key: cord- - sb ipab authors: lennemann, nicholas j.; rhein, bethany a.; ndungo, esther; chandran, kartik; qiu, xiangguo; maury, wendy title: comprehensive functional analysis of n-linked glycans on ebola virus gp date: - - journal: mbio doi: . /mbio. - sha: doc_id: cord_uid: sb ipab ebola virus (ebov) entry requires the virion surface-associated glycoprotein (gp) that is composed of a trimer of heterodimers (gp /gp ). the gp subunit contains two heavily glycosylated domains, the glycan cap and the mucin-like domain (mld). the glycan cap contains only n-linked glycans, whereas the mld contains both n- and o-linked glycans. site-directed mutagenesis was performed on ebov gp to systematically disrupt n-linked glycan sites to gain an understanding of their role in gp structure and function. all n-glycosylation sites of ebov gp could be removed without compromising the expression of gp. the loss of these glycosylation sites significantly enhanced pseudovirion transduction in vero cells, which correlated with an increase in protease sensitivity. interestingly, exposing the receptor-binding domain (rbd) by removing the glycan shield did not allow interaction with the endosomal receptor, npc , indicating that the glycan cap/mld domains mask rbd residues required for binding. the effects of the loss of gp n-linked glycans on ca( +)-dependent (c-type) lectin (clec)-dependent transduction were complex, and the effect was unique for each of the clecs tested. surprisingly, ebov entry into murine peritoneal macrophages was independent of gp n-glycans, suggesting that clec-gp n-glycan interactions are not required for entry into this important primary cell. finally, the removal of all gp n-glycans outside the mld enhanced antiserum and antibody sensitivity. in total, our results provide evidence that the conserved n-linked glycans on the ebov gp core protect gp from antibody neutralization despite the negative impact the glycans have on viral entry efficiency. ing diversification of this region of the protein. in contrast, the glycan cap and mld have extensive sequence diversity between the different ebola virus species. as the names suggest, both the glycan cap and the mld are highly glycosylated, resulting in the majority of the protein being masked by glycans (fig. a) ( ) . the glycan cap contains only n-linked glycan sites (ngs) and, despite generally poor sequence conservation within the glycan cap, these sites are well conserved among ebola viruses, suggesting functional significance (fig. c) . in contrast to the glycan cap, the glycosylation events found in the mld are highly variable between the different viruses and include both n-and o-linked glycans. despite extensive glycosylation, the mld remains highly targeted by neutralizing antibodies ( ) ( ) ( ) . several roles have been attributed to glycans attached to viral glycoproteins. glycans can serve as ligands for ca ϩ -dependent (c-type) lectins (clecs), facilitating viral attachment and internalization in a variety of cell types ( ) . additionally, glycans promote protein folding/stability and virion incorporation of gp, as demonstrated in studies with newcastle disease virus and lassa virus ( , ) . in the case of nipah virus g/f proteins, not only does glycosylation help protein expression, it also decreases membrane fusion efficiency, thereby controlling premature fusion events ( , ) . furthermore, the glycans on human immunodeficiency virus (hiv) gp /gp , nipah virus g/f, hepatitis c virus e /e , and influenza a virus hemagglutinin (ha) protect virions from antibody-mediated neutralization ( ) . despite the high degree of glycosylation found on filovirus gps, the importance of the n-linked glycans on ebov gp to the structure and function of the protein has not been well studied. here, we assess the structural and functional importance of n-linked glycans on ebov gp . to determine the role of gp n-glycans in ebov gp-dependent entry, a library of over individual and combinatorial mutations were made to disrupt the n-linked glycan sites (ngs; n-x-s/t sequons) within the gp subunit. we generated ngs mutations in gp of both gp and the mld-deleted gp (gp ⌬muc) (for details of all mutations, see table s in the supplemental material). ngs mutants were expressed in hek t cells and pseudotyped onto vsv⌬g-egfp (expressing enhanced green fluorescent protein). the relative expression levels of wild-type (wt) or ngs mutant gp were determined by assessing the gp-to-vsv matrix ratios present in cell supernatants by dot blot analysis. in addition, the transduction efficiencies of the pseudovirions were assessed in vero cells. initial studies were performed to disrupt the seven ngs within the gp core of gp ⌬muc and gp. reduced levels of glycosylation on mutant gps that were pseudotyped onto vsv were apparent in immunoblots, evidenced by faster migration of the mutant proteins (see fig. s a in the supplemental material). the expression of wt and mutant gps in supernatants and the transduction of vero cells mediated by these virions are summarized in fig. s . notably, removing more than glycans from the gp core significantly decreased the expression of gp ⌬muc and concomitantly reduced its transduction. however, all glycan sites within the gp core could be removed ( g) with no effect on gp expression and a significant enhancement in transduction over that of the wt, suggesting that the mld provides stability that allows the removal of glycans from the gp core ( fig. a) . furthermore, these mutant gps were incorporated into purified virions at a rate equivalent to the incorporation of the wt (fig. s b) . since complete deglycosylation of the core domains of gp did not negatively impact the expression of gp or the transduction of pseudotyped virions, we systematically combined n-linked glycan mutations in the mld with our g mutant. surprisingly, we were able to disrupt all ngs within gp without affecting expression ( gm g) ( fig. a ; see also fig. s e in the supplemental material). comparison of the gm g mutant and peptide n-glycosidase f (pngase f)-treated gp with immunoblot analysis confirmed that the gm g mutant lacked all n-linked glycans (fig. s c ). pseudovirion transduction of vero cells was significantly enhanced in all gp mutants that lacked the n glycan ( fig. b; fig. s d ), and is shown in light gray, rbd is shown in red, and mld structure that has not been solved is represented as a gray sphere. pbd id csy. (b) linear model of ebov gp. the disulfide bond between gp and gp is indicated, as well as the locations of n-linked glycans (marked with "ys") in the gp and - domains, and the known protease cleavage sites are noted. sp, signal peptide; rbd, receptor-binding domain; mld, mucin-like domain; ifl, internal fusion loop; hr and - , heptad repeats and ; tm, transmembrane domain. (c) alignment of predicted n-linked glycan sites within the gp core of the five ebola virus species. n-x-s/t sequons are highlighted with a black background. lennemann et al. virion transduction was further enhanced by the removal of all ngs within the gp core ( g) or the core and mld ( gm g). consistent with our findings with gm g, the elimination of n-glycans solely within the mld (gpm g) also led to a modest but consistent increase in transduction, suggesting that ngs in both the core and mld decreased transduction. in total, these results indicated that n-linked glycans are not required for entry into vero cells but, rather, decrease the efficiency of entry. gp n-glycans do not affect virion attachment to vero cells. as extensive n-linked deglycosylation of gp led to greater transduction, we sought to identify which step(s) in ebov entry were affected by the loss of n-linked sugars. removing the gp n-glycans within the gp core ( g) or throughout gp ( gm g) did not affect the binding of pseudovirions to the cell surface (see fig. s , top, in the supplemental material) and the binding of all virions tested was decreased to similar levels (~ % reduction) in the presence of a calcium chelator, egta (fig. s , middle). this finding was not unexpected, since ebov entry into vero cells has been shown to be mediated by the phosphatidylserine receptor, tim- , which binds to virion-associated phosphatidylserine in a calcium-dependent manner ( ) . removal of gp n-glycans imparts catb independence and increases protease sensitivity. endosomal processing of ebov gp by cathepsin b (catb) is an important step in ebov entry that results in the removal of both the mld and glycan cap, exposing the rbd ( ) ( ) ( ) ( ) . additionally, it has been shown that disruption of the n ngs (t a mutation) leads to catb independence in the absence of the mld ( ) . as the inclusion of a similar mutation (t v) in our glycoprotein mutants consistently led to enhanced transduction, we assessed whether the increase in transduction by our mutants correlated with increased catb independence. pseudovirion entry mediated by gp ⌬muc, gp, the g mutant, containing a fully deglycosylated glycan cap, and the gpm g mutant containing a mld that was fully deglycosylated for n-linked glycans were abrogated by treating vero cells with the catb inhibitor ca- , which profoundly blocked catb activity (fig. s a ). these data indicated that these gps were dependent upon catb processing for subsequent transduction steps (fig. a ). vsv pseudotyped with the native glycoprotein (vsv-g) served as a control in these studies and was completely resistant to the drug. as anticipated, the mutant with a mutation of t in the absence of the mld was significantly less sensitive to ca- ( fig. a , t v⌬muc); however, we did not observe complete catb independence as has been previously reported ( ) , possibly due to a val rather than ala substitution at this site. assessment of the t v mutant in the context of gp also demonstrated partial ca- independence. the catb independence of g was similar to that of the t v mutant, and removing all n-linked glycans in gp ( gm g) enhanced catb independence slightly more. our observed trend of enhanced transduction with increasing elimination of gp n-linked glycans was more robust than the trend of catb independence with our mutants. as glycosylation is known to protect from proteases ( ), we determined whether removing glycans increased the protease sensitivity of gp. previous work has shown that treatment with g/ml thermolysin (thl) results in an~ -fold increase in gp-mediated transduction associated with complete removal of the glycan cap and mld ( , ) . to initially assess thl sensitivity, we evaluated the transduction efficiency of gp and gm g following treatment with increasing concentrations of thl up to a high concentration ( g/ml). gm g was dramatically more sensitive to low concentrations of thl than gp, providing evidence that the deglycosylated gp was more readily proteolytically processed (fig. s b ). given these results, a panel of ngs mutants was evaluated for the relationship between entry and sensitivity of deglycosylated gp to low concentrations ( . g/ml) of protease (fig. b) . a correlation between the number of mutations and the thl sensitivity was observed. the results from g and gpm g indicated that the removal of glycans from the core has a greater effect on protease sensitivity, possibly due to the large number of o-glycans still present in the mld. however, gm g showed the greatest protease sensitivity, suggesting an additive effect when the mutations of g and gpm g were combined. these findings indicated that n-glycans in the glycan cap and, to a lesser extent, in the mld control the efficiency of proteolytic processing, thereby regulating subsequent steps in entry, such as binding to the endosomal receptor, npc . removal of glycans shielding the rbd does not allow for npc binding. enzymatic removal of the glycan cap and mld allows interaction of gp with the endosomal receptor npc ( , ) . consequently, we postulated that exposing the rbd by disrupting ngs within the gp core and mld might enhance the binding of gp to npc . in an npc binding assay, a soluble form of the second luminal domain (c loop) of npc , which directly interacts with residues in the rbd of ebov gp ( ) , was used to bind vsv pseudovirions. treatment of gp with thl, which is commonly used to mimic gp cleavage by catb/-l in removing the mld and glycan cap ( , ) , resulted in binding of c loop over a wide range of concentrations (fig. c) . however, exposure of the rbd through deglycosylation ( gm g) did not lead to binding of the npc c loop above the background level, indicating that loss of the n-linked glycans on gp does not abrogate the need for endosomal proteolysis that is required for receptor interactions. this result suggests that, while n-glycans on gp may interfere with the ability of the rbd to interact with this endosomal receptor, glycan interference is not the sole explanation for the inability of gp to bind to npc . domain-specific n-linked deglycosylation alters c-type lectin utilization. clecs, which mediate gp-dependent entry, are expressed on a wide variety of ebov target cell types, including macrophages, dendritic cells, and hepatic cells, and each binds to specific glycan moieties ( ) ( ) ( ) ( ) ( ) . to better understand the role of ebov gp n-linked glycans in clec-dependent tropism, we determined the receptor utilization of mutants that lack n-linked glycans on the gp core ( g), the mld (gpm g), or both ( gm g). constructs expressing myc-tagged clecs were transfected into poorly permissive hek t cells. to enhance expression, the ectodomains of clecs dc-sign and human mgl (hmgl) were fused to the transmembrane and cytoplasmic domain of l-sign (resulting in constructs l dc-sign and l hmgl, respectively). all constructs expressed well in transfected cells (fig. s ) . in these experiments, transfection of a tim- -expressing plasmid served as a transduction control, since tim- -dependent entry into endosomes is gp independent and should be independent of the gp glycosylation status ( , ) . consistent with this, the entry of all pseudovirions was similarly enhanced by transfection of tim- (fig. a) . while all five clecs enhanced the transduction of wt gp between -and -fold, the impact of the loss of gp n-glycans on transduction varied depending on the clec examined (fig. b to f). entry mediated by l-sign, l dc-sign, and lsectin was highly dependent on n-glycans present throughout gp ; however, lsectin-dependent entry was less dependent on n-glycans present in the gp core (fig. b to d) . asialoglycoprotein receptor (asgpri)-mediated entry was completely independent of n-glycans in the gp core. while the loss of ngs in the mld resulted in a trend of decreased transduction, this did not achieve statistical significance (fig. e) . furthermore, this clec was able to mediate entry independently of gp n-glycans. n-linked deglycosylation had a minimal impact on gp-mediated entry into cells expressing l hmgl (fig. f) . the results obtained with asgpri and l hmgl were consistent with previous reports indicating that ligands for these clecs are present in o-linked glycans present in the mld or, potentially, in n-linked glycans on gp . clec-expressing hek t cells were also used to evaluate the impact of n-linked glycans on our mld deletion mutant, gp ⌬muc. in these studies, we compared the transduction of gp ⌬muc to that of g⌬muc. while our g⌬muc mutant was poorly expressed and resulted in low levels of transduction compared to the transduction of gp ⌬muc (see fig. s in the supplemental material), the levels of g⌬muc transduction were about % of the levels observed with gp and sufficient to allow transduction studies to be performed (fig. s ) . all five clecs enhanced gp ⌬muc transduction, increasing entry by . -to fold ( fig. b to f) . in all cases, the loss of five of the n-linked glycans ( g⌬muc) on the glycan cap resulted in a decrease in transduction, with the reduction being more modest for transduction mediated by l dc-sign, asgpri, and l hmgl. as the better transduction of g⌬muc into cells expressing l dc-sign, asg-pri, and l hmgl cannot result from interactions with mld glycans, this enhancement must be due to either the two remaining intact n-glycans on the gp core at n and n or the two n-glycans on gp . additionally, the differences in g⌬mucmediated transduction observed between l-sign and l dc-sign indicate that these clecs have different ligand specificities, despite both binding to high-mannose oligosaccharides. n-linked glycans on gp are not required for entry into macrophages. macrophages are major, early targets during filovirus infections, and others have suggested that clecs may be important for entry into these cells ( ) ( ) ( ) . previous work has shown that murine mgl and signr , the murine homolog of dc-sign, are expressed on peritoneal macrophages ( , ) . we used our most extensively deglycosylated mutants to determine the role of n-linked and o-linked glycans in entry into these cells. since vsv is highly sensitive to the type i interferon response ( ), resident peritoneal cells were isolated from ifnar Ϫ/Ϫ mice for these studies. in addition, these mice lacked tim- , which eliminated the possibility for entry in epithelial cells that may have contaminated the preparations. cells were treated for days with murine macrophage colony-stimulating factor (m-csf) and their phenotypes were determined prior to use. eighty-five percent of adherent cells from the peritoneal cavity were cd b ϩ /f ϩ , indicative of matured macrophages (see fig. s in the supplemental