs (gibco, carlsbad, ca, united states) at • c in a humidified % co atmosphere. the pdcov-ch-ha - (mk ) strain, stored in our laboratory, was propagated in st cells. for rna-seq, st cells were infected with pdcov at a multiplicity of infection (moi) of ; the medium for pdcov infection was dmem containing . ug/ml trypsin that had been tpcktreated (millipore sigma, st. louis, mo, united states) for h. mock-infected cells were placed in the same volume of dmem, with the same concentration of tpck-treated trypsin. total rna was isolated from each group using superfectri tm total rna isolation reagent (pufei, shanghai, china) according to the manufacturer's instructions. the rna quality was checked by % agarose gel electrophoresis. the purity and concentration of rna were measured by nanophotometer r spectrophotometer (implen, münchen, germany) and qubit r rna assay kit in qubit r . fluorometer (life technologies, camarillo, ca, united states). rna integrity was assessed using the rna nano assay kit of the bioanalyzer system (agilent technologies, santa clara, ca, united states). for quantitative rt-pcr (rt-qpcr), st and ipec-j cells were infected or mock-infected with pdcov at an moi of and harvested at the indicated time. all experiments were conducted in triplicate. sequencing libraries were generated using the rrna-depleted rna with a nebnext r ultra tm directional rna library prep kit (new england biolabs, ipswich, ma, united states). after determining the quality of the library, rna-seq was performed using an illumina hiseq tm (illumina, san diego, ca, united states) to generate raw reads. after removing poly-n sequences, adapters, and low-quality reads, clean reads were obtained and the paired-end reads were aligned to ensemble pig genome (release ). lncrnas were identified using tophat (v . . ), and reads that were mapped to the pig genome were assembled using cufflinks v . . (trapnell et al., ) . cuffdiff (v . . ) was used to calculate the fpkms of both lncrnas and coding genes in each sample. gene fpkms were computed by summing the fpkms of transcripts in each gene group, and differentially expressed (de) transcripts were assigned where there was a statistically significant level of expression (p < . ). rna-seq and data analysis were completed by novogene. gene ontology (go) enrichment analysis of differentially expressed genes or lncrna target genes was conducted with respect to biological process, molecular function, and cellular component with the goseq r package, in which gene length bias was corrected. kyoto encyclopedia of genes and genomes (kegg) was used to perform pathway enrichment analysis . kobas software was used to test the level of statistical significance of enrichment of differentially expressed genes and/or lncrna target genes in kegg pathways (mao et al., ) . to determine the reliability of the rna-seq data, differentially expressed lncrnas were randomly selected to test the expression by rt-qpcr. total rna was extracted from st and ipec-j cells using superfectri tm total rna isolation reagent (pufei, shanghai, china). first-strand cdna was synthesized with a reverse transcriptase kit (vazyme, nanjing, china). rt-qpcr was performed with a sybr green master mix (vazyme, nanjing, china) on the lightcycler (roche, basel, switzerland). the pdcov m gene was detected by rt-pcr. all the primers are presented in table . relative expressions were calculated using the − ct method with gapdh as the internal control. comparisons between groups were made using two-way http://www.genome.jp/kegg/ table | primers used for rt-qpcr validation. sequence ( - ) amplicon anova. the data reported are the mean ± sem. differences were considered statistically significant when p < . . for each lncrna, the pearson correlation coefficient of its expression value with that of each protein-coding gene was calculated. under the conditions of an absolute value of the pearson correlation coefficient > . and p < . , the interaction network of the differentially expressed lncrnas and protein-coding gene co-expression pairs was then constructed using cytoscape (v . . ) (shannon et al., ) . to identify the lncrnas in pdcov-infected cells, we sequenced the transcriptomes of the st cells with or without pdcov infection using high-throughput rna sequencing. robust and reproducible data were obtained from all samples, and more than × clean reads per sample were retained after removing reads containing adapter or poly-n sequences and reads with low quality. afterward, all clean reads were aligned onto the pig reference genome (release ) using tophat and were compared and assembled with cuffcompare and cufflinks, respectively, and coverage analysis was performed on those clean reads on different annotated gene types. the distribution of each type of gene was counted according to the expression level. in total, eight categories of rna were identified, according to database annotation of those transcripts, in which the protein-coding genes were highly represented ( . % in ps and . % in st, respectively) ( figure a) . next, four software tools, cnci, cpc, phylocsf, and pfam, were used to calculate the protein-coding potential of assembledtranscripts to screen lncrnas, then taking the intersection of transcripts with no coding potential in these software products as the novel lncrna ( figure b) . in total, , annotated and , novel lncrna candidates were identified (supplementary tables s , s ). it has been reported that lncrnas, in comparison with protein-coding genes, usually share some common genomic features to their sequences. they are generally shorter in length, have fewer but longer exons, and there is lower evolutionary sequence conservation, with only ∼ % of mouse lncrnas having homologs in humans. lncrnas also demonstrate low expression levels (the median is ∼ % of that of protein-coding genes) (heward and lindsay, ) . to further determine the characteristics of the lncrnas identified in the present study, we compared the transcript length, exon number, and degree of conservation between protein-coding genes and lncrnas. conservation analysis of exons, introns, and promoters of lncrnas and protein-coding genes showed that the exons of protein-coding genes were the most conserved and the exons of lncrna were far less conserved ( figure c) . furthermore, fewer exons and shorter orfs were found in lncrnas, which was also consistent with the reported lncrnas (figures d,e) . long non-coding rnas sequences are poorly conserved and do not appear to form large homologous families, so it is difficult to infer their common ancestors by sequence similarity (ponting et al., ) . therefore, it is challenging to predict the functions of a type of lncrna on the basis of its sequence or structure. there have been reports of using genome-wide association analysis between lncrnas and the co-expressed and/or co-regulated protein-coding genes to characterize the function of the lncrna (huarte et al., ) . to investigate the putative role of lncrnas, we first analyzed the whole rna-seq profiles to identify target proteincoding genes whose location or expression was significantly correlated with the candidate lncrna. for co-located target gene prediction, we searched coding regions k upstream and downstream of lncrna. in total, , pairs of lncrna-proteincoding genes, containing , lncrnas and , proteincoding genes, were identified (supplementary table s ). for co-expressed target gene prediction, the expression correlation between lncrnas and protein-coding genes was evaluated. when the required pearson correlation coefficient was set above . , , , pairs of lncrna-protein-coding genes, containing , lncrnas and , protein-coding genes, were obtained (supplementary table s ). we next performed go and kegg pathway analysis for the target genes of lncrnas. the top go and kegg pathways with the highest representation of each term are reported (figure and supplementary tables s , s ) . kegg enrichment analysis revealed that pathways related to the immune system and metabolism were preferentially targeted. the go-term analysis was divided into three main categories: cellular component, biological process, and molecular function. significantly, a large number of biological processes, like protein-dna complex assembly, dna packaging and transcription, and the cellular macromolecule metabolic process, were enriched. furthermore, protein binding and nucleic acid binding and the nucleosome and organelles, belonging to molecular function and cellular component, respectively, were also enriched. go and kegg pathway enrichment analysis of target genes revealed that lncrnas may act in cis or trans to participate in the regulation of expression of multiple important genes in different processes including protein binding, dna transcription, metabolism, and immune response. to identify the pdcov-associated lncrnas, cuffdiff software was used to investigate the differentially expressed (de) lncrnas in pdcov-infected cells. the hierarchical clustering heat map in figure a shows the de lncrna expression profiling data. out of the , annotated and , novel lncrnas, we obtained annotated de lncrnas ( up-regulated and down-regulated) and novel de lncrnas ( up-regulated and down-regulated) after pdcov infection (p < . ; supplementary table s ) . importantly, we observed lncrnas whose expression levels were decreased to fpkm = after pdcov infection, while the fpkms of another lncrnas, all novel lncrnas, were before pdcov infection (supplementary table s ). this suggests that these lncrnas, though they have very low expression levels, might be strongly associated with the viral infection. furthermore, to evaluate the reliability of rna-seq data analysis, lncrnas were selected for rt-qpcr analysis