material). these cells were highly permissive for vsv pseudotyped with gp (fig. ) . entry mediated by gm g was en-hanced~ -fold over entry mediated by the wt, which is more consistent with our vero cell data than with our clec utilization data. furthermore, macrophages supported entry mediated by the g⌬muc mutation, although this was~ -fold decreased compared to the results for the wt. these findings demonstrated that neither o-or n-linked glycans on gp are absolutely required for entry into peritoneal macrophages. removal of n-linked glycans enhances antiserum sensitivity. our studies indicated that the most deglycosylated ebov gp mutants were expressed at wt levels and provided the highest levels of transduction into vero cells and peritoneal macrophages. nonetheless, the conservation of the glycan sites, particularly in the gp core sequences, across the species suggested that positive selection for these sites was occurring. therefore, our deglycosylated ebov gps were evaluated for their sensitivity to antiserum neutralization, since deglycosylation may increase the exposure of potential ebov gp epitopes. purified anti-ebov igg from convalescent cynomolgus macaques that were vaccinated prior to challenge with ebola virus (a gift from john m. dye, usamriid) was incubated with vsv pseudovirions that were normalized for the amount of matrix protein. little to no enhancement of antiserum sensitivity was observed with a mutant lacking three glycans in the gp core; however, the removal of six glycans from the glycan cap increased the neutralization sensitivity more than -fold ( fig. a and c) . in contrast, vsv pseudotyped with the marburg virus gp was not neutralized by anti-ebov igg. complete deglycosylation of the gp core ( g) led to a further increase in antiserum sensitivity ( fig. b and c) . since the mld is a major target of neutralizing antibodies ( - ), we anticipated that the removal of n-glycans from this domain would enhance anti-ebov igg sensitivity. surprisingly, gpm g was no more sensitive to neutralization than wt gp (fig. b) , indicating that ngs in the mld had little to no positive or negative effect on neutralization by the pooled anti-ebov igg. consistent with this, removal of all n-linked glycans on gp ( gm g) did not enhance antibody neutralization beyond that observed with deglycosylation of gp core ( g). we found similar but less pronounced neutralization sensitivity with pooled sera collected from mice surviving challenge with mouse-adapted ebov (see fig. s in the supplemental material). ebov gp is highly glycosylated, yet limited studies have investigated the role of these sugar chains on gp function to date. in this report, we determined the effects of removing the n-linked glycans present on the ebov gp subunit by examining the impact on protein expression/stability, viral entry, and antiserum/ antibody sensitivity. surprisingly, the elimination of all n-linked glycans on gp had no effect on the expression levels of gp, suggesting that, in the presence of the mld, n-linked glycans attached to gp are not critical for gp folding. however, as our future studies to test our deglycosylated gps in the context of recombinant filoviruses to verify our findings are warranted. the loss of gp ngs significantly decreased the utilization of four of the five c-type lectins known to be used by ebov gp for entry, while it enhanced ebov transduction into vero cells and peritoneal macrophages. the enhanced levels of transduction were associated with enhanced sensitivity to thermolysin cleavage, but deglycosylation did not alleviate the need for proteolysis prior to npc binding. finally, the loss of highly conserved glycans on the core of gp increased virus sensitivity to antibody neutralization. therefore, we propose that the strong conservation of individual glycosylation sites in the gp core across the ebola virus genus results, at least in part, from selective pressures to protect the virus against immune responses, despite the fact that these sites collectively can have a negative impact on gp-dependent entry. modeling n-linked glycans onto the gp ⌬muc structure suggests that the highly conserved rbd of ebola viruses is effectively masked and protected by glycan cap glycans attached to wellconserved ngs, likely protecting the rbd against selective immune pressures, as previously proposed ( , , , , , ) . however, we provide the first experimental evidence to support this hypothesis. our results suggest that there are neutralizing epitopes in the gp core that are normally masked by n-glycans. surprisingly, the removal of n-glycans from the mld, which is highly targeted by neutralizing antibodies ( - ), did not affect antiserum sensitivity. neutralizing epitopes within the mld that do not appear to be affected by the loss of n-linked glycans may be obstructed by the large number of o-glycans attached to the mld. alternatively, neutralizing epitopes within the mld may target o-linked glycan sequons. since filovirus hemorrhagic fevers are acute and often lethal infections in primates, it is likely that antibody-driven positive selection for ngs surrounding conserved regions occurs in nonprimate reservoirs, such as bats. thus, it is important to understand filovirus infection and persistence in bat populations. a role for clecs in ebov gp-dependent entry has been firmly established by others ( , , , ) . we have shown for the first time that the removal of n-glycans from either the glycan cap ( g) or mld (gpm g) dramatically decreases the utilization of dc-sign, l-sign, and lsectin. surprisingly, these same clecs enhanced the transduction mediated by gp ⌬muc, despite this form of the protein sharing exactly the same pattern of ngs as gpm g. another surprising finding was that hmgl utilization was largely unaffected by the loss of gp n-glycans. this provided indirect evidence that hmgl-dependent ebov transduction is strongly dependent on o-linked glycans in gp. however, despite the apparent utilization of o-linked glycans by hmgl, the transduction mediated by gp ⌬muc was greatly enhanced by the expression of this clec ( ) . in combination, these two sets of results suggest that deletion of the mld alters the species of glycans present on gp . such a conclusion is consistent with previous work showing that glycosylation differences exist between gp and the small, soluble form of gp (sgp), which shares the same ngs with gp ⌬muc ( ). more glycan processing was found on sgp, indicated by the higher percentage of galactose residues and lower percentage of high-mannose glycans. since the types of glycans present on proteins are largely dependent on the environment in which the protein is produced ( ) , it is possible that the cytotoxicity associated with overexpression of gp ( , ) or the large number of o-glycans present changes the glycosylation machinery in the er/golgi. previous work has shown that macrophages are important cellular targets during the early days of filovirus infection ( ) . the effective transduction of murine peritoneal macrophages by n-glycan-deficient gp -bearing pseudovirions, along with our findings that hmgl-dependent enhancement of transduction was relatively independent of the presence of gp n-glycans, supports the idea proposed by others ( ) that ebov entry into macrophages may be mediated by this clec. however, additional mechanisms of ebov entry into macrophages are also likely to be important, particularly in light of our findings that gm g pseudovirions have~ % better transduction of macrophages than gp, yet gm g transduces hmgl-expressing cells at~ % of the transduction level of the wt. we and others have identified that tim family members mediate virus uptake into cells by interacting with phosphatidylserine on the surface of virions ( , ) , and it is possible that this uptake mechanism is important for ebov entry into macrophages. future studies need to explore the role for phosphatidylserine receptors during in vivo filovirus infection. our findings that deglycosylation of gp pseudovirions enhances the transduction of vero cells and peritoneal macrophages provides evidence that the n-glycans on gp decrease the efficiency of the entry process. the removal of glycans masking the rbd enhanced proteolytic processing of gp but did not result in the ability of unprocessed ebov gp to bind the c loop of npc , consistent with an earlier study demonstrating that the removal of the mld did not unmask the rbd for npc interaction ( ) . this result suggests that there are residues that are critical for npc binding, such as f ( ) , that are masked by the glycan cap/mld polypeptide rather than being concealed by the heavy glycan shield. thus, it is likely that the increased sensitivity of our deglycosylated mutants to proteolytic processing results in more efficient transit through the endosomal compartments, leading to greater transduction efficiency. in these studies, we found that protease sensitivity and catb dependence are independently controlled within gp . for instance, gpm g, which retains the n glycosylation site, was completely dependent on catb but had enhanced thermolysin sensitivity. in contrast, the t v mutant was found to be partially catb independent but no more sensitive to thermolysin than wt gp. it was previously hypothesized that mutation of residue t , which eliminates the n-glycan at n , allows endosomal proteases other than catb to process gp within the endosome ( ) . extrapolating from these studies, we hypothesized that systematic deglycosylation would yield an ebov gp that was progressively more catb independent. however, this was not the case; extensive deglycosylation beyond loss of the n glycan sequon did not further alleviate dependence on catb but did increase thermolysin sensitivity. additionally, mutants with mutations of n and t were shown to display different protease sensitivities, suggesting that the glycan is not the only factor in determining catb dependence ( ) . previous work has shown that cellular receptors influence the intracellular trafficking and endosomal fate of viruses ( ) . ebov entry requires a specific intracellular compartment that requires catb, the hops complex, and npc ( , , , , ) . however, similar to our catb-independent ebov mutants, not all filoviruses require catb cleavage for entry ( ) . therefore, deglycosylated mutants with distinct protease dependence and sensitivity profiles may be useful in the further characterization of the endosomal compartments that allow for the efficient entry of filoviruses. pseudovirion matrix and glycoprotein quantitation. equal volumes of cell supernatants containing pseudovirions from three independent stocks were lysed in native lysis buffer (phosphate-buffered saline [pbs] with . % np- ) and then passed through a dot blot apparatus onto nitrocellulose (whatman). wells were washed ϫ with pbs before being blocked in pbs with % nonfat milk for h. dot blots were incubated with the mouse anti-vsv matrix monoclonal antibody (mab) h (a gift from douglas lyles) and the human anti-ebov gp mab kz (a gift from erica saphire and dennis burton) diluted in pbs with % nonfat milk and . % tween- overnight at °c. by using separate irdyeconjugated secondary antibodies (abs) (li-cor) directed toward the primary abs, we were able to detect both vsv matrix protein and ebov within a single well for each sample. the signals were visualized and quantified using an odyssey imaging station and image studio software (li-cor), which has been shown to be more sensitive and quantitative than enhanced chemiluminescence ( ) . thermolysin sensitivity assay. vsv pseudovirions, normalized for gp expression, were incubated with g/ml, -fold serial dilutions starting with g/ml, or a single concentration of . g/ml of thermolysin (thl) at °c for min. the reaction mixtures were immediately placed on ice and diluted -fold in growth medium containing m phosphoramidon (sigma), a thl inhibitor. thl-treated pseudovirions were evaluated for vero cell transduction. gfp-positive cells were analyzed by flow cytometry. antibody/antiserum neutralization assay. serial dilutions of fractionated igg pooled from convalescent, vaccinated cynomolgus macaques challenged with ebov (gift from john m. dye, usamriid) or pooled convalescent sera from mice challenged with mouse-adapted ebov (gift from gene olinger, usamriid) were incubated at °c for min with pseudotyped vsv bearing the glycoproteins studied and normalized for the amount of matrix protein. the mixtures were diluted -fold in growth medium and added to vero cells plated in a -well format. gfp-positive cells were enumerated with flow cytometry. the relative antiserum sensitivities were calculated as the reciprocals of the % infective concentration (ic ) values determined with graphpad prism . supplemental material for this article may be found at http://mbio.asm.org /lookup/suppl/doi: . /mbio. - /-/dcsupplemental. table s , pdf file, . mb. figure s , pdf file, . mb. figure s , pdf file, . mb. figure s , pdf file, . mb. figure s , pdf file, . mb. figure s , pdf file, . mb. figure s , pdf file, . mb. figure s , pdf file, . mb. figure s , pdf file, . mb. text s , doc file, . mb. ebola haemorrhagic fever antigenic analysis of strains of ebola virus: identification of two ebola virus serotypes characterization of filoviruses based on differences in structure and antigenicity of the virion glycoprotein proposal for a revised taxonomy of the family filoviridae: classification, names of taxa and viruses, and virus abbreviations covalent modifications of the ebola virus glycoprotein structure of the ebola virus glycoprotein bound to an antibody from a human survivor impact of ebola mucin-like domain on antiglycoprotein antibody responses induced by ebola virus-like particles influences of glycosylation on antigenicity, immunogenicity, and protective efficacy of ebola virus gp dna vaccines epitopes involved in antibody-mediated protection from ebola virus virus glycosylation: role in virulence and immune interactions carbohydrate modifications of the ndv fusion protein heptad repeat domains influence maturation and fusion activity the role of single n-glycans in proteolytic processing and cell surface transport of the lassa virus glycoprotein gp-c n-glycans on the nipah virus attachment glycoprotein modulate fusion and viral entry as they protect against antibody neutralization n-glycans on nipah virus fusion protein protect against neutralization but reduce membrane fusion and viral entry the secret life of viral entry glycoproteins: moonlighting in immune evasion role of the phosphatidylserine receptor tim- in enveloped virus entry biochemical and structural characterization of cathepsin l-processed ebola virus glycoprotein: implications for viral entry and immunogenicity the primed ebolavirus glycoprotein ( -kilodalton gp , ): sequence and residues critical for host cell binding endosomal proteolysis of the ebola virus glycoprotein is necessary for infection role of endosomal cathepsins in entry mediated by the ebola virus glycoprotein a forward genetic strategy reveals destabilizing mutations in the ebolavirus glycoprotein that alter its protease dependence during cell entry biological roles of oligosaccharides: all of the theories are correct ebola virus entry requires the host-programmed recognition of an intracellular receptor small molecule inhibitors reveal niemann-pick c is essential for ebola virus infection a novel mechanism for lsectin binding to ebola virus surface glycoprotein through truncated glycans structural basis for distinct ligandbinding and targeting properties of the receptors dc-sign and dc-signr dc-sign and l-sign: the signs for infection structural basis for selective recognition of oligosaccharides by dc-sign and dc-signr crystal structure of the carbohydrate recognition domain of the h subunit of the asialoglycoprotein receptor tim-family proteins promote infection of multiple enveloped viruses through virion-associated phosphatidylserine an analysis of features of pathogenesis in two animal models of ebola virus infection human macrophage c-type lectin specific for galactose and n-acetylgalactosamine promotes filovirus entry c-type lectins dc-sign and l-sign mediate cellular entry by ebola virus in cis and in trans the role of signr and the beta-glucan receptor (dectin- ) in the nonopsonic recognition of yeast by specific macrophages generation of mice deficient for macrophage galactose-and n-acetylgalactosamine-specific lectin: limited role in lymphoid and erythroid homeostasis and evidence for multiple lectins convenient assay for interferons the envelope glycoprotein of ebola virus contains an immunosuppressive-like domain similar to oncogenic retroviruses lsectin interacts with filovirus glycoproteins and the spike protein of sars coronavirus the asialoglycoprotein receptor is a potential liver-specific receptor for marburg virus identification of n-glycans from ebola virus glycoproteins by matrix-assisted laser desorption/ionisation time-of-flight and negative ion electrospray tandem mass spectrometry differences in the glycosylation of recombinant proteins expressed in hek and cho cells the erk mitogenactivated protein kinase pathway contributes to ebola virus glycoproteininduced cytotoxicity full-length ebola glycoprotein accumulates in the endoplasmic reticulum characteristics of the cellular receptor influence the intracellular fate and efficiency of virus infection ebola virus entry requires the cholesterol transporter niemann-pick c filoviruses require endosomal cysteine proteases for entry but exhibit distinct protease preferences quantitative analyses reveal the importance of regulated hdmx degradation for p activation we thank richard roller (university of iowa) and john m. dye (usam-riid) for critical evaluation of the manuscript. we would like to thank erica saphire and dennis burton (scripps institute) for providing the kz mab, gene olinger (usamriid) for providing the convalescent mouse sera, and john m. dye (usamriid) for providing the cynomolgus macaque anti-ebov igg.the work described in the manuscript was supported by the following nih/niaid grants: r ai (w.m.), t ai (n.l.), and r ai (k.c.). the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. key: cord- -kqcx lrq authors: ladner, jason t.; beitzel, brett; chain, patrick s. g.; davenport, matthew g.; donaldson, eric; frieman, matthew; kugelman, jeffrey; kuhn, jens h.; o’rear, jules; sabeti, pardis c.; wentworth, david e.; wiley, michael r.; yu, guo-yun; sozhamannan, shanmuga; bradburne, christopher; palacios, gustavo title: standards for sequencing viral genomes in the era of high-throughput sequencing date: - - journal: mbio doi: . /mbio. - sha: doc_id: cord_uid: kqcx lrq thanks to high-throughput sequencing technologies, genome sequencing has become a common component in nearly all aspects of viral research; thus, we are experiencing an explosion in both the number of available genome sequences and the number of institutions producing such data. however, there are currently no common standards used to convey the quality, and therefore utility, of these various genome sequences. here, we propose five “standard” categories that encompass all stages of viral genome finishing, and we define them using simple criteria that are agnostic to the technology used for sequencing. we also provide genome finishing recommendations for various downstream applications, keeping in mind the cost-benefit trade-offs associated with different levels of finishing. our goal is to define a common vocabulary that will allow comparison of genome quality across different research groups, sequencing platforms, and assembly techniques. v iruses represent the greatest source of biological diversity on earth, and with the help of high-throughput (ht) sequencing technologies, great strides are being made toward the genomic characterization of this diversity ( ) ( ) ( ) . genome sequences play a critical role in our understanding of viral evolution, disease epidemiology, surveillance, diagnosis, and countermeasure development and thus represent valuable resources which must be properly documented and curated to ensure future utility. here, we outline a set of viral genome quality standards, similar in concept to those proposed for large dna genomes ( ) but focused on the particular challenges of and needs for research on small rna/ dna viruses, including characterization of the genomic diversity inherent in all viral samples/populations. our goal is to define a common vocabulary that will allow comparison of genome quality across different research groups, sequencing platforms, and assembly techniques. despite the small sizes of viral genomes, complications related to limited rna quantities, host "contamination," and secondary structure mean that it is often not time-or cost-effective to finish every genome, and given the intended use, finishing may be unnecessary ( ) . therefore, we have used technology-agnostic criteria to define five standard categories designed to encompass the levels of completeness most often encountered in viral sequencing projects. each viral family/species comes with its own challenges (e.g., secondary structure and gc content); therefore, we provide only loose guidance on the depth of sequence coverage likely required to obtain different levels of finishing. in reality, a similar amount of data will generate genomes with different levels of finishing for different viruses. to alleviate any reliance on particular aspects of the different sequencing technologies, we have made two assumptions that should be valid in most viral sequencing projects. the first assumption is a basic understanding of the genomic structure of the virus being sequenced, including the expected size of the genome, the number of segments, and the number and distribution of major open reading frames (orfs). fortunately, genome structure is highly conserved within viral groups ( ) , and although new viruses are constantly being uncovered, the discovery of a novel family or even genus remains relatively uncommon ( ) . in the absence of such information, the defined standards can still be applied following further analysis to determine genome structure. the second assumption is that the genetic material of the virus being described can be accurately separated from the genomes of the host and/or other microbes, either physically or bioinformatically. depending on the technology used, it is critical that the potential for crosscontamination of samples during the sample indexing/bar coding process and sequencing procedure be addressed with appropriate internal controls and procedural methods ( ) . for a summary of the proposed categories for whole-genome sequencing of viruses, see fig. and table . the "standard draft" category is for whole shotgun genome assemblies with coverage that is low and/or uneven enough to prevent the assembly of a single contig for Ն genome segments. genomes in this category are likely to result from samples with low viral titers, such as clinical and environmental samples, or to be those containing regions that are difficult to sequence across (e.g., intergenic hairpin regions) ( ) . to distinguish standard drafts from targeted amplification of partial viral sequences, standard drafts should contain at least contig for each genomic segment and should be prepared in a manner that allows the possibility of sequencing the vast majority of a virus's genome. to avoid the inclusion of small pieces of genomes as "drafts," there needs to be some type of minimum cutoff for breadth of coverage. therefore, we suggest that at least a majority (Ն %) of the genome be present for a set of sequences to be considered a draft genome. high quality (hq). genomes should be considered high quality if no gaps remain (i.e., a single contig per genome/segment), even if one or more orfs remain incomplete due to missing sequence at the ends of segments. an hq genome can often be achieved with modest levels of ht sequencing coverage (~ to ϫ) or through sanger-mediated gap resolution of an sd. coding complete (cc). the "coding complete" category indicates that in addition to the lack of gaps, all orfs are complete. this level of completion is typically possible with high levels of ht sequencing coverage (Ͼ ϫ) or may require the use of conserved pcr primers targeting the ends of the segments. complete. a genome is complete when the genome sequence has been fully resolved, including all non-protein-coding sequences at the ends of the segment(s). this is typically achieved through rapid amplification of cdna ends (race) or similar procedures. finished. this final category represents a special instance in which, in addition to having a completed consensus genome sequence, there has been a population-level characterization of genomic diversity. typically this requires~ to , ϫ coverage (see below). this provides the most complete picture of a viral population; however, this designation will apply only for a single stock. additional characterizations will be necessary for future passages. population-level characterization. ht sequencing technologies provide powerful platforms for investigating the genetic diversity within viral populations, which is integral to our understanding of viral evolution and pathogenesis ( , ) . population-level characterization requires very high levels of ht sequencing coverage ( , ); however, the exact level will depend on the background error profiles of the sequencing technology and the desired level of sensitivity. as an example, wang et al. ( ) determined that for pyrosequencing data,~ ϫ coverage is necessary to identify minor variants present at % frequency with . % confidence, and~ , ϫ coverage is needed for variants with a frequency of . %. targeted amplification of the viral genome is often necessary to achieve these coverage requirements. due to the modest sequence lengths of most ht technologies, the state of the art for population-level analysis has been the characterization of unphased polymorphisms. however, single-molecule technologies, with maximum read lengths of Ͼ kb, are opening the door for complete genome haplotype phasing ( ) . identification of contaminants or adventitious agents. after isolation, viruses are often maintained as stocks, which are propagated within host cells in tissue culture and thus amplified and preserved for future use. despite careful laboratory practices, it is possible for these stocks to become contaminated with additional microbes. contaminating microbes are often detrimental to subsequent applications such as vaccine development or the testing of therapeutics, making it imperative to monitor the purity of viral stocks. ht sequencing provides a powerful method for not only detecting the presence of contaminants within a sample but also for identification and characterization of any contaminants. the level of sequencing required for contamination analysis is dependent on the desired sensitivity, with more sequencing required to ensure detection of contaminants present at very low levels. for most approaches, hq-level sequencing should be sufficient. depending on the intended applications, analysis may need to be repeated after further passaging to ensure that no additional contaminants have been introduced. description of novel viruses. despite the rapidly growing collection of viral sequences, the description of novel viruses is likely to remain an important aspect of viral genome sequencing ( , , ) . this is true in part because viruses evolve rapidly and are capable of recombining to form novel genotypes ( , ) . it is also true that most of the viruses that are currently circulating remain uncharacterized ( ) . particularly lacking are representatives from groups that are not currently known to infect humans or organisms of economic importance. it would be imprudent, however, to continue to ignore these uncharacterized reservoirs of diversity, because it is difficult to predict the source of future emerging diseases ( ) ( ) ( ) . additionally, with the current suite of primarily sequence similarity-based pathogen identification tools, the ability to detect novel pathogens is wholly dependent on highquality reference databases ( ) . there is a trend toward requiring a complete genome sequence when a description of a novel virus is being published, and we agree that this is a good goal; however, the amount of time and resources required to complete the last to % of a viral genome is often cost and time prohibitive for projects sequencing a large number of samples, and in most cases the very ends of the segments are not essential for proper identification and characterization. therefore, for the majority of viral characterization projects, we recommend, at a minimum, a cc genome. this will ensure a complete description of the viral proteome and will allow accurate phylogenetic placement. molecular epidemiology. one of the most common and important applications for viral genomes is in the study of viral epidemiology, which encompasses our understanding of the patterns, causes, and effects of disease. early studies of molecular epidemiology targeted small pieces of viral genomes; however, this type of analysis is likely to miss important changes elsewhere in the genome. therefore, there has been a strong focus in recent years toward the sequencing of "full" viral genomes. institutes such as the broad institute and the j. craig venter institute (jcvi) have been instrumental in breaking ground in the collection of large numbers of good-quality viral sequences. their newly identified genomes typically fall within our cc category. this is likely to remain the gold standard for studies involving a large number of genome sequences, especially when some samples come from lowtiter clinical samples, often necessitating amplicon-based sequencing methods. cc genomes allow for interrogation of changes throughout the coding portion of the viral genome and often include partial noncoding regions. in the absence of highthroughput race alternatives, the time and resources required to complete hundreds or thousands of genomes are likely to continue to outweigh the potential information gained from completing the terminal sequences. countermeasure development. advancements in our capabilities to sequence viral genomes are changing the way we counteract global pandemics and acts of bioterrorism. there are two important aspects of countermeasure development that can benefit strongly from the availability of genome sequences and ht sequencing data: the detection of the infectious agent and the treatment of the disease caused by the agent. taxonomic classification and detection through dna/rna-based inclusivity assays (i.e., using techniques such as pcr to detect the presence of a pathogen) can be designed using fragmented and incomplete genomes (e.g., sd and hq sequences). fully resolved orfs (cc) further enable the development of immunological assays, such as enzyme-linked immunosorbent assays (elisa) and immunofluorescence assays (ifa), for protein-based detection, and obtaining a complete genome opens the door to a plethora of additional downstream applications, including the design of exclusivity tests, the establishment of reverse genetics systems, and the design of robust forensics protocols. however, for effective development and testing of animal models, therapeutics, vaccines, and prophylactics, it is necessary to obtain a complete picture of the variability present within both the challenge stock and postinfection populations, thereby necessitating finished genomes. in these medical applications, it is also important to demonstrate the absence of adventitious agents. in addition to standardizing the vocabulary of viral genome assemblies, it is also critical for researchers to routinely provide raw sequencing reads. without these, it is impossible for others to independently verify the quality of an assembly. data repositories such as genbank already provide a platform for depositing ht sequencing reads, but this is not a requirement for the submission of a genome, nor is this option typically utilized. wider analysis of data will ultimately result in higher-quality assemblies. it is worth considering broader implementation of a wiki-like, crowdsourcing strategy to genome assembly, similar to the annotation strategies that have been adopted for specific genomes of high interest ( , ) . this approach would allow multiple parties to work on genome assembly and annotation at the same time and would provide instant updates for the entire community to evaluate and utilize in their own research. our primary goal here is to initiate a conversation. the rate at which viral genomes are being sequenced is only going to increase in the coming years, and without some standardization, it will be impossible for these valuable resources to be utilized to their full potential. we present these categories as a starting point, with the goal of adjusting and refining them over time as our capabilities and needs continue to change. crystal ball. the viriosphere: the greatest biological diversity on earth and driver of global processes metagenomic analysis of coastal rna virus communities the search for meaning in virus discovery genome project standards in a new era of sequencing next generation sequencing of viral rna genomes . virus taxonomy. ninth report of the international committee on taxonomy of viruses human viruses: discovery and emergence double 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middle east respiratory syndrome coronavirus: the "knowns" and "unknowns relationship between domestic and wild birds in live poultry market and a novel human h n virus in china computational tools for viral metagenomics and their application in clinical research web apollo: a web-based genomic annotation editing platform pseudomonas genome database: improved comparative analysis and population genomics capability for pseudomonas genomes key: cord- -b czrq authors: ielasi, francesco s.; alioscha-perez, mitchel; donohue, dagmara; claes, sandra; sahli, hichem; schols, dominique; willaert, ronnie g. title: lectin-glycan interaction network-based identification of host receptors of microbial pathogenic adhesins date: - - journal: mbio doi: . /mbio. - sha: doc_id: cord_uid: b czrq the first step in the infection of humans by microbial pathogens is their adherence to host tissue cells, which is frequently based on the binding of carbohydrate-binding proteins (lectin-like adhesins) to human cell receptors that expose glycans. in only a few cases have the human receptors of pathogenic adhesins been described. a novel strategy—based on the construction of a lectin-glycan interaction (lgi) network—to identify the potential human binding receptors for pathogenic adhesins with lectin activity was developed. the new approach is based on linking glycan array screening results of these adhesins to a human glycoprotein