in pdcov-infected cells. as shown in figure b , the expression levels of the selected lncrnas, though exhibiting no significant differences at h post-infection (hpi), were all significantly changed at hpi in st cells. also, different expression patterns of lncrnas were detected in ipec-j cells. as shown in figure c , out of the selected rna were significantly altered at hpi, and all of them were differently expressed at hpi. for both st and ipec-j cells, they had a strong expression pattern consistent with the rna-seq results ( table ) . the lncrna in the genome is not randomly distributed, so locus classification will be an effective first step in analyzing its regulatory functions at the genome level (luo et al., ) . in general, lncrnas function either in cis or in trans to affect the transcription of genes within or far from the same genomic locus (clark and blackshaw, ) . to understand the potential functional association between lncrnas and cognate genes, we investigated their genomic distribution pattern relative to protein-coding loci and classified all de lncrnas to ascertain their potential biological roles. all de lncrnas were classified into six categories comprising sense-upstream lncrna, sense-downstream lncrna, sense-overlapping lncrna, antisense-upstream lncrna, antisense-downstream lncrna and antisense-overlapping lncrna. as shown in figure a , % of de lncrnas were located in the same strand but upstream of protein-coding genes and % were located downstream, while antisense-upstream and antisense-overlapping comprised and %, respectively, and the remaining % were antisensedownstream lncrnas. next, in order to define the lncrna functions more precisely, go enrichment analysis of the colocated genes of up-and down-regulated lncrnas were analyzed independently. the results showed that protein-coding genes associated with de lncrnas were mainly enriched in terms of molecular function and cellular component, primarily under the category of nucleic acid binding and intracellular membranebounded organelle ( figure b) . notably, by analyzing the relative expression level, we found that antisense lncrna and proteincoding genes were specifically co-expressed, in which two pairs showed a positive and three pairs showed a negative correlation in their expression patterns ( figure c and supplementary table s ). we speculated that these antisense lncrnas act in cis to modulate the expression of their cognate genes. the functional association between regulatory lncrna and protein-coding gene transcripts can be determined by performing expression correlation analysis coupled with ascertaining their putative role in related physiological processes. to further investigate the potential mechanism of action of the pdcov-associated lncrnas, the de lncrnas and their predicted target de protein-coding genes were investigated by delineating lncrna-protein-coding gene functional interactions. here we identified , , pairs of de lncrna-de proteincoding genes, containing lncrnas and , protein-coding genes (p < . ). next, kegg pathway analysis was repeated once again (figure a) , and we found that metabolic and tnf signaling pathways were significantly enriched. the interaction network involving the metabolic and tnf signaling pathways was then constructed. several key genes in metabolism were positively or negatively regulated by lncrnas ( figure b ). of the significantly enriched genes, atp l and atp f , two of the mitochondrial membrane atp synthase subunits, were regulated by lnc- , lnc- , aldbssct , and aldbssct . in addition, three lncrnas, lnc- , lnc- , and aldbssct , might regulate acyl-coenzyme a thioesterase four expression. these results suggest that these lncrnas might be involved in the regulation of metabolic processes particularly involving energy and lipid metabolism. meanwhile, an inducible program of inflammatory gene expression is central to antiviral defense. many of them, i.e., ccl , ccl , cxcl , cxcl , map k , nf-κb , and interleukin (il- ), were protein-coding genes known to have roles in the inflammatory response. in the network (figure c ), eight lncrnas have putative regulatory roles in il- expression. six of them, lnc- , lnc- , lnc- , lnc- , aldbssct , and aldbssct , might exert positive regulation, while lnc- and aldbssct showed the opposite effect. this suggests that these lncrnas might act as the regulatory module of the circuit that is involved in the inflammatory response. numerous studies have shown that lncrnas play a key role during viral infection. the lncrnas thril, nest, neat, and lincrna-cox can participate in immune responses against viral infection mainly through regulating the expression of tnf-α, ifn-γ, il , and inflammatory response, respectively (carpenter et al., ; gomez et al., ; imamura et al., ; li et al., ) . pdcov is an important enteric virus mainly causing diarrhea in suckling pigs. infection with pdcov causes changes in the expression levels of several host cell proteins of host innate immune response, but little is known about the critical roles of lncrnas in these processes. here, we performed rna-seq to identify the lncrnas involved in pdcov infection. the results of comparing clean reads to the genome showed that more than % of reads are protein-coding genes, and no lncrna classifications were identified due to the limited lncrna database annotation in pig. in our results, , novel lncrnas were identified. further analysis showed that the basic characteristics of these novel lncrnas are consistent with the known ones. our rna-seq results further enrich the pig lncrna database. in total, we found annotated and novel lncrnas that were differentially expressed during pdcov infection. these lncrnas were classified as sense-upstream lncrna, sense-downstream lncrna, sense-overlapping lncrna, antisense-upstream lncrna, antisense-downstream lncrna, and antisense-overlapping lncrna. many antisense-overlapping lncrnas have inverse expression patterns with their sense transcript counterparts. this suggests that these antisenseoverlapping lncrnas may have a negative regulatory effect on them. in contrast, many lncrnas that do not contain overlapping sequences display expression patterns correlated with their neighboring protein-coding gene transcripts. in the present study, two out of five antisense overlapping lncrnas were found to have high consistency in their expression (figure ) . similarly, the lncrna evx as, which initiates within the first exon of the gene evx , has an overlap of eight nucleotides with the evx mrna and promotes transcription of its neighbor gene by increasing the binding affinity of histone h lysine tri-methylation (h k me ) and histone h lysine acetylation (h k ac) at the promoter region. considering that most lncrnas might function through their secondary structure rather than the primary one, this suggests that the regulation of antisense transcripts by antisense-overlapping lncrna may not be mediated through base-complementary pairing. correlation analysis of de lncrna and protein-coding genes identified a number of de lncrna-de protein-coding gene pairs. the main enriched kegg pathways of these protein-coding genes were in metabolism and oxidative phosphorylation. in a recent report, -day-old neonatal pigs were infected with pdcov, and transcriptome profile and kegg pathway enrichment analysis were performed at different stages of infection (wu et al., ) . in our study, we found that the lncrna targeted genes enriched in those pathways that were perturbed during the late stage of infection. in addition, the expression level of transglutaminase (tgm ) and apolipoprotein a- (apoa ) in a wu et al. ( ) study were significantly changed. similarly, we also found that tgm was up-regulated, and apoa , apoa , and apoa were down-regulated during pdcov infection (data not shown). moreover, our data show that many cytokines and chemokines, which elicit an inflammatory response, were differentially expressed in the infected cells compared to mock cells. the inflammation causes injury to the intestinal tissues, resulting in diarrhea or even death. raised ccl and cxcl levels were associated with the severity of virus infection (betakova et al., ; masood et al., ) . here, we identified a number of lncrnas that may regulate the expression of these inflammatory molecules. to the best of our knowledge, this is the first study focusing on the expression profile of cellular lncrnas after pdcov infection. our data show the expression landscape of lncrnas, with special emphasis on the lncrna-protein modules operating in response to pdcov infection. moreover, this study provides a comprehensive genome-wide resource for exploring the molecular and cellular regulatory functions of lncrnas. this study will also be useful for identifying lncrnas as potential biomarkers for the diagnosis of pdcov infection and designing better prophylactic and therapeutic tools against virus infection. in the present study, the expression profiles of lncrnas were determined in pdcov-infected st cells. in total, , novel lncrnas were identified. a total of lncrnas were differentially expressed between pdcov-infected or mockedinfected st cells. kegg pathway analysis of de lncrna coexpressed genes revealed that they might be primarily involved in regulating metabolism and tnf signaling pathways. our study systematically characterizes lncrna expression during pdcov infection and provides a useful resource for identifying and functionally characterizing the cognate gene products of those lncrnas. this study will also be useful for assigning lncrnas as potential biomarkers of pdcov infection and designing better preventive and therapeutic measures against the virus infection, which would be economically beneficial for the pig farming community. the raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualified researcher. jll, jg, and jz conceived and designed the experiments. fw, ld, and jl performed the experiments. jll, yy, yj, and ty analyzed the data. jll drafted the manuscript. all authors read and approved the final manuscript. herdlevel prevalence and incidence of porcine epidemic diarrhoea virus (pedv) and porcine deltacoronavirus (pdcov) in swine herds in ontario cytokines induced during influenza virus infection a long noncoding rna mediates both activation and repression of immune response genes long non-coding rna-dependent transcriptional regulation in neuronal development and disease the gencode v catalog of human long noncoding rnas: analysis of their gene structure, evolution, and 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antagonizes type i interferon signaling by cleaving stat the supplementary material for this article can be found online at: https://www.frontiersin.org/articles/ . /fmicb. the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © liu, wang, du, li, yu, jin, yan, zhou and gu. this is an openaccess article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- -siwzjeea authors: forni, diego; cagliani, rachele; pontremoli, chiara; pozzoli, uberto; vertemara, jacopo; de gioia, luca; clerici, mario; sironi, manuela title: evolutionary analysis provides insight into the origin and adaptation of hcv date: - - journal: front microbiol doi: . /fmicb. . sha: doc_id: cord_uid: siwzjeea hepatitis c virus (hcv) belongs to the hepacivirus genus and is genetically heterogeneous, with seven major genotypes further divided into several recognized subtypes. hcv origin was previously dated in a range between ∼ and years ago. hepaciviruses have been identified in several domestic and wild mammals, the largest viral diversity being observed in bats and rodents. the closest relatives of hcv were found in horses/donkeys (equine hepaciviruses, ehv). however, the origin of hcv as a human pathogen is still an unsolved puzzle. using a selection-informed evolutionary model, we show that the common ancestor of extant hcv genotypes existed at least years ago (ci: – years ago), with the oldest genotypes being endemic to asia. ehv originated around ce (ci: – ce). these time estimates exclude that ehv transmission was mainly sustained by widespread veterinary practices and suggest that hcv originated from a single zoonotic event with subsequent diversification in human populations. we also describe a number of biologically important sites in the major hcv genotypes that have been positively selected and indicate that drug resistance-associated variants are significantly enriched at positively selected sites. hcv exploits several cell-surface molecules for cell entry, but only two of these (cd and ocln) determine the species-specificity of infection. herein evolutionary analyses do not support a long-standing association between primates and hepaciviruses, and signals of positive selection at cd were only observed in chiroptera. no evidence of selection was detected for ocln in any mammalian order. these results shed light on the origin of hcv and provide a catalog of candidate genetic modulators of hcv phenotypic diversity. hepatitis c virus (hcv) belongs to the hepacivirus genus and is genetically heterogeneous, with seven major genotypes further divided into several recognized subtypes. hcv origin was previously dated in a range between ∼ and years ago. hepaciviruses have been identified in several domestic and wild mammals, the largest viral diversity being observed in bats and rodents. the closest relatives of hcv were found in horses/donkeys (equine hepaciviruses, ehv). however, the origin of hcv as a human pathogen is still an unsolved puzzle. using a selection-informed evolutionary model, we show that the common ancestor of extant hcv genotypes existed at least years ago (ci: - years ago), with the oldest genotypes being endemic to asia. ehv originated around ce (ci: - ce). these time estimates exclude that ehv transmission was mainly sustained by widespread veterinary practices and suggest that hcv originated from a single zoonotic event with subsequent diversification in human populations. we also describe a number of biologically important sites in the major hcv genotypes that have been positively selected and indicate that drug resistance-associated variants are significantly enriched at positively selected sites. hcv exploits several cell-surface molecules for cell entry, but only two of these (cd and ocln) determine the species-specificity of infection. herein evolutionary analyses do not support a long-standing association between primates and hepaciviruses, and signals of positive selection at cd were only observed in chiroptera. no evidence of selection was detected for ocln in any mammalian order. these results shed light on the origin of hcv and provide a catalog of candidate genetic modulators of hcv phenotypic diversity. hepatitis c virus (hcv, genus hepacivirus, family flaviviridae) is a hepatotropic human pathogen with an estimated worldwide seroprevalence around . % (mohd hanafiah et al., ) . the seven major hcv genotypes display remarkable antigenic variability and are classified into several recognized subtypes . a small number of "epidemic" subtypes ( a, b, a, and a) account for the overwhelming majority of infections worldwide (simmonds, ; messina et al., ) . their spread occurred recently (in the last - years), following the development of practices that cause parenteral exposure (pybus et al., ; simmonds, ; jackowiak et al., ) . the epidemic subtypes represent a small fraction of hcv diversity. in sub-saharan africa and south-east asia, the pattern of hcv diversity is characterized by highly divergent subtypes of the same genotype dominating transmissions across geographically contiguous areas ("endemic" transmission) (simmonds, ; jackowiak et al., ) . hepaciviruses have been identified in several domestic and wild mammals, the largest viral diversity being observed in bats and rodents (kapoor et al., (kapoor et al., , burbelo et al., ; drexler et al., ; quan et al., ; corman et al., ; scheel et al., ; walter et al., ; van nguyen et al., ) . the closest relatives of hcv were found in horses/donkeys and dogs (equine and canine hepaciviruses, ehv, and chv) (pybus and gray, ; pybus and theze, ) , but the origin of hcv as a human pathogen is still an unsolved puzzle. some authors envisaged the possibility that hcv evolved from a horse-tohuman transmission event (pfaender et al., ; scheel et al., ) , others suggested that hcv originated in relatively recent times from one or multiple cross-species transmission events from a still to be defined species (pybus and gray, ; scheel et al., ; pybus and theze, ) . however, its strict speciesspecificity, as well as the ability of hcv to persist lifelong in humans, led to the alternative hypothesis whereby hcvrelated viruses have been infecting humans and other primates throughout their evolutionary history (simmonds, ; scheel et al., ) . another open question is whether hcv genotypes differ in terms of transmissibility or disease outcome. conversely, the viral genotype is known to represent a predictive factor for antiviral treatment success. treatment with pegylated interferon and ribavirin (pegifn/rbv) is less likely to be successful in patients infected with genotypes and compared to those infected with genotypes and (bartenschlager et al., ) , and variants at the human ifnl /ifnl locus were reproducibly associated with pegifn/rbv treatment outcome (o'brien et al., ) . interestingly, the same polymorphisms are strongly associated with spontaneous hcv clearance (o'brien et al., ) . recently, direct-acting antivirals (daas) have greatly improved the efficacy of treatment strategies for hcv. however, daas have limited pan-genotypic activity and tend to select for resistance-associated amino acid variants (ravs) (lontok et al., ) . most ravs naturally occur in treatment-naive patients and in some instances rav prevalence differs among genotypes or subtypes (rai and deval, ; bartenschlager et al., ; lontok et al., ; cannalire et al., ; chen et al., ; patino-galindo et al., ) . herein, we apply an evolutionary approach to shed light into hcv origin, to analyze its adaptation to human populations, and to verify if the emergence of drug-resistant variants is a result of positive selection. we also analyzed the most important cell-surface molecules that mediate hcv cell entry to determine whether hcv or related hepaciviruses exerted a selective pressure on these receptors and whether selection was particularly strong in specific mammalian orders or superorders. coding sequences for mammalian ocln, cd , cldn , and scarb (encoding srb ) were retrieved from the ncbi database. a list of species is available in the supplementary table s . dna alignments were performed with the revtrans . utility , mafft v . as an aligner) (wernersson and pedersen, ) , which uses the protein sequence alignment as a scaffold for constructing the corresponding dna multiple alignment. all alignments were screened for the presence of recombination using genetic algorithm recombination detection (gard) (kosakovsky pond et al., ) and two methods (rdp and geneconv) (sawyer, ; martin and rybicki, ) implemented in the rdp program (martin et al., ) . rdp and geneconv were selected because they showed good power in previous simulation analyses (posada and crandall, ; bay and bielawski, ) and only breakpoints identified by both methods were accepted. the cutoff p-value was set to . in both gard and rdp . no method detected recombination in any alignment. after running a codon model selection analysis