database via the construction of an lgi network. this strategy was used to detect human receptors for virulent escherichia coli (fimh adhesin), and the fungal pathogens candida albicans (als p and als p adhesins) and c. glabrata (epa , epa , and epa adhesins), which cause candidiasis. this lgi network strategy allows the profiling of potential adhesin binding receptors in the host with prioritization, based on experimental binding data, of the most relevant interactions. new potential targets for the selected adhesins were predicted and experimentally confirmed. this methodology was also used to predict lectin interactions with envelope glycoproteins of human-pathogenic viruses. it was shown that this strategy was successful in revealing that the fimh adhesin has anti-hiv activity. , -glcnac-␤ is the preferential binding receptor for fimh-ld. fimbriated e. coli expressing fimh is able to bind uroplakins ia and ib, which are two glycoproteins of the apical urothelial plaques carrying high-mannose glycans ( ) and the main urothelial receptors for type fimbriae ( ) . candidiasis is a fungal infection caused by the adhesion of candida yeast species to host cells. candida albicans and c. glabrata are commensal yeasts of the human gastrointestinal tract, but they are also the major causes of opportunistic candida infections in susceptible hosts ( , ) . the als (agglutinin-like sequence) family is the best-characterized adhesin family of c. albicans ( ) . the binding of als proteins to human epithelial tissues has been attributed to the n-terminal part of the protein, which contains tandem immunoglobulin-like domains that are able to adhere to host proteins ( , ) . among the best-studied als proteins are als p and als p, both of which are responsible for the mediation of cellular adhesion to a broad range of ligands, such as fibronectin (fn), laminin, and collagen iv, as well as fibrinogen and gelatin ( ) ( ) ( ) ( ) . recently, we showed that n-als p has a lectin-like activity, since it interacts with fucose-containing carbohydrates ( ) . despite the vast amount of information available on als-mediated adhesion, there is still little data available on the als molecular binding mechanisms mediated by host carbohydrates. another prominent yeast adhesin family is the epa (epithelial adhesin) family, since it has been reported to be mainly responsible for the adherence of c. glabrata to human cells ( ) ( ) ( ) . the n-terminal domains of epa proteins (n-epa-p) do not share sequence homology with the adhesins of the als family, which are not present in c. glabrata. rather, n-epa proteins are classified as pa -like lectins ( ) ( ) ( ) because of their sequence homology and structural similarity to the pa fragment of the anthrax toxin protective antigen. they mediate adherence to human epithelial and endothelial cells by recognizing glycans containing terminal galactose residues ( ) and show the highest affinity for the thomsen-friedenreich (t or tf) antigen (gal␤- , -galnac), which likely mediates n-epa-p adherence to highly glycosylated proteins such as mucins ( ) . although we recently demonstrated that wild-type n-epa p binds to fn from human plasma ( ) , no experimental data on the potential host glycoprotein binding receptors of epa p or other c. glabrata adhesins are available. in viral host-pathogen interactions, lectin carbohydratebinding agents (cbas) can bind to viral envelope glycans and thereby inhibit the entry of, e.g., the human immunodeficiency virus (hiv) into host cells ( ) ( ) ( ) ( ) . a strong feature of lectin cbas as potential antiviral drugs is their multifarious mechanism of action. they can inhibit viral replication and cell-cell transmission of viral particles and induce partial deletion of the envelope glycan shield, with consequent exposure of immunogenic epitopes to neutralizing antibodies. moreover, these antiviral compounds do not need to be internalized by host cells to be effective against the virus ( ) . various mannose-specific lectins endowed with potent antiretroviral activity have been discovered. they have been isolated from cyanobacteria, actinobacteria, algae, higher plants, and worms ( ) ( ) ( ) . antiviral activity of lectin cbas against viruses other than hiv with high-mannose glycosylated envelope proteins, such as influenza virus, herpesvirus, hepatitis c virus, dengue virus, marburg virus (marv), severe acute respiratory syndrome (sars) coronavirus, measles virus, and ebola virus, has been discovered ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . in this report, we present a novel glycan array-based network strategy aimed at identifying the potential biological binding receptors for adhesin lectins. first, the glycan determinants of the lectins are determined from the experimentally evaluated glycanbinding specificities of the lectins by glycan array analysis. next, the glycosuitedb glycoproteomic database of the unicarbkb platform ( , ) is searched for these determinants to obtain a set of human glycoproteins expressing the glycan determinants that are of interest and are considered potential targets for lectin recognition. by performing additional queries of the glycosuitedb database, these potential target glycoproteins can be further linked to the cell types on which they are present, the tissues and body systems, and the disease state (if applicable). finally, the network is analyzed in order to profile the potential adhesin binding receptors in the host with prioritization, based on experimental binding data, of the most relevant interactions. new potential targets for the selected adhesins were predicted and experimentally confirmed. the constructed networks are referred to as lectin-glycan interaction (lgi) networks. we explored this strategy and constructed lgi networks for two classes of pathogenic microbial adhesins that are characterized by lectin-like properties, i.e., the bacterial upec adhesin fimh-ld, as well as the yeast c. albicans and c. glabrata adhesins (i.e., als p and als p and epa p, epa p, and epa p, respectively). the lgi networks constructed were corroborated by comparison with interaction data available in the literature, and some links in the networks were experimentally confirmed by quantitative lectin-glycan interaction analysis (by surface plasmon resonance [spr] and atomic force microscopysingle-molecule force spectroscopy [afm-smfs]). this networking strategy was also used to predict lectin interactions with envelope glycoproteins of human-pathogenic viruses. it was successful in anticipating the molecular recognition of the hiv gp envelope protein by the bacterial adhesin fimh-ld, and it led to the discovery of the anti-hiv activity of fimh. lgi network has been modeled as a weighted, undirected graph composed of a set of nodes (i.e., lectin, glycan, glycan determinant, glycoprotein, disease, tissue, body system) and a set of edges connecting the pair of nodes (see materials and methods and the supplemental material). an edge connecting the two nodes can represent biochemical interactions, as well as biological and/or hierarchical associations between the nodes. with each node, we associate a node relevance, and with each edge, we associate an edge relevance, which indicates the relevance of the interaction between the connected nodes. the proposed representation allows visualization of the network in multiple ways in order to (i) highlight a set of glycoproteins as promising receptor candidates that were obscured in the huge amount of data in the lgi network and (ii) predict the potential binding receptors for several lectins. in our specific case, the network involved a combination of both experimental data and the data from the publicly available glyco-suitedb database. the binding specificity and strength of microbial lectins (fimh-ld, n-als, and n-epa) were experimentally determined by glycan array screening. the measured binding strengths were exported into a spreadsheet, whereas those glycan structures and determinants that are recognized by the adhesins were queried in the glycosuitedb database. the query provided a list of hundreds of potential target proteins (cases of glycan deter-minants not attached to any protein were also taken into account), and they were further linked to cell types, tissues, body systems, and diseases (if available) through additional queries in the same database. the resultant nodes and edges/links (associations) provided the lgi network definition in its initial state, where all of the nodes and edges were considered equally relevant; a relevance quantification stage followed. the relevance quantification process was achieved in two steps. we first associated each edge with a relevance depending on the type of its connecting nodes (i.e., protein-glycan) according to the proposed weights (see equations to in the supplemental material) as a function of the lectin binding intensity measured via glycan array screenings. in the second step, the node's relevance was estimated by using a network analysis centrality measure ( ) that involves both the number of edges (associations) connected to the node and their weights (lectin binding intensity) (see the supplemental material). the influence of the number of edges (or associations) versus the binding intensity is regulated by the parameter (see the supplemental material for a discussion of its influence), and its value can be interactively adjusted during the visualizations to better study the different associations (see fig. and ). determination of the relevance values allowed us to rank or establish priorities among the potential targets, enabling a convenient visualization of the most important proteins (or disease, glycan, etc.) as the bigger nodes and the most important associations (i.e., protein-disease, disease-glycan) as thicker edges/links. in particular, three complementary visualization types were used, namely, hierarchical, circular, and cluster views ( ) . this allowed an interactive analysis of the resultant network in great detail, highlighting the most relevant nodes in the overall network, as well as within specific clusters, at the same time (see the supplemental material). the hierarchical view (see fig. , , and ) allowed linking of the experimentally determined lectin specificities (i.e., the glycan determinants) with the potential receptors (human or viral glycoproteins) and then linking of these glycoproteins to cell types/tissues and body systems. the circular view allowed easy identification of all of the relevant nodes according to their size, and the most important associations were made as the thickest arrows (see fig. and ). the cluster view revealed cluster-like visualizations ( ) , thus allowing us to explore groups of nodes belonging to the same local cluster (see fig. and ). some glow and shading effects can be (optionally) applied in order to distinguish between the groups of nodes according to their types within the same cluster (see fig. a and b and a and b). the results of network analysis both exhibited known associations and predicted novel ones, proposing novel candidates (i.e., glycoproteins and glycan determinants) as promising targets for later adhesin binding experiments. notably, the same network analysis can be performed to predict the binding receptors of any carbohydrate-binding protein in other host organisms once the experimental carbohydrate-binding characterization of the lectin has provided the molecular binding strength and the association data. the lgi network of e. coli fimh-ld with predicted human receptors. the fimh lgi network ( fig. ) was generated by using fimh-ld experimental glycan array data that are perfectly consistent with the previously published microarray results ( ) and reflect the high specificity of fimh-ld for high-mannose glycans, i.e., the oligomannose , oligomannose , mannotriose, and mannopentaose structures. a hierarchical view of the network shows the predicted receptors (human glycoproteins) for the fimh adhesin and their classification in terms of body tissues and systems (fig. a) . a circular view of the same lgi network (fig. b) focuses on the glycan determinants that are bound by fimh-ld, the potential glycoprotein ligands, and also lists the diseases correlated with the expression of the glycosylation determinants indicated. only a few of the predicted glycoprotein binding receptors are associated with specific disease states (mainly malignancies), which is the case for several immunoglobulins (such as, ige, igm, and iga), cg-a, cg-b, the epidermal growth factor receptor (egfr), and psap (see table s in the supplemental material for a list with abbreviated protein names). among these, immunoglobulin mu (igm) and the egfr are reported by the network to be particularly relevant, while tf is highlighted in the group of non-disease-associated glycoproteins. the latter are all linked to the determinants containing either man␣- , -man or man␣- , -man as terminal moieties. the network displays other human glycoproteins, such as umod, cd e/cd , c , t-pa, and plg, for which a link with fimh or type fimbriae has already been established (see discussion) and for which any defined relationship with the bacterial adhesin has not been found yet, for example, bace , vwf, and gamma interferon. the cluster view of the network shows the identification of the host tissues that could be potentially targeted by the adhesin and the disease states that could promote the binding of the adhesin to the predicted glycoprotein receptors (fig. ) . two main clusters can be identified in the cluster views of the network. cluster contains glycoproteins and diseases linked to the tissues of the urogenital system, and cluster is associated with the hemic system. in cluster are proteins such as umod, cd e/cd , cg-a, and cg-b, which are found in several tissues of the urogenital tract and constitute a relevant subcluster. the latter two glycoproteins (cg-a, cg-b) are particularly highlighted in the second view of the network (fig. b ) and are associated with choriocarcinoma, hydatidiform moles, and diabetes mellitus (fig. , cluster b) . the same cluster also contains glycoproteins that are present in a healthy uterus and ovary. cluster is mainly characterized by the presence of links between immunoglobulins, namely, iga, igm, ige, and igg / / , and different forms of blood cell cancer, such as myelomas and waldenström's disease (fig. , cluster b). the lgi network of candida n-als-p and n-epa-p with predicted human receptors. (i) modeling and visualization of the lgi network of n-als and n-epa lectins from candida. the hierarchical view of the overall epa/als network (fig. ) is extended, since it is based on the glycan specificities of six adhesins. mucins (several proteins containing the abbreviation muc) appear as the most relevant human glycoproteins. in the circular view of the epa network ( fig. a) , mucins are mainly connected to the glycan determinants gal␤- , -galnac, gal␤- , -glcnac, and gal␤- , -glcnac␤- , [gal␤- , -]galnac. these three determinants, together with the sialyl-t antigen, are prioritized on the basis of the related lectin binding intensities, especially in the case of the gal␤- , -containing moieties (large arrow width), and the number of connections, i.e., for gal␤- , -glcnac, the largest node size in the network but unlabeled because of the low binding intensity (the label was filtered out in the program for better visualization of the more relevant nodes [see the supplemental material]). in the als network (fig. b) , mucins are mainly linked to the glcnac␤- , -gal determinant, which is also the most relevant (high binding table s in the supplemental material. (b) circular view of the network (generated with the lgi network algorithm). the glycan determinants queried in the unicarbkb database, the human glycoproteins bearing the glycan determinants, and the related diseases are depicted as blue, green, and red nodes, respectively. connections between nodes are depicted with blue arrows (to indicate the determinants expressed on each glycoprotein) or green arrows (to indicate the diseases associated with the altered glycosylation of each glycoprotein). a closeup view of the network is shown on the left. the size of each node (and the font size of the node label) in the circular view is proportional to the number of connections with other nodes and the associated lectin binding intensity, which were experimentally determined by glycan array analysis; the arrow thickness is correlated to the lectin binding intensity. ielasi et al. intensity) in this network. other interesting links with glycoproteins that have already been discussed in the literature (k-casein, egfr, cd , ltf, tf, igm) or need to be confirmed (tumor necrosis factor alpha, psgl- , cd , cd , vwf, bace , lamp / , plg) emerge from the two networks ( fig. ) (see discussion). among the als glycan determinants, the di-lacnac determinant (gal␤- , -glcnac␤- , -gal␤- , -glcnac␤- , -man) emerges and is based on experimental high binding intensity (fig. b ), while the fuc␣- , -bearing structures, especially fuc␣- , -gal (largest unlabeled node), are relevant because of their large number of connections. several disease nodes are present in both networks, such as cystic fibrosis, diverticulosis, and different forms of cancer, while adenocarcinoma is highlighted in the epa network and is associated mainly with mucins ( fig. a) . four main clusters are identified in the