in hyphy (delport et al., ) , gene trees were generated by maximumlikelihood using phyml with the approximate likelihood-ratio test (alrt) method (guindon et al., ) . to search for positive selection, we applied the site models implemented in paml (yang, ) . specifically, a model (m , positive selection model) that allows a class of sites to evolve with dn/ds > was compared to two models (m and m a, neutral models) that do not allow dn/ds > (yang, ) . positively selected sites were identified through bayes empirical bayes (beb) analysis (anisimova et al., ; yang et al., ) from model m (with a posterior probability cutoff of . ) and through mixed effects model of evolution (meme), with a default p-value cutoff of . ) (murrell et al., ) . only sites detected by both methods were considered as positively selected. very similar results were obtained using the gene tree obtained with phyml or the species tree as inputs for paml analysis. to estimate the time to the most common ancestor (tmrca) of the hcv genotypes, we used alignments of hcv sequences with known isolation dates (supplementary table s ). viral sequences were retrieved from the ncbi database. we used prank (loytynoja and goldman, ) to generate multiple sequence alignments and guidance (sela et al., ) for filtering unreliably aligned codons (we masked codons with a score < . ), as suggested (privman et al., ) . the ns b region we used for dating is non-recombining, as assessed by gard and rdp analyses of the entire nonstructural portion of the genome. this region was selected because it is one of the most conserved across hcv genotypes (kapoor et al., ) and required very minor filtering. the tmrca of ehv/chv was estimated on a phylogeny of sequences with complete coding sequence information: ehv isolated from horses or donkeys and chv (kapoor et al., ; burbelo et al., ; walter et al., ) (supplementary table s ). guidance detected no uncertainty in the alignments (all codons had a score > . ) and gard detected a breakpoint at position . in good agreement, rdp detected a breakpoint at position . the terminal nucleotides were thus removed and the resulting alignment was used for tree construction and tmrca estimate. to determine whether there was sufficient temporal structure in the ns b and ehv phylogenies to estimate divergence times, we calculate the correlation coefficients (r) of regressions of rootto-tip genetic distances against sequence sampling times (murray et al., ) . a method that minimizes the residual mean squares of the models, rather than one that maximizes r , was applied, as suggested (murray et al., ) . the p-values were calculated by performing , clustered permutations of the sampling dates murray et al., ) . evidence for temporal structure was obtained for both phylogenies (ns b: r = . , r = . , p-value = . ; ehv: r = . , r = . , p-value = . ). the action of saturation and purifying selection can underestimate the tmrca, and selection-informed models can improve branch length estimation (wertheim and kosakovsky pond, ; wertheim et al., wertheim et al., , . we thus applied a branch-site test (abs-rel, adaptive branch-site random effects likelihood) to estimate branch lengths while taking into account the effect of different selective pressures among lineages in the phylogeny. previous works showed that, in the presence of saturation and selection, abs-rel estimates branch lengths more reliably than other commonly used models such as the general time-reversible substitution model with a four-bin gamma rate distribution (gtr+ ) (wertheim and kosakovsky pond, ; wertheim et al., ) . estimate of divergence times was performed by a penalizedlikelihood (pl) method implemented in r s (sanderson, ) , using the abs-rel tree as the input tree. in the case of extremely long (i.e., more than substitution/site) terminal branches, most likely resulting from low precision in point estimates of dn/ds, mg branch lengths were used instead of abs-rel lengths to avoid biases in the tmrca estimates. mg (muse and gaut, ) is the baseline model used by abs-rel and it models synonymous and nonsynonymous substitution rate variation across branches but not across sites (muse and gaut, ) . the pl method in r s employs a smoothing parameter, which represents how much the assumption of a molecular clock has been relaxed. cross-validation was run to determine the best smoothing value for the abs-rel tree (sanderson, ) . a latin hypercube sampling scheme (lhc) was used to sample from the abs-rel parameter distributions so as to estimate the confidence interval, as previously suggested (wertheim and kosakovsky pond, ; wertheim et al., wertheim et al., , . briefly, samples were drawn from abs-rel analyses to estimate branch length variance, trees were generated, and then used as input trees for r s. the upper and the lower % bounds are used as confidence intervals. as a comparison, branch lengths were also estimated using phyml with a maximum-likelihood approach and a gtr+ model (guindon et al., ). we used coding sequence information for the recognized hcv subtypes (supplementary table s ). viral sequences were retrieved from the ncbi database. as above, we used prank (loytynoja and goldman, ) to generate multiple sequence alignments and guidance (sela et al., ) for filtering unreliably aligned codons (privman et al., ) . gard (kosakovsky pond et al., ) and the above-mentioned methods implemented in rdp (sawyer, ; martin and rybicki, ) were used to screen for recombination. phylogenetic trees were generated by maximum-likelihood using phyml (guindon et al., ) after codon model selection in hyphy (delport et al., ) . to investigate whether episodic positive selection acted on the internal branches of hcv phylogenies, we applied the branch-site tests from paml (zhang et al., ) and busted (branch-site unrestricted statistical test for episodic diversification) . the p-values from both methods were fdr-corrected to account for multiple tested branches. a branch was considered positively selected when statistically significant evidence was obtained with both methods. positively selected sites were then identified through the beb analysis from model ma (with a posterior probability cutoff of . ) (zhang et al., ) and with busted (with a p-value cutoff of . ) . sites were called as positively selected if they were detected by both methods. beb and busted were in good general agreement. on selected branches, . % of beb sites were also detected by busted and . % of busted sites were also identified by beb. we used single-likelihood ancestor counting (slac) and fixed effects likelihood (fel) (kosakovsky pond and frost, ) to calculate the rates of nonsynonymous and synonymous changes at each site in the structural and in the non-structural region alignments (analyses were performed on alignments split on the basis of the recombination breakpoint detected by gard). both slac and fel estimate the probability of selection at each site in an alignment through the dn-ds metric (rate of nonsynonymous changes-rate of synonymous changes) (kosakovsky pond and frost, ) . this metric is preferred over the conventional dn/ds ratio as this latter is rendered to infinite for ds values equal to . although slac and fel use different methodologies to estimate substitution rates (kosakovsky pond and frost, ) , they yielded very similar results (for all sites in the hcv genome, spearman's correlation coefficient = . , p-value < − ). slac and fel were used to estimate the evolutionary rate at rav vs. non-rav positions. the dn-ds statistics was also exploited to provide an overall view of hcv genome evolution. in this case, only slac results are shown (those obtained with fel were very similar). a total of ravs in ns , ns b, ns a, and ns b were included in the study (supplementary table s ). ravs were obtained by merging six recently compiled lists of naturally occurring variants associated with daa resistance (rai and deval, ; bartenschlager et al., ; lontok et al., ; cannalire et al., ; chen et al., ; patino-galindo et al., ) . specifically, a list of positions where ravs have been reported was compiled, irrespective of the viral genotype carrying the rav. for statistical analysis, six ravs that occur at codons pruned from the alignment by guidance were removed. the overall probability of rav occurrence was calculated over the number of non-pruned codons in ns , ns b, ns a, and ns b (total length = codons). for the ns a-oas docking, the crystal structure of the ns a domain i of a subtype b strain (pdb: zh ) and the structure of human oas (pdb: ig ) were obtained from the protein data bank (pdb) (tellinghuisen et al., ; donovan et al., ) . the b subtype was used in the original study describing interaction between oas and ns a (taguchi et al., ) . two docking methods were applied and their results compared for consistency. specifically, we used the patchdock server (schneidman-duhovny et al., ) and imposed that the f residue in ns a is located at the binding interface, as described (taguchi et al., ) . we next used cluspro (comeau et al., ; kozakov et al., ) without any assumption on the residues involved in binding. the best models from cluspro and patchdock were superimposed and revealed an almost identical binding pose. we next checked that the two oas regions necessary for ns a binding (taguchi et al., ) were located at the interface: one of these two regions (amino acids - ) defines most of the contact area in the models. overall, these observations indicate that the binding interaction pose obtained with the two docking methods is reliable. the c sphingomyelin ligand structure was prepared for simulation using the ligprep module of maestro (schrodinger release, - : maestro schrodinger, llc, new york, ny, united