epa/als network (fig. ) . the most relevant links are found in cluster , and it involves mucins; the diseases associated include lung and intestinal adenocarcinomas (muc, muc , muc , muc , muc ac/ b), diverticulosis (muc , muc , muc , muc ), cystic fibrosis (muc, muc ), chronic bronchitis, breast cancer (muc ), and kartagener's syndrome (bronchiectasis) (muc). in cluster , the glycoproteins associated with the urogenital system, such as choriogonadotropin (cg), and some related malignant states, such as choriocarcinoma and diabetes mellitus, are identified. in cluster , glycoproteins cd and cd are strongly associated with wiskott-aldrich syndrome (was) (cd ), together with different forms of leukemia (cd /cd ) affecting cells of the immune system and also coexisting with hiv infections (cd ). another important link is established by glycan determinants, which are not attached to any protein (mainly fucosyl-capped oligosaccharides for als adhesins and nonreducing terminal gal-glcnac moieties for epa adhesins). they are connected to chronic kidney failure, gangliosidosis, fucosidosis, and sialidosis. additionally, associations were found in cluster , mainly linking myelomas with several immunoglobulins and other plasmatic glycoproteins and glycoproteins of the secretion system. (ii) validation of als/epa predicted interactions: binding of n-als p to fn and laminin and of n-epa p to mucin. glycan array screening was performed to determine the glycan specificities and affinities of n-als p (see fig. s a to d in the supplemental material). spr experiments confirmed the binding of n-als p to glcnac (fig. a) , as well as the binding of n-als p to fn and laminin (fig. a) , two proteins of the extracellular matrix (ecm) also recognized by n-als p ( ) . the n-als p dissociation constant at the equilibrium state (k d ) for the bovine serum albumin (bsa)-glcnac glycoconjugate was estimated in the micromolar systems (orange nodes), and diseases (red nodes) associated with glycan determinants (generated with the lgi network algorithm). the tissue clusters are depicted with a pink glow (a), the system clusters are depicted with an orange glow (a), the protein clusters are depicted with a green glow (b), and the disease clusters are depicted with a red glow (b). two main clusters are present in the cluster view: cluster , containing glycoproteins and diseases linked to tissues of the urogenital system, and cluster , which is mostly associated with the hemic system. closeup views of these clusters (b) are shown to indicate the most relevant nodes (lower panels). in panel a, the nodes related to the tissues are important, since they are the most connected ones (the larger the number of connections, the larger the node and font size of the label). in panel b, the disease and protein nodes are most important, since the node (and font) sizes are proportional to the lectin binding intensities, and they are the most connected ones. the thickness of the arrows is proportional to the associated lectin binding intensity. range (k d ϭ Ϯ m). the k d constants were Ϯ m for the n-als p-fn interaction and Ϯ m for the n-als plaminin interaction. the affinity of n-epa p for mucin was also determined by spr (fig. b) , and the k d constant was . Ϯ . m. in order to verify that the observed interactions were specifically mediated by galactose-containing glycans attached to fn and mucin, binding inhibition experiments were performed (fig. b ). the binding of n-epa p to mucin could be blocked by lactose in a concentration-dependent manner, but it was not affected by the presence of glucose. the lgi network of e. coli fimh-ld with viral envelope glycoproteins. (i) modeling and visualization of the fimh-ld lgi network with viral envelope glycoproteins. the fimh-viral network displays the connections of fimh-ld and the glycans (recognized by the lectin on the glycan array) to several viral glycoproteins through glycan determinants (fig. a ). connected viruses include hiv, sendai virus, friend murine virus, marv, and influenza a virus. the gp envelope glycoprotein of hiv is included in the network, and this interaction was explored further (see below). interestingly, fimh-ld is also linked to influenza a virus hemagglutinin (ha) (from insect cells and chicken isolates) and to the marv envelope glycoprotein (gp; from monkey iso-lates). this may anticipate the possible interactions between the ld of fimh and the envelope of these critical viral pathogens. the circular view (fig. b ) predicts the most relevant adhesion epitopes for fimh on viral glycoproteins, such as influenza a virus ha and marv gp, that are connected to several high-affinity glycan epitopes (especially to man␣- , -[man␣- , -]man). (ii) validation of e. coli fimh-ld predicted interactions with viral envelope glycoproteins. the affinity of the ld of fimh for gp from hiv- (iii b ) and hiv- (yu ) was kinetically characterized by spr (fig. a ). fimh-ld shows very low association rate constants (k on values to m Ϫ · s Ϫ ), but also very low dissociation rate constants (k off values Յ Ϫ s Ϫ ). the difference between the calculated k d parameters for the two interaction couples is related mainly to the -fold higher k on in their binding with baculovirus-derived gp . this discrepancy can be explained after considering that protein glycosylation processes are different in baculovirus and cho expression systems. to confirm that the recognition of gp by fimh-ld is mediated by the glycan moieties on the viral protein, spr inhibition experiments were performed with man␣- , -man and man␣- , -man, which mimicked different moieties of the viral protein glycosylation sites. fifty percent inhibitory concentrations (ic s) table s in the supplemental material. ielasi et al. were calculated by using the response values at equilibrium (r eq ) at a constant fimh-ld concentration (fig. a) . the interaction of fimh-ld with the viral envelope protein could be blocked in a dose-dependent manner by both disaccharides that exhibited a similar inhibitory concentration but a slightly higher specificity for the ␣- , -linked mannobiose. an additional confirmation of the interaction of fimh-ld with gp was obtained by afm-smfs. adhesion force histograms resulting from fimh-ld-gp (yu ) unbinding events showed a broad range of interactions occurring at forces that are relatively high for a noncovalent bond (see fig. s in the supplemental material). unbinding force distributions with an average peak force of Ϯ pn (see fig. s c ) were obtained. this interaction force value is comparable with that observed for fimbrial tip adhesion to bsa-mannose ( ) . the fimh-ld-gp interactions could be blocked almost completely in the presence of ␣- , -mannobiose (see fig. s b ). fimh-ld anti-hiv activity was assessed in different in vitro lgi network strategy developed pointed out a limited number of human glycoproteins, mainly situated in the urogenital and hemic systems of the human body, as the potential binding receptors for the fimh adhesin. only a few of the predicted links have been experimentally confirmed in the literature, and these deliver the first proof of the validity of our strategy for the prediction of lectin binding receptors. type fimbriated e. coli is able to recognize tamm-horsfall glycoprotein, also known as uromodulin (umod) ( ) . the binding to this protein was confirmed by the network strategy (fig. ) . umod is a urinary defense factor, since it can prevent the interaction of bacterial fimbriae with the uroplakin receptors through its single glycan chain. fimh is also involved in the invasion of uroepithelial cells because of its direct binding to highmannose glycans of integrins ␤ and ␣ ( ). other integrin subunits could also be involved in the entry of fimh-expressing bacteria into host cells ( ) . one of these subunits could be the ␣ integrin, which was also predicted in the network, together with the ␤ integrin as a ␣ /␤ heterodimer (cd e/cd ) (fig. ) that was already reported to mediate host cell invasion through the pathogenic bacterium staphylococcus aureus ( ) . type fimbriae also have a role in complement-dependent bacterial internalization ( ) . synergy between the expression of fimh on bacterial fimbriae and binding of the c complement protein increased e. coli internalization through epithelial cells of the urinary tract. this effect was abrogated by mannose or in the absence of the mannose-specific adhesin on the bacterial fimbriae. the same synergistic effect could not be correlated with the expression of p fimbriae, which would be carrying the papg adhesin. these data and the presence of c in the fimh lgi network suggest that the glycosylated complement protein, scavenged by and directly associated with the fimh lds, could facilitate bacterial entry into uroepithelial host cells. a genetically engineered pseudomonas aeruginosa strain that expresses type fimbriae can specifically adhere to breast cancer cells overexpressing the egfr and block the egfr signaling pathway ( ) . the effect of fimbriae on receptor signaling has been described as mannose sensitive. indeed, the lgi network suggests the recognition by fimh-ld of the egfr from carcinoma cells. we can thus hypothesize that fimh-ld may also mediate bacterial adhesion and have the same effect on growth factor receptor signaling. the fimh-ld lgi network displays several immunoglobulin families that are associated with different malignant diseases, including iga from myeloma cells (fig. ) . immunoglobulin preparations containing secretory iga or iga from myeloma cells can induce mannose-dependent agglutination of type fimbria-expressing e. coli ( ) . weaker agglutination of these cells can also be achieved through certain igm isotypes and prevented by the presence of d-mannose. other connections are established in the network between the fimbrial adhesin and disease states affecting protein glycosylation. for example, a link with diabetes mellitus is corroborated by the observation that female patients with this disease are more susceptible to utis than are healthy patients because of the greater adherence of type fimbriated e. coli to bladder cells ( ) . the lgi network of candida als and epa adhesins n-als p has been shown to interact with fucose-containing glycans that are present in blood group antigens and preferentially with antigen h type ( ) . therefore, we performed glycan array screening to also determine the carbohydrate-binding specificity of n-als p (see fig. s a to d in the supplemental material). among the strongest binders, we found long chains of repeated lacnac (gal␤- , -glcnac), and a micromolar affinity was determined for the interaction of the adhesin with bsa-glcnac. glcnac constitutes of a part of the type lacnac (gal␤- , -glcnac) and type lacnac (gal␤- , -glcnac) structures that build the scaffold for blood group h and lewis-type units ( ) . some human pathogens use the glcnac residue as a binding receptor; e.g., the fimbrial adhesin f -g of enterotoxigenic e. coli binds to n-acetylglucosamine-presenting receptors on the microvilli of the intestinal epithelium of ruminants ( ) . our n-als p glycan array data, together with data related to n-als p and the epa adhesins, were used for the generation of lgi networks (fig. to ) . these networks revealed that a large set of potential binding receptors that may be recognized by these adhesins through their interaction with carbohydrates are displayed on human cells or present in body fluids. the most interesting ones are summarized in table s in the supplemental material. several glycan determinants are linked to mucins ( fig. and ) . mucins are the main constituents of the extracellular secreted mucus and cell surface glycocalyx, which is rich in the galnac-containing structures commonly used by many pathogens for adhesion. c. albicans adhesion to human cells has been previously linked to mucins ( , ) . the binding of epa adhesins to mucin-type o-glycans has also been described, especially the ability of epa p, epa p, and epa p to recognize the t antigen ( , , ) . this disaccharide constitutes of the core structure of mucin-type o-glycans, and it is mainly exposed on the surface of colon cancer tissues in a truncated form. the t and sialyl-t antigens are also found on breast cancer cells ( ) . the three epa proteins are linked in the network to the mucins found in several tissues or fluids, such as saliva, lung tissue, stomach tissue, mammary gland tissue, milk, colon tissue, and uterine tissue. a consistent fraction of these mucins is associated with diseased states, i.e., colon adenocarcinoma (muc , muc , muc , muc a/b/c), breast and uterine cancers (muc ), and lung diseases, which may cause bronchiectasis (muc) (fig. ) . these mucins carry the t antigen, the sialyl-t antigen, or both. a link between epa adhesins and the colon mucosa has been demonstrated by showing adhesion of saccharomyces cerevisiae cells expressing epa p and epa p to human colorectal carcinoma (caco- ) cells ( ) . a connection between the epa proteins and cd (leukosialin)/cd (receptor-type tyrosine-protein phosphatase c) is also present in the lgi network (fig. ) , and this is justified by the presence of the t antigen that is linked to leukemia-and hiv-associated cd and to was-associated cd , as well as other glycans related to a healthy condition. cd and cd receptors are commonly expressed on leukocytes ( ) . they are involved in lymphocyte activation and may present altered glycosylation in hiv-infected cells ( ) ( ) ( ) or in diseases such as leukemia and was. lysosome-associated membrane glycoproteins and (lamp , lamp ), which are as well predicted, are expressed in macrophages and are essential for the fusion of phagosomes and lysosomes during phagocytosis ( ) . they may also present altered glycosylation in leukemia cells. epa p is able to mediate yeast adhesion to human leukemic macrophages and healthy pbmcs, in order to trigger cytokine expression and induce phagocytosis ( ) . the inhibitory effect of phagocytosed c. glabrata on the fusion between phagosomes and lysosomes in macrophages is an efficient immune evasion strategy ( ) . possibly, epa p adhesion to human pbmcs is mediated by cd / . this is consistent with cd / -mediated adherence of actinomyces naeslundii, expressing gal␤- , -gal(nac)-specific adhesins, to polymorphonuclear leukocytes or promyelocytic leukemia cells ( ) and the involvement of cd in the induction of cytokine production, as for the t-antigen-specific jacalin (plant lectin) that recognizes cd on t lymphocytes and induces cytokine secretion ( ) . epa proteins are linked to fn (fig. ) ; i.e., epa p and epa p are linked to fn of fibroblasts by lacnac-terminated n-glycan branches, suggesting again the specificity of only these two epa adhesins for the ecm protein. the link of n-epa p with fn is corroborated by our recent results ( ) , which show that the adhesin domain is able to bind fn with submicromolar affinity in a carbohydrate-sensitive manner. on the other hand, a connection between als proteins and fn was not found in the network, although we demonstrated here and previously that n-als p and n-als p both recognize fn ( ) . the network shows that als and epa adhesins may bind to ceruloplasmin (cp), (sero)transferrin (tf), and lactotransferrin (or lactoferrin [ltf]) ( fig. and ). the first two are blood plasma glycoproteins, while the latter is present in different exocrine secretions. all of them are involved in iron metabolism. c. albicans is able to acquire iron from tf ( ) . thus, c. albicans iron acquisition from host tf may be als mediated, as is iron acquisition from human ferritin ( ) . inhibitory effects of tf, igm, and another components of cohn fraction iv (most probably ceruloplasmin) on the growth of c. glabrata have been discussed ( ) , as has the capacity of a special ltf formulation to impair yeast adherence to vaginal epithelial cells ( ) . the egfr and cadherin- (cd ) are also indicated in the network as potential ligands of both als proteins (fig. ) . for the first glycoprotein, there is agreement with a study describing the egfr and her as interaction receptors for als p ( ) . the interaction of als p with these receptors triggers their autophosphorylation, which leads to endocytosis of c. albicans by host cells. it has also been demonstrated previously that als p binds to n-and e-cadherins, which are present on endothelial and epithelial cells, respectively ( ) . as shown in table s in the supplemental material, additional reported experimental results confirmed some als lgi network nodes, such as for chondroitin sulfate proteoglycan , -casein, and cg-a/b. fn, laminin, and mucin recognition by candida adhesins. using spr, we characterized the interaction of n-epa p with mucin (fig. b) . a comparable k d value was found for the n-epa p-fn interaction ( ) . the specific binding inhibition by lactose corroborated the specificity of glycan recognition by n-epa p. these data confirmed, for the first time, the ability of n-epa p to bind mucins. as shown in the lgi network, adhesion of epa adhesins to mucins is extremely relevant in the context of host adherence and is mediated by multiple o-glycan determinants. the binding of n-als p to ecm glycoproteins, such as fn and laminin, was also characterized by spr (fig. a) . compared to n-als p-fn binding ( ) and the n-epa p-fn interaction ( ) , the n-als p-fn interaction was Ͼ and -fold weaker, respectively. also, the n-als p-laminin dissociation constant was