states, ) to determine the d structure and ionization states at ph . ± . . the ns b structure of a subtype b strain (pdb id: mk chain a) was processed with protein preparation wizard module of maestro to remove crystal water molecules, add hydrogen atoms, assign bond orders, and optimize the orientation of hydroxyl groups. docking simulation was carried out with ifd protocol (sherman et al., ) in extra precision mode. the receptor grid was generated around the sphingomyelin binding domain (residues e to g ). all the simulations were performed using the opls force field (harder et al., ) . the docked ligand-receptor poses were analyzed in terms of glide score, a scoring function that estimate protein-ligand binding affinities . the d structures were rendered using pymol (the pymol molecular graphics system, version . . . schrödinger, llc). host receptors that mediate virus entry often evolve under positive selection (a situation that favors amino acid replacements over silent substitutions) to avoid viral recognition (sironi et al., ) . four cell-surface molecules, cd , occludin (ocln), claudin (cldn ), and scavenger receptor class b member (srb ), are particularly important for hcv cell entry, although only cd and ocln determine the species-specificity of infection (bartenschlager et al., ; lindenbach and rice, ) . we thus reasoned that if primates experienced long-term interactions with hcv or closely related hepaciviruses, signals of positive selection should be detectable at the genes encoding hcv receptors. conversely, if selection is evident in other mammalian orders, these may represent the ancestral hcv reservoirs. we retrieved coding sequences of the four genes for mammalian species belonging to different orders, superorders or clades (supplementary table s ). due to the hypothesized role of bats as reservoir hosts for mammalian hepaciviruses, chiroptera were analyzed separately from other species in the laurasiatheria superorder. evidence of positive selection was searched for using models that allow dn/ds to vary among sites in the alignment (yang, ) . no evidence of positive selection was detected for cldn and srb (table ) . also, selection was not detected at ocln or cd in primates (table ) , an observation that does not support a long-standing selective pressure exerted by hepaciviruses on these hosts. however, positive selection was not detected for rodents, either, although these mammals are known to host a wide diversity of hepaciviruses (drexler et al., ; kapoor et al., ; scheel et al., ) . for laurasiatherian ocln, we detected one positively selected site located in the extracellular loop , which is not directly involved in hcv binding. notably, evidence of positive selection for cd was detected only in chiroptera (figure and table ). the five positively selected sites are located at the binding interface with the hcv glycoprotein. single point mutations in the corresponding region of the human receptor were shown to reduce or abolish the interaction with the hcv e protein and/or hcv infectivity (figure and table ) (drummer et al., ; bertaux and dragic, ; flint et al., ) . conversely, the cd region required for infection of hepatocytes by plasmodium yoelii (a rodent parasite) sporozoites is unaffected by selection (figure ) (yalaoui et al., ) . the observation that the positively selected sites are involved in hcv binding and infectivity suggests that hepaciviruses contributed to shape the genetic diversity of cd in bats. previous studies provided estimates of the time to the most recent common ancestor (tmrca) of hcv genotypes in a range between ∼ and years ago, with one single study indicating that hcv origin may date years back (smith et al., ; pybus et al., ; kapoor et al., ; simmonds, ; li et al., ; lu et al., ) . the tmrca of equine/canine hepaciviruses (ehv/chv) was estimated to be recent, dating around ce (pybus and theze, ) . it is well known that the temporal variation in rates of nucleotide substitutions often results in underestimation of the age of viral lineages (duchene et al., ; aiewsakun and katzourakis, ) . purifying selection and substitution saturation are strongly associated with temporal rate variation (duchene et al., ) . simulations experiments indicated that "classic" models (e.g., gtr+ ) tend to underestimate branch lengths in the presence of purifying selection (wertheim and kosakovsky pond, ) and substitution saturation (wertheim et al., ) . because both phenomena are more pronounced for internal branches, length underestimation is more severe for these branches and dating inferences are consequently affected (wertheim and kosakovsky pond, ; wertheim et al., ) . the use of models that allow site-and branch-specific variation in selective pressure can improve branch length estimates in the presence of both purifying selection and substitution saturation (wertheim and kosakovsky pond, ; wertheim et al., ) . we thus applied the abs-rel model (adaptive branchsite random effects likelihood) to calculate the tmrca of relevant nodes in a phylogeny of hcv strains (supplementary table s ). this approach was previously applied to revise the dating of other rna viruses (wertheim and kosakovsky pond, ; wertheim et al., ) . we constructed a phylogeny for the ns b region and we found that only . % of branches showed saturation of ds. as expected, branch lengths estimated with abs-rel were generally longer than those obtained with a gtr+ model, and this was particularly true for internal branches (figure a and supplementary figure s ). using the abs-rel model, we estimated the tmrca of the seven hcv genotypes to be years ago ( % ci: - ) ( figure b) . the deepest tmrca was obtained for genotype (endemic in south-east asia), with an origin dating at least years ago (figures b,c) . the tmrcas of genotype (endemic in south asia) and of the ancestor of genotypes and (both widespread in central africa) were estimated to be in the bce- ce range (figures b,c) . results for genotype (endemic in west africa) indicated an origin around years ago (figures b,c) . horse-to-human hepaciviral transmission was hypothesized as the origin of hcv (pfaender et al., ; scheel et al., ) . ehv isolates show limited divergence and high levels of purifying selection (simmonds, ; pfaender et al., ) . we thus used the same approach described above to obtain the tmrca of extant ehv/chv strains (figure a ) (supplementary table s ), producing an estimate of years ago ( % ci: - ). a number of studies have explored the recent selective events that generated intra-genotype and within-host genetic diversity (jackowiak et al., ) , whereas the deeper evolutionary history of hcv has remained largely unexplored. we thus used complete sequence information for the recognized hcv subtypes to search for selective events that occurred during the radiation of the seven genotypes. the structural and non-structural coding regions were analyzed separately, and the core region was excluded due to the presence of a putative alternative reading frame (xu et al., ) . we next tested for the presence of recombination using gard and rdp (see the section "materials and methods"). none of the rdp methods detected recombination either in the structural or in the non-structural regions. conversely, gard detected a possible breakpoint at the end of ns . the lack of consistency among recombination detection methods is somehow expected as these approaches have distinct performances depending on the amount of recombination, the genetic diversity of analyzed sequences, the tree topology, and the rate variation among sites (posada and crandall, ; bay and bielawski, ) . because our aim was to avoid the inflation of positive selection inference caused by unrecognized recombination, the alignment was split into two sub-alignments (based on the gard-detected breakpoint) with slightly different topologies (figure ) . evidence of episodic positive selection along the internal branches of the phylogenies was searched for using two different branch-site methods, which rely on different assumptions of dn/ds variation among branches (zhang table s ). analysis of positively selected sites was performed by inspection of literature reports describing the effect of specific mutations, as well as by performing docking analyses. overall, positively selected sites could be categorized on the basis of the functional effect they are likely to entail, as follows: cell entry, interaction with the host immune system, and association with membrane/lipids. the details of these sites are reported below, as well as in figures , . as expected, none of the positively selected sites in e involved the conserved residues at the cd binding site (lavie et al., ) . interestingly though, a mutation at residue in e was previously obtained through in vitro adaptation of hcv to mouse cells ( figure a ) (bitzegeio et al., ) . specifically, the l f mutation (as well as two other mutations in the hypervariable region , hvr ) allow infection of cells expressing cd of rodent origin (bitzegeio et al., ) , suggesting that hcv adaptation to new hosts does not necessarily entail changes at the direct binding interface with cd . several positively selected sites were detected in the transmembrane (tm) regions of both e and e (figure a) . a positively selected site in the e tm domain (residue ) was shown to reduce the dependency on scarb for cell-to-cell spread in vitro, but increases viral susceptibility to antibody-mediated neutralization (catanese et al., ) . interestingly, a similar phenotype was described for a t v mutant in the jfh viral background (corresponding to the positively selected site in strain h ) ( figure a ) (zuiani et al., ) . using a site-directed saturation mutagenesis approach, the t v mutation was shown to confer growth advantage in vitro via reduced dependency on scarb and probably stronger binding to cd (zuiani et al., ) . several mechanisms for hcv resistance to ifn have been proposed. the ns a protein displays a double-stranded rnaactivated protein kinase r (pkr) binding site that overlaps with the ifn sensitivity determinant (isdr) region but includes additional amino acids essential for pkr binding (gale et al., ) . two positively selected sites were detected within this amino acid region ( figure a ). mutations at the second selected site (position i in h , corresponding to c in the jfh sequence) arise when hcv is passaged in the presence of ifn-alpha (perales et al., ) . the c i change is positively selected on the genotypes / branch and both genotypes are relatively resistant to ifn therapy (jackowiak et al., ) . the binding of ns a to oas also contributes to inhibition of ifn antiviral activities (taguchi et al., ) . based on previous biochemical data (taguchi et al., ) , we performed protein-protein docking of ns a domain i and human oas . results showed that three of the positively selected sites in ns a domain i, positions , , and are at the direct binding interface with oas ( figure b) . notably, besides being described as ravs for daas, substitutions at the y site in the subtype b background occur at different frequency depending on the host's genotype at the ifnl /ifnl locus (akamatsu et al., ; peiffer et al., ) . a similar trend was observed for variants at ns a positions (not included in the crystal), (which modulates oas binding) (taguchi et al., ) , (close to the binding interface), and (a positively selected rav close to the binding interface) ( figure b ) (akamatsu et al., ) . interestingly, sites and are positively selected on the genotype and branches, respectively (supplementary table s ). both these figure | selected sites in hcv proteins. (a) schematic representation of the hcv structural region. regions that were not analyzed (i.e., core region) or filtered due to poor alignment quality are colored in gray. the location of positively selected sites is shown and residues with known functional significance (see text and below) are underlined. sites are numbered based on the sequence of the h strain (af . ). the ectodomain region of e contains two short regions with sequence similarity to class ii fusion peptides (drummer et al., ) : in vitro mutagenesis indicated that changes at positively selected residues , , and abolish or reduce viral entry (drummer et al., ; li et al., ; russell et al., ) . a amino acid motif in e (the pkr-eif α phosphorylation site homology domain, pephd) is required for pkr and perk (pkr-like er-resident kinase) inhibition (taylor et al., ; pavio et al., ) . an amino acid alignment for the pephd domain is reported (representative hcv sequences only), with positively selected sites in red. tm: transmembrane domain. (b) topological structure of the ns and ns b proteins. positively selected sites are mapped (red) on the ns (pdb id: hd ) and ns b (pdb id: lvg, jxf, kdr) protein structures. protein segments of unresolved structure are represented as cylinders (transmembrane domains) or dotted lines. an amino acid alignment of the second amphipathic helix of ns is reported for representative strains (positively selected sites in red): mutagenesis of positively charged residues at positions or (depending on the genotype) affect ns membrane association, protein stability, and efficient hcv polyprotein processing (lange et al., ) . the presence of at least one positively charged residue at these positions is sufficient to allow proper membrane localization (lange et al., ) and indeed, the two positions evolve in concert in the hcv phylogeny with a charged residue always observed at either position or , but never at both sites. er: endoplasmic reticulum. genotypes are endemic in asia, where the frequency of the protective human ifnl /ifnl variant (rs ) is highest ( figure c) . recently, a large-scale analysis of patients mostly infected with hcv genotype a, identified sites in the viral polyprotein showing association with one or more hla alleles (ansari et al., ) . we found three of these sites ( in e , in ns , and in ns b) to be positively selected, with site in ns (position in the polyprotein) representing one of the strongest association detected in the study (ansari et al., ) . all hcv proteins are tethered to intracellular membranes either via transmembrane domain or through amphipathic alpha helices or both (bartenschlager et al., ) . we identified several positively selected sites within amphipathic alpha helices (figure b ). in the case of ns b, virtually all sites are located in these regions, where several ravs also map (supplementary table s ). among these, w (selected on the genotype branch) is essential for ns b association to the membrane and to lipid droplets (gouttenoire et al., ; tanaka et al., ) . we found positively selected sites within the sphingomyelin binding domain (sbd) of the hcv rna polymerase (ns b). binding of sphingomyelin to ns b allows localization of the polymerase to lipid rafts and activates the enzymatic activity in a genotype-dependent manner (sakamoto et al., ; weng et al., ) . remarkably, mutagenesis experiments indicated that the positively selected sites and modulate sphingomyelin binding and activation, respectively (weng et al., ) . we thus docked a sphingomyelin molecule onto the d structure of subtype b ns b (figure d) . docking results confirmed a salt bridge interaction between residue and the sphingomyelin (along with residues , , and ) and the localization of residue at the interaction surface. moreover, analysis of atomic distances indicated that two additional selected sites regions that were filtered due to poor alignment quality are colored in gray. the location of positively selected sites is shown and residues discussed in the text are underlined. positions and are also underlined, as a single lysine insertion between these two sites strongly increases viral replication (pflugheber et al., ) . (b) superimposition of ns a-oas binding pose obtained using two different docking programs. for clarity, one oas molecule is shown (green); the binding poses of the ns a dimer obtained with cluspro (yellow) and patchdock (light orange) are shown. the f residue, known to modulate ns a binding to oas , is marked in orange. positively selected sites are in red and labeled based on the sequence of h . oas regions that are essential for ns a binding are in dark green. (c) world map showing rs allele frequency in human populations (data from https://www.ncbi.nlm.nih.gov/variation/tools/ genomes/ and https://alfred.med.yale.edu/alfred/high throughput.asp). (d) docking pose of ns b (pdb id: mk , white) with sphingomyelin (green). positively selected sites are colored in red and labeled when located at the ns b-sphingomyelin binding interface. (e) ligand interaction diagram of best docked pose of ns b-sphingomyelin. residues within a Å distance and hydrogen bonds are shown (see legend). ( and ) are likely involved in the binding of sphingomyelin ( figure e ). a recent analysis indicated that several ravs occurred de novo on external branches of hcv phylogenies, although a minority appeared on internal branches, indicating a common origin in multiple strains (patino-galindo et al., ) . however, the evolutionary history of ravs has remained a poorly investigated issue. up to now, direct-acting antivirals (daas) have been developed to inhibit the function of the ns , ns b, ns a, and ns b proteins. to obtain a codon-wise measure of selective pressure acting on these proteins across the hcv phylogeny, we calculated dn-ds at each site. comparison of rav and non-rav sites revealed no statistically significant difference in dn-ds calculated with either slac (wilcoxon's rank-sum test p-value = . ) or fel (wilcoxon's rank-sum test p-value = . ) ( figure a ). this suggests that ravs do not originate and are not maintained in viral populations as a result of relaxed selective constraints. this overall trend does not imply that all rav sites evolve at the same rate. thus, we tested whether positions where ravs occur are more likely to diversify through the action of natural selection. nine rav positions coincided with positively selected sites, a number higher than expected by chance (binomial test, p-value = . ) ( supplementary table s ). moreover, three of these rav positions were targeted by positive selection on two distinct branches of the phylogeny, a situation observed for only . % of selected sites ( figure a) . to gain a comprehensive view of the action of selection, we plotted dn-ds along the hcv genome and we superimposed the location of positively selected sites and of ravs ( figure b ). in agreement with a previous work that analyzed conservation and selection in hcv- a and hcv- b genomes (patino-galindo and gonzalez-candelas, ), dn-ds tended to be higher in the e and e regions, compared to the non-structural portions. a non-negligible fraction of codons showed almost complete conservation: about % displayed ds = and most of these ( %) also had dn = . both rav positions and positively selected sites displayed variable dn-ds values. we note that this is not unexpected for positively selected sites as they were identified using branch-site tests and are thus not selected across the entire hcv phylogeny. the worldwide spread of hcv epidemic strains started recently, in the s- s, but the existence of genetically diverse endemic hcv strains that circulate in specific geographic locations implies a long-standing association of the virus with human populations (simmonds, ) . using an evolutionary model that accounts for variation in the