higher than that previously determined for n-als p. the fulllength als and als adhesins showed significant binding to fn and laminin ( ) . it seems that the interaction of als proteins with fn is determined mainly by the protein-protein interactions. indeed, fucose failed to inhibit n-als p binding to the glycoprotein, while glucose and galactose only partially inhibited it ( ) . additionally, we could not find a connection between als adhesins and fn in the lgi network. recently, it was shown that nt-als - p recognizes the c-terminal sequence of the fibrinogen ␥ peptide ( ) . these results indicate that protein-protein interactions may dominate the binding of n-alsp to fn. the lgi network of the e. coli fimh-ld adhesin with viral envelope proteins and the anti-hiv activity of fimh-ld. several viral pathogens-such as hiv, influenza virus, sars virus, hepatitis c virus, marv, and ebola virus-contain highmannose glycans attached to the envelope proteins ( ) . to predict interactions of fimh-ld with viral envelope glycoproteins, we generated a viral lgi network by employing fimh-ld glycan array data and the viral glycoproteomic data available in the gly-cosuitedb database. the network suggested the recognition of different viruses, including three human-pathogenic viruses (fig. ) . one of these predictions, i.e., the link with hiv, was experimentally and thoroughly validated by using biomolecular and cellular assays. the gp -fimh-ld spr affinity and inhibition results are compatible with a prevalent recognition of oligomannose glycans on cho-derived gp . the related k d ( nm) is, indeed, very similar to the affinity of the ld for the same carbohydrate structure (~ nm) ( ) . by interacting with this longer oligosaccharide, the lectin would recognize the terminal ␣- , -linked mannose units rather than the man-␣- , -man-␤- , -glcnac, which is only recognized in a terminal configuration. on the other hand, we can explain the higher affinity for baculovirus-derived gp by hypothesizing the binding to a mixture of "long" and "short" glycans, including oligomannose / . the ic s reflect the actual preference of fimh-ld for ␣- , -linked mannosides over the ␣- , -linked oligosaccharides. envelope glycans are becoming increasingly promising targets for lectins as viral entry inhibitor proteins ( , ) . the kinetic analysis results of fimh-ld-gp interactions revealed association constants to orders of magnitude lower than those for other antiviral lectins, such as cyanovirin (cvn), actinohivin, and griffithsin (grft), but also, the dissociation rates are slower than those for the other antiviral lectins ( , ) . accordingly, the resulting equilibrium constants for the same interactions were much higher than the k d of the best-characterized lectins cvn and grft, but they are still in the nanomolar range. this affinity of fimh-ld for the viral envelope protein justifies the moderate in vitro antiviral activity of the lectin. comparable anti-hiv activity values were obtained for fimh-ld, and this in cellular assays using cd ϩ t lymphocytes, tzm-bl cells, and pbmcs against cxcr -using (x ) and ccr -using (r ) hiv- strains (fig. b ). especially the pbmc data are very relevant, since these are the real target cd ϩ t cells for hiv. envelope glycoproteins from marv and influenza a virus are also connected in the network as potential targets for fimh-ld (fig. a) . the mannose-binding lectin, a protein belonging to the innate immune system and specific for mannose-containing glycans, is able to hamper both influenza a virus ( , ) and marv ( ) infectivity and spreading in vitro, which represents the body's first line of defense against infection. moreover, cvn activity against ebola virus, marv ( ) , and influenza a virus h n isolates ( ) and antiviral properties of mannose-specific lectins from algae against influenza a virus ( ) have been demonstrated. on this basis, it would be worth further exploring the antiviral properties of fimh-ld not only against hiv but also against other viral species, especially influenza a virus and marv, in order to evaluate the possibility of the development of a multivalent drug that would be effective in the prophylaxis for different pathogens. conclusions. we developed and successfully employed a novel lgi network in which the carbohydrate-binding properties of the e. coli adhesin fimh and candida adhesins from the epa and als adhesin families were explored. this lgi network strategy, based on glycan array screening results and a glycoprotein database inquiry, allowed the profiling of potential glycoprotein binding targets for the selected adhesins. we confirmed some of these potential targets either experimentally or by reference to the literature. additionally, this strategy was adapted for the prediction of adhesin-viral glycoprotein interactions, validated by the discovery of anti-hiv activity of fimh. this study shows the potential of the new strategy for the study of some microbial and viral interactions. the lgi networks presented were based on the glycan array results coming from the database of the consortium for functional glycomics (cfg). experimental data could also be extracted from other databases (such as the glycosciences laboratory database, imperial college london). care should be taken to check the quality of the extracted data. although the glycosuite database of unicarbkb used is a curated glycoproteomic database, it has some limitations, since it is not exhaustive. we expect future improvements in the content of this database and, consequently, an enhancement of the lgi network's prediction quality and accuracy. the lgi network strategy could be connected to additional bioinformatic resources that could support the validation of the lgi network generated, for example, the sugarbind database (http://sugarbind.expasy.org), which provides information on known carbohydrate sequences to which pathogenic organisms or substances (bacteria, toxins, and viruses) specifically adhere to. the network strategy developed could also be easily extended to other lectin-glycan interactions, where-besides human or viral interactions-other mammalian, plant, or protozoan glycan interactions could be explored, and also used, for example, for the discovery of new antimicrobial agents. furthermore, the strategy could be used to predict the binding of viral proteins to human glycoproteins or carbohydrate structures, with the aim to facilitate the design of novel antiviral drug compounds, especially in the case of emerging viral pathogens. test compounds. recombinant gp from hiv- (iii b ), produced in cho cells, and hiv- (yu ), produced in insect cells, were purchased from immunodiagnostics (woburn, ma). ␣- , -mannobiose and ␣- , mannobiose were purchased from dextra (united kingdom). laminin, from human placenta, and mucin, partially purified from porcine stomach, were purchased from sigma, while bsa-glcnac was purchased from dextra laboratories (united kingdom). viruses, cell lines, and cell cultures. hiv- r strain bal and hiv- x strain nl . were originally obtained through the aids research and reference reagent program (division of aids, niaid, nih). mt- cells were a gift from l. montagnier (during that time at the pasteur institute, paris, france) and cultured in rpmi medium supplemented with % fetal calf serum (fcs; hyclone, perbio science) and mm l-glutamine (invitrogen) at °c in a % co controlled atmosphere. pbmcs from healthy donors were isolated from buffy coats obtained from the blood transfusion centre (uz leuven, belgium). pbmcs were cultured in rpmi containing % fcs, mm l-glutamine, and ng/ml interleukin- (il- ; roche molecular biochemicals). the cells were activated with g/ml phytohemagglutinin (pha; sigma-aldrich) for days before infection with hiv- . tzm-bl cells were obtained from the aids research and reference reagent program (division of aids, niaid, nih). expression and purification of fimh-ld, n-als p, n-als p, and n-epa p. the sequence of fimh-ld from e. coli k- (strain k ) was used for this work (residues to ; uniprot entry p ). the cloning, expression, and purification of fimh-ld have been previously described ( ) . the n-terminal parts of the als , als , and epa p proteins were expressed in and purified from s. cerevisiae and e. coli as previously described ( , ) . glycan array screening. the n-terminal parts of als p and als p were subjected to glycan array screening for binding to glycans printed on a glass slide microarray (version . ) developed by the cfg ( ). screening of als p was performed at concentrations of and g/ml. the adhesins were labeled with nt- dye via an amine-coupling method (nanotemper) (see the supplemental material). spr. spr experiments were performed with a biacore instrument (ge healthcare) at °c. the recombinant gp envelope proteins, bsa-glcnac, fn, mucin, and laminin were covalently immobilized on a cm sensor chip by amine-coupling chemistry. a reference flow cell chemically treated in the same way as the ligand flow cell was used as a control. for fimh-ld-gp kinetic analysis, fitting of experimental curves and calculation of kinetic parameters were performed by using the biaevaluation software version . (ge healthcare) and a : (langmuir) binding model. in all of the other cases, dissociation constants in the equilibrium state (k d ) were determined. the results were then analyzed with the biaevaluation software and with prism software (graph-pad) (see the supplemental material). afm-smfs. afm-smfs experiments to determine the unbinding force between hiv- (yu ) gp and fimh-ld were performed as described in the supplemental material. lgi network construction. the results of the glycan array screenings for fimh-ld (l. wyns, , cfg database, glycan array version . ) n-als p (first screening, glycan array version . [ ] ; second screening, cfg glycan array version . [this work]), n-als p, n-epa p ( ), n-epa p, and n-epa p ( ) were retrieved from the cfg-core h database (http://www.functionalglycomics.org/) and used to generate the als/ epa-glycan interaction networks. the results were filtered by removing the data of three times the standard error of the mean (sem) and using a signal-to-noise ratio cutoff value that was larger than the average number of relative fluorescence units (rfu). the signal-to-noise ratio cutoff value was visually selected on the glycan array screening graphs. the rfu values were normalized by dividing the values by the maximal rfu value of the screening. the mean rfu value was used when more than one value was detected in different screenings for the same glycan interaction. glycanbinding protein binding sites may accommodate glycan determinants made up of two to six linear monosaccharides together with their potential side chains containing other sugars and modifications ( ) . therefore, glycan determinant structures containing oligosaccharides composed of , , , , and carbohydrate residues present at the nonreducing end were submitted to the glycosuitedb database in the unicarbkb platform ( , ) . in order to obtain the glycoproteomic data, we developed a set of three perl scripts that collaborated to extract the data from the glycosuitedb website (see the supplemental material). lgi network modeling and visualization. the interactions were modeled as a weighted undirected graph g(v, e, w), where v is the set of vertices, e is the set of edges connecting the pairs of vertices, and w is the set of weights associated with each edge. the edges' weights were calcu-lated according to the interaction type (see the supplemental material). the gephi open-source graph visualization and manipulation software was used ( ) . within gephi, the force-directed visualization method of hu ( ) , which is known to be accurate when visualizing local clustering and symmetry ( ) , was used. this approach allowed us to obtain relatively well-defined spatial distributions and local clusters according to the network structure ( fig. and ) . the lgi networks were also visualized as a hierarchical structure by using cytoscape . ( ) and the cerebral app ( ) (see the supplemental material). mts-pes antiviral assays. the anti-hiv- activity of each compound, both alone and in combination, in mt- cell cultures was determined by a tetrazolium-based colorimetric assay. briefly, -fold dilutions of the test compounds were added to a -well plate, and it was preincubated for min at °c with mt- cells. five days postinfection, cpes were scored microscopically and antiviral activity was measured by the mts-pes method with a spectramax -well plate reader (molecular devices) as described previously ( ) . pha-stimulated pbmcs were resuspended in cell culture medium supplemented with ng/ml il- and seeded into -well plates (iwaki glass) containing various concentrations of test compounds. after min of preincubation at °c, infection with hiv- was performed. il- was added at days and postinfection. supernatant was collected at day , and viral replication was measured with an hiv- p ag elisa (perkinelmer) according to the manufacturer's guidelines. tzm-bl-hiv- infectivity luminescence assay. firefly luciferaseand e. coli ␤-galactosidase-expressing cd ϩ cxcr ϩ ccr ϩ tzm-bl cells were resuspended in cell culture medium (dulbecco's modified eagle's medium with % fcs and % hepes) supplemented with g/ml deae-dextran (sigma-aldrich, diegem, belgium) and preincubated for min at °c in cell culture medium-diluted test compounds in -well plates. next, a laboratory hiv- strain (x nl . or r bal) was added according to the % tissue culture infective dose of the viral stock. two days postinfection, cpes were first scored microscopically, and afterward, viral replication was measured by luminescence. steadylite plus reagent (perkinelmer) was mixed with lyophilized substrate in accordance with the manufacturer's guidelines. supernatant was removed, and the steadylite plus substrate solution was added to the -well plates. next, the plates were incubated in the dark for min in a closed plate shaker. finally, cell lysis was scored microscopically and aliquots were transferred to white lumitrac -well plates (greiner bio-one) to measure the relative luminescence units with a spectramax l microplate reader and softmax pro software (molecular devices), an integration time of . s, and a dark adaptation time of min. supplemental material for this article may be found at http://mbio.asm.org/ lookup/suppl/doi: . /mbio. - /-/dcsupplemental. text s , docx file, . mb. figure s , tif file, . mb. figure s , tif file, . mb. the endothelial glycocalyx: composition, functions, and visualization role of glycosides as epithelial cell receptors for candida albicans fucose-specific adhesins on germ tubes of candida albicans the repertoire of glycan determinants in the human glycome printed covalent glycan array for ligand profiling of diverse glycan binding proteins uropathogenic escherichia coli virulence and innate immune responses during urinary tract infection exploring the d molecular architecture of escherichia 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high-quality force-directed graph drawing fimh forms catch bonds that are enhanced by mechanical force due to allosteric regulation tamm-horsfall protein binds to type fimbriated escherichia coli and prevents e. coli from binding to uroplakin ia and ib receptors integrin-mediated host cell invasion by type -piliated uropathogenic escherichia coli src kinase has a central role in in vitro cellular internalization of staphylococcus aureus synergy between type fimbriae expression and c opsonisation increases internalisation of e. coli by human tubular epithelial cells inhibition of egfr pathway signaling and the metastatic potential of breast cancer cells by pa-msha mediated by type fimbriae via a mannose-dependent manner secretory immunoglobulin a carries oligosaccharide receptors for escherichia coli type fimbrial lectin adherence of type -fimbriated escherichia coli to uroepithelial cells: more in diabetic women than in control subjects o-linked oligosaccharides from salivary agglutinin: helicobacter pylori binding sialyl-lewis x and lewis b are terminating moieties on hyperfucosylated oligo-n-acetyllactosamine the fimbrial adhesin f -g of enterotoxigenic escherichia coli has an immunoglobulin-like lectin domain that binds n-acetylglucosamine analysis of candida albicans adhesion to salivary mucin characterization of binding of candida albicans to small intestinal mucin and its role in adherence to mucosal epithelial cells mucin-type o-glycans in human colon and breast cancer: glycodynamics and functions identification of markers that distinguish monocyte-derived fibrocytes from monocytes, macrophages, and fibroblasts altered glycosylation of leukosialin, cd , in hiv- -infected cells of the cem line altered sialylation of cd in hiv- -infected t lymphocytes altered t cell surface glycosylation in hiv- infection results in increased susceptibility to galectin- -induced cell death lamp proteins are required for fusion of lysosomes with phagosomes the candida glabrata adhesin epa p causes adhesion, phagocytosis, and cytokine secretion by innate immune cells the facultative intracellular pathogen candida glabrata subverts macrophage cytokine production and phagolysosome maturation identification of polymorphonuclear leukocyte and hl- cell receptors for adhesins of streptococcus gordonii and actinomyces naeslundii glycosylation-dependent interaction of jacalin with cd induces t lymphocyte activation and th /th cytokine secretion iron acquisition from transferrin by candida albicans depends on the reductive pathway the hyphal-associated adhesin and invasin als of candida albicans mediates iron acquisition from host ferritin the inhibitory effect of serum on the growth of torulopsis glabrata activated lactoferrin's ability to inhibit candida growth and block yeast adhesion to the vaginal epithelial monolayer egfr and her receptor kinase signaling mediate epithelial cell invasion by candida albicans during oropharyngeal infection als is a candida albicans invasin that binds to cadherins and induces endocytosis by host cells structural basis for the broad specificity to host-cell ligands by the pathogenic fungus candida albicans a recombinant allosteric lectin antagonist of hiv- envelope gp interactions actinohivin, a broadly neutralizing prokaryotic lectin, inhibits hiv- infection by specifically targeting high-mannose-type glycans on the gp envelope human mannan-binding lectin inhibits the infection of influenza a virus without complement anti-influenza a virus activities of mannan-binding lectins and bovine conglutinin mannosebinding lectin binds to ebola and marburg envelope glycoproteins, resulting in blocking of virus interaction with dc-sign and complementmediated virus neutralization treatment of influenza a (h n ) virus infections in mice and ferrets with cyanovirin-n structural hotspots determine functional diversity of the candida glabrata epithelial adhesin family gephi: an open source software for exploring and manipulating networks an experimental study on distance-based graph drawing cytoscape . : new features for data integration and network visualization cerebral: a cytoscape plugin for layout of and interaction with biological networks using subcellular localization annotation cada, a novel cd -targeted hiv inhibitor, is synergistic with various anti-hiv drugs in vitro social network analysis: methods and applications power and centrality: a family of measures a new status index derived from sociometric analysis key: cord- -krvei r authors: holmes, kathryn v.; dominguez, samuel r. title: the new age of virus discovery: genomic analysis of a novel human betacoronavirus isolated from a fatal case of pneumonia date: - - journal: mbio doi: . /mbio. - sha: doc_id: cord_uid: krvei r a new human betacoronavirus in lineage c, tentatively called hcov-emc, was isolated from a patient from the kingdom of saudi arabia who died from acute severe pneumonia and renal failure. the viral rna has been detected in eight additional cases. sequencing and bioinformatic analysis of the viral genomic rna showed that it is a novel virus not previously detected in any other species and that its closest relatives are two asian bat coronaviruses. hcov-emc may represent a sporadic spillover to humans from an unknown animal reservoir. in a recent article, van boheemen et al. demonstrated how state-of-the-art sequencing technology and bioinformatic analysis can quickly provide critical insight into the viral genome sequence, phylogeny, replication strategy, and potential drug and vaccine targets and generate tools to evaluate the possible epidemic risk associated with this novel human virus. d uring the past decade, at least novel human respiratory viral pathogens have been discovered, primarily by using highly sensitive nucleotide sequencing and new virus detection technologies. these human viruses include human metapneumovirus, rhinoviruses in clade c, bocavirus, polyomaviruses wu and ki pandemic h n influenza virus, and new human coronaviruses (hcov). until , only two coronaviruses, hcov-oc and hcov- e, were known to cause human disease, primarily upper respiratory tract infections. the discovery of severe acute respiratory syndrome-cov (sars-cov) as the cause of the sars pandemic of to demonstrated the epidemic potential of this large family of rna viruses and emphasized their importance in human respiratory diseases. after the sars pandemic, two additional human coronaviruses, nl and hku , were identified and found to cause both upper and lower respiratory tract disease. although these coronaviruses were only recently discovered, they have probably been circulating in the human population worldwide for a long time. hcov-oc apparently jumped from a bovine host into humans more than years ago and has become endemic worldwide. in contrast, the sars pandemic was caused by a novel human virus that had very recently emerged into the human population from its zoonotic reservoirs, chinese horseshoe bats (suborder microchiroptera, family rhinolophidae, genus rhinolophus) ( , ) . indeed, extensive phylogenetic studies suggest that all alpha-and beta coronaviruses may be derived from bat coronaviruses ( ). in june , a novel coronavirus, tentatively named hcov-emc (for erasmus medical center where the initial genome sequence was done), was isolated in a monkey kidney cell line from a sputum specimen from a -year-old man from the kingdom of saudi arabia who died of acute severe pneumonia and renal failure. results of standard diagnostic tests of his clinical samples for known respiratory viruses were all negative, which prompted testing of infected cell culture supernatants using a pan-coronavirus reverse transcription-pcr (rt-pcr) assay to amplify a short, highly conserved region of the rna-dependent rna polymerase gene. sequencing the amplicon from the patient's specimen revealed the novel human coronavirus ( ) . the discovery of a new coronavirus in a patient who died of severe respiratory disease was promptly reported to the world health organization and posted on promed to alert physicians and diagnostic laboratories to look for additional cases. eight additional patients from the middle east with hcov-emc infection and severe respiratory disease have been identified, bringing the total of laboratory-confirmed cases up to , with deaths ( ). in a recent article, van boheemen et al. described the characterization of the hcov-emc genome using unbiased high-throughput next-generation sequencing and state-of-the-art bioinformatics to mine a wealth of information about this new human virus ( ) . what can analysis of its rna genome tell us about this new human coronavirus? analysis of the hcov-emc genome was able to distinguish whether hcov-emc was a known human coronavirus with mutations that increased its virulence or a product of recombination between two or more known coronavirus species or, like sars-cov, a spillover of a zoonotic coronavirus that was transmitted, either directly or indirectly, to humans from an animal reservoir. the genomic analysis showed that hcov-emc is a new coronavirus that had never before been detected in any other species. what is the relationship between hcov-emc and known coronaviruses? according to the current classification by the international committee on taxonomy of viruses, coronaviruses are in the order nidovirales, family coronaviridae, and subfamily coronavirinae. there are at least genera, alpha-, beta-, gamma-, and deltacoronaviruses, and the many virus species within each genus are grouped in multiple lineages called a, b, c, etc. the genome sequence revealed that hcov-emc is a betacoronavirus in lineage c, significantly different from sars-cov, a betacoronavirus in lineage b, and human betacoronaviruses oc and hku , which are placed in lineage a. the closest known relatives of hcov-emc in lineage c are two betacoronaviruses of asian bats, but because hcov-emc and the bat viruses have only % to % amino acid identity in the key conserved replicase domains used for phylogenetic analysis, hcov-emc is classified as a novel species within betacoronavirus lineage c ( ) . the genomic analysis suggests that hcov-emc may be a zoonotic virus that spilled over to infect the laboratory confirmed patients, either directly or indirectly, from an unknown reservoir, possibly a bat. this discovery justifies the recent worldwide surveillance of wildlife for coronavirus infection and the extensive genome sequencing and phylogenetic analysis that have been done since the sars pandemic to identify potential risk to humans from zoonotic coronavirus infections. what features of the genome and its translational strategy are similar to or different from those of other coronaviruses? like all coronaviruses, hcov-emc has a single-stranded, plus-sense, polyadenylated rna genome about kb in length. as expected, the -kb sequence at the = end of the genome is translated to yield a huge polyprotein that is cotranslationally cleaved in cis by two viral proteases into functional nonstructural proteins that cooperatively form the complex machinery for viral rna synthesis and rna recombination. the -kb sequence in the = region of coronavirus genomes uses a different translational strategy. this region encodes structural proteins with features common to all covs as well as several so-called accessory proteins that are different for each coronavirus and whose origins and functions are unknown. a nested series of = coterminal polyadenylated subgenomic mrnas is generated in the cytoplasm, and only the gene at the = end of each of these mrnas is translated. the genome reveals conservation in hcov-emc of several potential targets for drugs and vaccines being developed for other covs, including the viral spike glycoprotein (s), virus-encoded proteases, and essential enzymatic functions such as the rna-dependent rna polymerase and helicase. how does the genomic analysis of hcov-emc expedite further research on this new human virus? as soon as partial nucleotide sequences of hcov-emc were available, sensitive rt-pcr tests were developed to specifically detect rna of this virus in tissues and body fluids of humans and animals. these tests are being used to screen for hcov-emc rna in patients with severe respiratory disease of unknown etiology and in wildlife surveillance. based on the predicted amino acid sequences of the hcov-emc proteins, plasmids can be engineered to express the proteins for structural, antigenic, and functional studies. recombinant viral proteins are being used in enzyme-linked immunosorbent assays (elisas) to detect hcov-emc-specific antibodies in sera. hcov-emc spike protein in retrovirus pseudotypes can be used to identify the virus receptor and study virus entry. antisera raised against the recombinant hcov-emc proteins can detect viral antigens in infected cell cultures and infected tissues of humans or animals for studies on virus tissue tropism and pathogenesis. the viral genome can now be reconstructed by synthetic biology to create a manipulable cdna copy that can be mutated for analysis of virus replication, pathogenesis, virulence factors, host range, and vaccines. when additional virus isolates from other patients or animal reservoirs become available, genomic analysis can be used to analyze variants to detect amino acid substitutions in the spike or other proteins that are associated with adaptation to cell culture or changes in antigenicity, host range, and virulence. what questions must be answered to show whether hcov-emc is likely to cause an epidemic or adapt to become endemic in humans? first, it is necessary to prove whether the hcov-emc cultured from the first patient actually caused his fatal disease ( ) . several bacterial pathogens were also cultured from his respiratory tract, although this is not uncommon in severely ill patients on respirators. development of an animal model for hcovinduced respiratory disease would fulfill koch's postulates and be useful for testing candidate coronavirus drugs and vaccine strategies. sera from humans in the middle east and elsewhere are being tested by elisa for hcov-emc-specific antibodies to determine the prevalence of infection of humans in different regions and estimate the percentage of clinically apparent hcov-emc infections and the case/fatality ratio. surveillance of bats and other animals in the middle east and elsewhere for the presence of rna from betacoronavirus lineage c viruses or antibodies to these viruses will help to identify reservoir hosts and possible intermediate hosts that could transmit the virus to humans. so far, no epidemiological connections have been found between animals and the hcov-emc patients. this virus is apparently much less easily transmitted from human to human than sars-cov, although three of the cases in saudi arabia were within one family and several health care workers who cared for two of the confirmed cases in jordan also developed pneumonia and are now considered probable cases ( ) . it is possible that as the virus replicates in sporadic cases, mutations, particularly in the genes encoding the spike protein and any immunomodulatory proteins, might be selected that increase human-to-human transmissibility. sporadic hcov-emc infections in epidemiologically unlinked individuals in the middle east suggest that it is a zoonotic virus. where encounters between humans and wildlife have become common, sporadic zoonotic infections may occur without human-to-human transmission. this so-called "virus chatter" was obvious in retrospective studies of sars-cov infections in china which showed that isolated cases occurred well before the virus began to spread from human to human during the sars pandemic ( ) ( ) ( ) . mutations in the sars-cov spike protein and other viral proteins were associated with the transition from "chatter" to epidemic. it is possible that hcov-emc is not yet a human-adapted virus and has simply been detected in rare cases of "chatter" by increasingly sophisticated surveillance. the work by van boheemen et al. demonstrates the ability of state-of-the-art technology and bioinformatics to obtain full-genome sequence data within days and provide critical insight into the potential risks associated with novel viral pathogens ( ) . over the past decade, other advances, including the development of enhanced virus detection technologies, surveillance programs, increased global awareness and communication through internet-based technologies such as promed, real-time sharing of genome sequences in genbank, and internet-based publications and the posting of publications in advance of printing, as well as increasing collaborations between researchers and medical and veterinary practitioners such as the one health initiative, have greatly facilitated rapid and effective responses to threats of new emerging infectious diseases. bats are natural reservoirs of sars-like coronaviruses severe acute respiratory syndrome coronavirus-like commentary virus in chinese horseshoe bats 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 isolation of a novel coronavirus from a man with pneumonia in saudi arabia world health organization global alert and response. background and summary of novel coronavirus infection-as of genomic characterization of a newly discovered coronavirus associated with acute respiratory distress syndrome in humans microbe hunting. microbiol cross-host evolution of severe acute respiratory syndrome coronavirus in palm civet and human cross-species virus transmission and the emergence of new epidemic diseases. microbiol bushmeat hunting, deforestation, and prediction of zoonoses emergence key: cord- - owcqt d authors: iketani, sho; shean, ryan c.; ferren, marion; makhsous, negar; aquino, dolly b.; des georges, amedee; rima, bert; mathieu, cyrille; porotto, matteo; moscona, anne; greninger, alexander l. title: viral entry properties required for fitness in humans are lost through rapid genomic change during viral isolation date: - - journal: mbio doi: . /mbio. - sha: doc_id: cord_uid: owcqt d human parainfluenza viruses cause a large burden of human respiratory illness. while much research relies upon viruses grown in cultured immortalized cells, human parainfluenza virus (hpiv- ) evolves in culture. cultured viruses differ in their properties compared to clinical strains. we present a genome-wide survey of hpiv- adaptations to culture using metagenomic next-generation sequencing of matched pairs of clinical samples and primary culture isolates (zero passage virus). nonsynonymous changes arose during primary viral isolation, almost entirely in the genes encoding the two surface glycoproteins—the receptor binding protein hemagglutinin-neuraminidase (hn) or the fusion protein (f). we recovered genomes from hpiv- primary culture isolates and hpiv- strains directly from clinical samples. hn mutations arising during primary viral isolation resulted in substitutions at hn’s dimerization/f-interaction site, a site critical for activation of viral fusion. alterations in hn dimer interface residues known to favor infection in culture occurred within days (h and n ). a novel cluster of residues at a different face of the hn dimer interface emerged (p and r ) and imply a role in hpiv- -mediated fusion. functional characterization of these culture-associated hn mutations in a clinical isolate background revealed acquisition of the fusogenic phenotype associated with cultured hpiv- ; the hn-f complex showed enhanced fusion and decreased receptor-cleaving activity. these results utilize a method for identifying genome-wide changes associated with brief adaptation to culture to highlight the notion that even brief exposure to immortalized cells may affect key viral properties and underscore the balance of features of the hn-f complex required for fitness by circulating viruses. altered to suit cultured cells and could no longer be considered clinical strains. there