pressure of natural selection across sites and branches, we show that the common ancestor of extant hcv genotypes existed at least years ago, with a lower bound estimate of ∼ years before present. the same approach placed the origin of ehv/chv around ce, with a lower bound at ce. although we applied a selectioninformed approach that accounts for the effect of purifying selection and is relatively robust in the presence of substitution saturation (wertheim et al., ) , we most likely failed to fully correct for time-dependent substitution rate variation . thus, the time of hcv and ehv/chv origin might be underestimated. however, these time estimates rule out the possibility that the use of horse serum to obtain therapeutic antitoxins originated hcv and, more generally, seem to exclude the hypothesis that the human virus derived from a cross-species transmission event from horses (pfaender et al., ; scheel et al., ) . nonetheless, the sampling of ehv is still sparse. the isolation of additional ehv sequences, possibly from a wider geographic range, may date the origin of this virus further back. horse domestication begun around years ago in central asia (outram et al., ) : where ehv found to be older, close contacts between horses and humans may have resulted in zoonotic cross-species transmission or in the acquisition of hcv and ehv from a common source (possibly bat or rodent). active sampling of horse and donkeys worldwide will be required to address this important point. the time frame of hcv origin and the geographic distribution of the endemic strains are not consistent with the possibility that hcv dispersed with humans following the major out-of-africa colonization routes across the old world ( - years ago) (nielsen et al., ) . the dating we estimated for hcv origin instead suggests that the (possible) zoonotic transmission and subsequent spread in human populations occurred in a timeframe when long-distance trade routes were being established. the deepest tmrca for hcv genotypes was obtained for genotype , possibly indicating an asian origin of extant strains. several historical situations may help explain the diffusion of hcv in asia and africa. maritime trading routes across the south chinese sea were already developed in the first millennium bce (hung et al., ) , and regular sea connections between south-east asia and india were established by - bce (pearson, ) . more extensive human movements connecting asia with africa occurred starting around ∼ bce as a result of alexander the great expansion ( - bce) and the full development of the silk road (since bce), this latter comprising maritime and land routes that reached africa (lawler, ) . around this time, indian slaves, mostly women, were regularly imported to egypt (mark, ) . moreover, archaeological evidences suggest that trade circuits connected the east coast of africa with south asia in the first millennium ce . whereas these represent possible transmission routes, it remains unclear how, in a time when parenteral exposure was limited, hcv spread and was maintained in human populations. vertical transmission of hcv is rare and sexual contact is also thought to be a scarcely efficient route for hcv dissemination. it was thus proposed that cultural practices (e.g., circumcision, tattooing, or acupunture) (shepard et al., ; simmonds, ) or natural routes such as arthropod biting (pybus et al., ) might account for endemic hcv transmission. this issue is not addressed by the analyses herein. however, our time estimates place the origin of ehv in a time frame when invasive veterinary procedures and parenteral exposure were most likely rare. although we cannot exclude that some forms of human intervention (e.g., the use of spurs and animal housing in crowded stables) facilitated viral transmission, natural routes are likely to account for the spread of ehv. whether the same mechanisms are responsible for the endemic transmission of hcv and of ehv remains an open question. in this respect, we note that sexually transmitted infections (stis) that disrupt mucosal integrity have been implicated in increased sexual transmission of hcv, at least in high-risk groups (chan et al., ) . stis have probably been common throughout human history and several such diseases are known in horses (dobson and carper, ; samper and tibary, ) . the association between hcv sexual transmission and specific stis might help explain the geographic distribution http://www.sealinksproject.com/ of endemic hcv strains. for instance, lymphogranuloma venereum and granuloma inguinale are confined to tropical and subtropical regions (richens, ; white, ) and their presence may have facilitated the maintenance and diversification of endemic hcv strains in these areas. although the present-day distribution of these stis may not necessarily reflect the historical prevalence of the causative bacteria, the hypothesis of sti-facilitated hcv transmission warrants further consideration. although we found that the tmrca for hcv is greater than the previously inferred time ranges (smith et al., ; pybus et al., ; kapoor et al., ; simmonds, ; li et al., ; lu et al., ) , hcv can be thought of as a recently emerged pathogen, largely postdating the origin of our species. this observation, as well as the presently limited evidence of hepacivirus infection in non-human primates, does not support a long-standing association between these viruses and primates (simmonds, ; scheel et al., ) . the absence of selection signatures at primate cd and ocln, the two molecules that determine the species-specificity of hcv infection, further supports this view. however, as noted above, we also failed to detect positive selection for hcv receptors in glires, even though the wide variety of rodent hepaciviruses suggests that these mammals have been infected by hepaciviruses for a long time (drexler et al., ; kapoor et al., ; scheel et al., ) . thus, results obtained for the hcv receptors are clearly not conclusive and should be regarded as preliminary. indeed, the power of the positive selection tests depends not only on the strength of selection, but also on the number and divergence of the analyzed sequences (anisimova et al., (anisimova et al., , . considering these two parameters, we have most likely a power lower than % although accuracy is higher than % (anisimova et al., (anisimova et al., , . given this premise, it is nonetheless interesting that the only mammalian order showing evidence of selection at cd was chiroptera, which host a wide diversity of hepaciviruses (kapoor et al., (kapoor et al., , drexler et al., ; quan et al., ; corman et al., ; scheel et al., ; walter et al., ) . known bat hepaciviruses share very little similarity to the hcv e protein, and this also applies to the e regions involved in cd binding. this raises the possibility that these animal viruses engage cellular receptors distinct from cd , although previous studies on coronaviruses showed that the same region of the cellular receptor can be bound by viruses that share little sequence or structural similarity in their glycoprotein receptor binding domains (forni et al., ) . also, recent studies have indicated that hcv adaptation to the usage of rodent cd molecules is not necessarily paralleled by changes at the direct binding interface (bitzegeio et al., ) . thus, the receptor preferences of non-human hepaciviruses will require experimental investigation. nonetheless, the observation that the cd positively selected sites in chiroptera are located in a small region that corresponds to the e binding site is consistent with the view that cd -binding hepaciviruses have been infecting bats for a long time. however, we note that cd was also shown to interact with other pathogens. for instance, cd is required for human plasmodium falciparum and rodent plasmodium yoelii sporozoite hepatocyte infection (silvie et al., ) . even though the protein region necessary and sufficient for p. yoelii infection is distinct from that involved in hcv binding (yalaoui et al., ) , we cannot exclude that batinfecting plasmodium parasites (schaer et al., ) bind different regions of cd . also, cd was shown to be important for influenza virus infection (he et al., ) , but the molecular details of the interaction with this virus are unknown. thus, we cannot rule out the possibility that pathogens other than hepaciviruses were responsible for the selection signatures we detected at cd in bats. zoonotic events are commonly associated with bursts of positive selection whereby the pathogen adapts to successfully infect and be transmitted in a new species (longdon et al., ) . a common trade-off of the adaptation to new hosts is the loss or reduction of infectivity in the original host (longdon et al., ) . indeed, hcv infection is now restricted to humans (and to experimentally infected chimpanzees). because the ancestor of hcv has not been identified, knowledge of the molecular events that allowed hcv adaptation to humans remains elusive. we identified several positively selected sites in the e and e regions and some of these were shown to modulate the viral requirement of specific cellular factors (i.e., cd and scarb ). whereas these changes are not expected to represent specific adaptations to the human host, they may confer a selective advantage by increasing viral titers or cell-to-cell spread, a process possibly implicated in the establishment of a persistent infection (ding et al., ) . an interesting possibility is that, during its spread in asia and africa, hcv has adapted in response to the genetic background of distinct human populations, as expected given the distinctive geographic localization of viral genotypes for most of their evolutionary history. a paradigmatic example of this is the distribution of the ifnl /ifnl polymorphism most strongly associated with spontaneous clearance of hcv: the frequency of the protective genotype differs dramatically among populations and this variant explains part of the ethnic variance in the probability to clear hcv infection (o'brien et al., ) . we identified two positively selected sites (h and y in ns a) on the branches of the asian endemic genotypes ( and , respectively). analysis of patients infected with hcv subtype b showed that substitutions at the y sites and, to a lesser extent at the h position, vary depending on the host ifnl /ifnl genotype (akamatsu et al., ; peiffer et al., ) . this observation implies viral adaptation to the host depending on ifnl /ifnl allelic status. although these findings are presently limited to genotype b viruses (akamatsu et al., ; pedergnana et al., ; peiffer et al., ) , they provide a proof of principle for the hypothesis that human genetic diversity exerted a selective pressure on hcv and possibly contributed to the radiation of the seven genotypes. clearly, it remains to be evaluated whether changes at positions y and h exert their effects via modulation of oas binding, as the docking analysis suggests, at least for position . alternatively, other mechanisms may be at play: a lysine insertion between ns a positively selected sites k and n , which are not at the binding interface with oas , regulates pkr and irf- activity (pflugheber et al., ; sumpter et al., ) . in general, the positively selected sites we identified herein represent excellent candidates for future functional studies as they are expected to modulate viral phenotypes. for instance, selected sites in ns b account for genotype differences in terms of sphingomyelin-driven polymerase activation (sakamoto et al., ; weng et al., ) , and the c i change that arose in the common ancestor of genotypes and may help explain the poor response of these genotypes to ifn therapy. exposure to treatment cannot be regarded as the selective force underlying the evolution of the hcv sequences analyzed herein, as all strains derive from treatment-naive subjects. it was recently reported that several ravs have a recent origin and occurred independently on distinct viral lineages (patino-galindo et al., ) . this observation is in line with the notion that substitutions at rav positions can reduce viral fitness (bartenschlager et al., ) and thus fail to be maintained in viral populations or transmission clusters. indeed, by calculating the strength of selective pressure acting on hcv proteins targeted by daas, we observed that most rav positions are subject to a degree of purifying selection similar to non-rav positions. this suggests that ravs do not arise via relaxed selection but rather that these positions are functionally constrained. however, we also found that rav positions are targeted by positive selection more frequently than expected by chance. variation at these sites must therefore be adaptive for the virus, raising the possibility that daa treatment further selects for daa-resistant viruses with high fitness. this pattern contrasts with observations in hiv- infection, as positive selection at codons that confer drug resistance was only observed in patients receiving antiretroviral therapy (de s leal et al., ) . we note, though, that most ravs investigated here were described for genotype and might confer little or no drug resistance to other hcv genotypes. conversely, most rav sites were positively selected on branches different than that leading to genotype . their functional relevance will thus need to be analyzed further. ms conceived the study, with inputs from df and mc. ms and mc supervised the project. rc and cp performed the evolutionary analysis in mammals. df performed the molecular dating analyses. rc, cp, and df performed the evolutionary analysis of hcv phylogenies. ms and df performed the rav analyses. jv and ldg performed the docking analyses. rc and df produced the figures, with input from all authors. up provided support during the bioinformatic analyses. ms, mc, and df wrote the manuscript, with critical input from all authors. the supplementary material for this article can be found online at: https://www.frontiersin.org/articles/ . /fmicb. . /full#supplementary-material time-dependent rate phenomenon in viruses association between variants in the interferon lambda locus and 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subtypes, including the newly sequenced d and e mutagenesis of the fusion peptide-like domain of hepatitis c virus e glycoprotein: involvement in cell fusion and virus entry the ins and outs of hepatitis c virus entry and assembly the evolution and genetics of virus host shifts hepatitis c virus drug resistance-associated substitutions: state of the art summary an algorithm for progressive multiple alignment of sequences with insertions full-length genomes of hepatitis c virus genotype isolates representing subtypes c, d, e, g, h, i, j and k, and two new subtypes m and n, and four unclassified variants reveal ancestral relationships among subtypes alexander the great, seafaring, and the spread of leprosy rdp: detection of recombination amongst aligned sequences detecting and analyzing genetic recombination using rdp global distribution and prevalence of hepatitis c virus genotypes global epidemiology of hepatitis c virus infection: new estimates of age-specific antibody to hcv seroprevalence the effect of genetic structure on molecular dating and tests for temporal signal gene-wide identification of episodic selection detecting individual sites subject to episodic diversifying selection a likelihood approach for comparing synonymous and nonsynonymous nucleotide substitution rates, with application to the chloroplast genome tracing the peopling of the world through genomics ifn-lambda : the paradoxical new member of the interferon lambda family the earliest horse harnessing and milking comparative analysis of variation and selection in the hcv genome comprehensive screening for naturally occurring hepatitis c virus resistance to direct-acting antivirals in the ns , ns a, and ns b genes in worldwide isolates of viral genotypes to protein synthesis and endoplasmic reticulum stress can be modulated by the hepatitis c virus envelope protein e through the eukaryotic initiation factor alpha kinase perk trade, circulation, and flow in the indian ocean world 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virus drug discovery: targeting ns b donovanosis (granuloma inguinale) mutational analysis of the hepatitis c virus e glycoprotein in retroviral pseudoparticles and cell-culture-derived h /jfh chimeric infectious virus particles host sphingolipid biosynthesis as a target for hepatitis c virus therapy disease transmission in horses r s: inferring absolute rates of molecular evolution and divergence times in the absence of a molecular clock statistical tests for detecting gene conversion high diversity of west african bat malaria parasites and a tight link with rodent plasmodium taxa surveying the global virome: identification and characterization of hcv-related animal hepaciviruses patchdock and symmdock: servers for rigid and symmetric docking guidance : accurate detection of unreliable alignment regions accounting for the uncertainty of multiple parameters global epidemiology of hepatitis c virus infection novel procedure for modeling ligand/receptor induced fit effects hepatocyte cd is required for plasmodium falciparum and plasmodium yoelii sporozoite infectivity the origin of hepatitis c virus evolutionary insights into host-pathogen interactions from mammalian sequence data expanded classification of hepatitis c virus into genotypes and subtypes: updated criteria and genotype assignment web resource the origin of hepatitis c virus genotypes less is more: an adaptive branch-site random effects model for efficient detection of episodic diversifying selection viral evolution and interferon resistance of hepatitis c virus rna replication in a cell culture model hepatitis c virus ns a protein interacts with ' , '-oligoadenylate synthetase and inhibits antiviral activity of ifn in an ifn sensitivitydetermining region-independent manner hepatitis c virus ns b targets lipid droplets through hydrophobic residues in the amphipathic helices inhibition of the interferon-inducible protein kinase pkr by hcv e protein structure of the zincbinding domain of an essential component of the hepatitis c virus replicase detection and characterization of homologues of human hepatitis viruses and pegiviruses in rodents and bats in vietnam differential infection patterns and recent evolutionary origins of equine hepaciviruses in donkeys sphingomyelin activates hepatitis c virus rna polymerase in a genotypespecific manner revtrans: multiple alignment of coding dna from aligned amino acid sequences a case for the ancient origin of coronaviruses purifying selection can obscure the ancient age of viral lineages evolutionary origins of human herpes simplex viruses and manifestations and management of lymphogranuloma venereum synthesis of a novel hepatitis c virus protein by ribosomal frameshift hepatocyte permissiveness to plasmodium infection is conveyed by a short and structurally conserved region of the cd large extracellular domain paml : phylogenetic analysis by maximum likelihood bayes empirical bayes inference of amino acid sites under positive selection evaluation of an improved branch-site likelihood method for detecting positive selection at the molecular level a library of infectious hepatitis c viruses with engineered mutations in the e gene reveals growth-adaptive mutations that modulate interactions with scavenger receptor class b type i