has been a relative dearth of sequence information available for hpiv compared to other respiratory viruses ( ) , and more importantly a lack of information about strains prior to passaging in the laboratory and thereby being subjected to selective pressure. to address the question of which features associated with fitness in humans may be lost in routine viral culture, we sought to understand the genomic changes associated with brief exposure of hpiv- from clinical samples to culture during the isolation process. we used metagenomic next-generation sequencing (mngs) of field strains of hpiv- isolated from humans to analyze the diversity and commonalities of circulating hpiv- strains at the genomic level, and we simultaneously sequenced paired specimens that were subjected to culture for viral isolation. the sequencing strategy we used to sequence clinical strains directly without passage in immortalized cells does not rely on designing sequence-specific primers that may amplify only a subset of samples (in case of mutations) ( ) . deep genomic sequencing of nine sets of paired clinical samples (primary nasal swabs in viral transport medium) and culture isolates (culture harvest from zero passage virus) led to discovery of a number of hn mutations associated with rapid evolution in culture. to assess the frequency of mutations identified earlier, we also performed deep sequencing of hpiv- clinical samples and culture isolates from the university of washington virology laboratory, allowing us to confirm that the alterations associated with brief exposure to culture for viral isolation were almost entirely found in the sequences of culture isolates and found commonly within populations of viruses in those isolates. functional characterization of the effects of several of the specific hn gene mutations found in the cultured isolates in a clinical isolate (ci) background-in expressed hn-f complexes or recombinant viruses bearing the mutated genes-reveals that the culture-associated mutations all increase fusion promotion by the hn-f complex. these results support the notion that clinical strains differ from cultured viruses in the balance of fusion properties. the locations of the residues that differ after brief culture of clinical viruses point to specific domains that are relevant to the control of viral entry in vivo. the sequencing data, when correlated to functional data, help define the flexibility and constraints of the hpiv- fusion complex. these data provide the first characterization of culture-associated hn mutations in the background of hpiv- clinical strains directly from patient samples. we show that, even during transient exposure to culture ("zero passage" virus), the act of isolating a virus is associated with genomic evolution that may significantly affect viral protein function and not reflect in vivo dynamics. mutation and mixed allele frequency comparison between clinical samples and their matched cultured specimens. to compare hpiv- sequenced directly from patients with hpiv- sequenced after brief growth in culture, nine hpiv- -positive (hpiv- ϩ) nasal swabs were sequenced metagenomically, and their paired viral isolates were sequenced after initial isolation in rhesus monkey kidney (rhmk) cells. we investigated the variant alleles with a minor allele frequency of Ͼ % that arose in culture. the nine swabs included one clinical sample that was independently cultured twice, such that ten viral isolates were sequenced. clinical respiratory samples that tested positive for hpiv- by quantitative reverse transcription-pcr (qrt-pcr) with cycle thresholds under were either sequenced directly by metagenomic nextgeneration sequencing (mngs) without exposure to culture or grown on rhmk cells and viral isolates were harvested and sequenced. the choice of rhmk cells for this study was based on the fact that these are the standard cells used for viral isolation by many or most diagnostic microbiology laboratories and would allow us to answer the question of whether this standard process leads to alteration in the viruses. seven of the nine cultured isolates were harvested before day of culture, and no viruses were passaged beyond initial isolation. sequencing reads for the cultured viruses were analyzed for mutational changes relative to the hpiv- consensus genome obtained from metagenomic sequencing of the original clinical sample (without expo-sure to any cell culture), and both assemblies were analyzed for variants with Ͼ % minor allele frequency (maf) ( ) . a total of nonsynonymous mutational changes with maf change of Ͼ % in response to exposure to cell culture were found across the nine sets of hpiv- viral isolates, with of the in the hn protein (p Ͻ . by fisher's exact test) (fig. ) . of the ten mutations found outside the hn protein, four were found in the p protein, four in the l protein, and two in the f protein. of the two l variants that increased in allele frequency in cultured specimens, one (g d) was not found in a separate viral isolation performed on the same clinical sample, while the other (p l) was found at a high allele frequency in the original clinical sample (fig. d to g). evolution of the polymerase complex will be explored separately, as the focus of this current paper is on the entry complex. during viral isolation from sample sc , we noted the appearance of the previously described hn h q mutation at % maf after only days in culture (fig. g) . hn h q has previously been isolated in laboratory-adapted hpiv- strains and is possible cpe day -cpe, had+ sc l n h f m p n r c % r , , , , , , , , , , , , , , mutational changes and allele frequencies in matched clinical samples and cultures. blue genomes represent clinical samples, and the green genomes below the blue genomes represent the same sample after it was inoculated into culture and harvested several days later. the number of days cultured, hemagglutinin adsorption assay results (had), and observed cytopathic effect (cpe) are shown for each viral isolate. virus from sample sc in panel g was isolated twice. coverage is averaged across the entire genome and depicted underneath each genome. allele frequencies are plotted to scale above the genome organization. mutational changes are described relative to the amino acid in the consensus genome of the original patient sample. variants indicated in italics were found exclusively on separate reads and are considered unlinked. iketani et al. ® associated with enhanced fusion promotion as a result of hn's f-triggering function ( , ( ) ( ) ( ) . two independent viral isolations in rhmk cells of this clinical sample resulted in selection for the hn h q variant allele. during viral isolation in rhmk cells from sample sc , an hn n d mutation emerged from % to % allele frequency in only days, the greatest change in allele frequency during the brief exposure to culture (fig. e ). previous comparison of direct clinical isolates with laboratory-adapted strains showed that every clinical strain bore asparagine (n) at position , while our laboratory strains bore aspartic acid (d) at position ( ). the hns derived from clinical isolates (cis) showed from -to -fold-higher neuraminidase activity than hns derived from the lab-adapted strains, and the substitution of the aspartic acid at this position in the laboratory strains led to an approximately -fold-lower receptor cleavage in the laboratory-adapted strains, suggesting that the asparagine at position is essential for the high propensity for receptor cleavage in field strains. the rapid emergence of this adaptation to culture by reducing receptor cleavage is remarkable. during viral isolation from both samples sc and sc , an hn l s mutation, adjacent to residues and mentioned above, arose to % and % maf ( fig. a to i). during isolation of sample sc , the same site contained an l f mutation that was unlinked to the h q mutation that also arose during that isolation (i.e., was found exclusively on separate reads). the sc isolation also resulted in selection of novel mutations hn s n and hn t i, comprising a cluster from positions to with significant alterations in the cultured viruses (fig. h) . while the region of hn corresponding to the cluster at positions to above was known to be of interest for entry in vivo (based on features of h and n ), during independent viral isolations from sample sc , a set of mutations emerged in hn at residues previously unrecognized to be relevant to fitness: p and r (hn p l and hn r k). these mutations were not linked to each other. the p l mutation described above was also recovered during viral isolations of sc and sc ( fig. c to h). adjacent to these residues, another previously undescribed hn mutation l f was selected at a high allele frequency ( %) during isolation of sc and at a low level during isolation of sc ( % maf in k and % maf in cul ). as discussed further below, these three residues describe a region of interest based on our previous structural analysis of the hn dimer interface ( ) . phylogeny and hn mutation history in original samples. to dissect the influence exerted by background hn sequence of the clinical virus on subsequent culture adaptation, we generated a phylogenetic tree based on amino acid substitution distance in the hn protein of our clinical samples (fig. ) . none of the phylogenydetermining mutations among direct clinical sample strains were located in hn's receptor binding site ii or second dimer interface domain that emerged during rapid culture adaptation. independent viral isolates from sample sc ( k and cul ) produced p l, r k, and h q. the hn mutations l f, p l, s n, and l s also arose among separate lineages. large-scale whole-genome sequencing of hpiv- direct-and culture-exposed strains shows that culture-associated mutations are rarely found in clinical strains. to place the alleles that arose in the paired clinical-culture specimens above in the broader context of hpiv- sequence information, we used mngs to obtain whole genomes from an additional hpiv- strains, of which were primary viral isolates (passage zero virus; brief exposure to culture only during viral isolation) and the remaining were directly from patient samples. all specimens were collected in or (see table s in the supplemental material). recombination analysis using rdp ( , ) revealed no recombination among these strains similarly to other nonsegmented negative-strand rna viruses ( , ) . phylodynamic analysis of the concatenated coding regions of the hpiv- sequenced strains revealed a mean mutation rate of . eϪ substitutions/site/year ( % confidence interval, . eϪ to . eϪ ). analysis of concatenated coding regions from all hpiv- sequences with complete coding sequences (cds) in the genbank nt database as of september revealed a similar mean mutation rate of . eϪ substitutions/site/year ( % confidence interval, . eϪ to . eϪ ) similar to those of other paramyxoviruses ( ) . phylogenetic analysis of the newly sequenced hpiv- coding sequences revealed multiple lineages of hpiv- present in - clinical samples and clustering by time of collection as the dominant feature rather than by sequence obtained from viral isolate versus clinical sample (fig. ) . however, examination of the deep sequencing data for of the viral isolates revealed mutations with Ͼ % maf in residues and and residues to selected during the paired sample-isolate analysis above. while the original clinical samples for these isolates are not available, none of the clinical samples sequenced metagenomically contained variant residues at these loci. of the strains with variant residues, only viral isolates and laboratoryadapted strains contained variant residues with Ͼ % maf that would be expected to be reflected in consensus sequence available publicly. in addition to the newly derived hpiv- genome sequences, we also examined all complete and partial consensus hpiv- hn sequences available in the genbank nt database as of august -a total of , sequences-in a search for the culture-associated mutations isolated here. of the culture-associated mutations in the hn protein described above, p l ( ), s n ( ), l f ( ), l s ( ), n d ( ) , and t i ( ) appeared exclusively in sequences from cultured isolates ( , ( ) ( ) ( ) ( ) ( ) ( ) . none of the consensus sequences in ncbi with a p l mutation had a mutation in the to residue range (consensus sequence, -hkslnt- ). the h q mutation appeared previously in sequences, with the vast majority of these ( / ) occurring in culture isolates; note that as above, even sequences listed as clinical may have experienced brief exposure to culture conditions. the l f mutation has not been reported in any consensus hpiv- hn sequence in ncbi. three instances of the r k alteration were found in ncbi genbank ( , ) . one of these was recovered using rt-pcr directly from clinical specimens during an hpiv- outbreak in a children's hospital cancer ward in barcelona, spain, in summer ( ) . the remaining two r k mutations reported were from a likely culture-adapted college of american pathologists proficiency testing sample (strain f , genbank accession no. ky ) and a primary culture isolate from japan (ddbj accession no. ab ) ( ) . hpiv- culture adaptations localize to the dimer interface of hn. we mapped all the residue changes in the hn protein that arose during adaptation to brief culture onto the hpiv- hn protein crystal structure wef (fig. ). all alterations localized to the dimer interface. we previously described critical residues at the hn dimer interface for hn's roles in receptor engagement and activation of f at h -which we identified as the secondary sialic acid binding site-and in receptor cleavage at n , shown at the top of the hn globular head dimer interface in the diagram (fig. ) . the diagram highlights the fact that n likely makes contact with the nac modification of n , and glycosylation could therefore have a significant effect on binding. we have shown that alterations that enhanced dimer contacts were associated with culture adaptation, while host fitness was correlated with reduced dimer contacts at these residues ( ) . alterations in residues and have been associated with other culture conditions parainfluenza virus fitness for host tissues ® as well ( - ), supporting the notion that this site is intimately involved in the mechanism whereby hn activates f in the natural host. the mutations p l and r k have not been described before and appear in a cluster at the opposing side of the dimer interface. the rapid emergence of alterations here during adaptation of a patient's hpiv- to culture suggests a role in viral entry in the natural host. of note, p does not make specific contact with the opposing monomer, but a mutation would change the conformation of the loop significantly, thereby altering the contacts that the loop can have. hn mutations in site ii and dimer interface modulate fusion promotion. to understand the selection forces that affect hpiv- fitness, we next analyzed the biological properties conferred by the culture-associated mutations in hn. here we characterized the influence of each specific residue on the function of the hn-f fusion complex. to investigate whether the identified mutations altered hn's fusion promotion ability we had previously observed in clinical isolates of hpiv- , we introduced seven of these rapidly emerging mutations into the background of a well-characterized clinical isolate hn, clinical isolate (ci- ) ( , ). we paired these mutated hn proteins with a lab-adapted f to ensure that we could observe adequate levels of fusion to compare function, since clinical isolate hn and f pairs result in nearly unobservable levels of fusion ( ) . fusion promotion by each of these mutant hn-f pairs was quantified with a ␤-galactosidase complementation assay and compared to the wildtype clinical isolate hn (ci wt) paired with the lab-adapted f (fig. ) . we included an influenza a virus hemagglutinin (ha) and f pair as a positive control for fusion, as it had been previously shown that hn has a stabilizing property that prevents f triggering; that is, an increase in fusion relative to the ci wt would be expected and was seen ( ) . the fusion exhibited by all mutant hn-f pairs was considerably higher than that of the ci wt hn-f pair, ranging from a -fold increase (hn s n) to a -fold increase (hn l f). neuraminidase activity is reduced by hn's adaptations to culture except for h q. after observing that fusion promotion was increased by all hn mutants, we examined several interacting properties of hn (see introduction) that are involved in this process. we assessed the neuraminidase (receptor cleavage) activity and also quantified the release of receptor-bearing erythrocytes (rbcs) from hn-expressing cells as a measure of the balance between avidity and receptor-cleaving properties. neuraminidase activity was measured through the cleavage of =-( -methylumbelliferyl)-␣-d-n-acetylneuraminic acid, sodium salt hydrate ( -munana) by hn (fig. a) . as previously described, almost all the culture-adapted hn mutants displayed lower neuraminidase activity than ci wt, ranging from roughly % (l s) to % (r k) relative activity ( ). the h q mutant had increased neuraminidase activity, with roughly % activity relative to ci wt. we have previously shown that this mutation at h on hn confers both higher receptor avidity and intrinsically enhanced triggering of f and thereby augments fusogenicity, an effect that overrides the increase in receptor cleavage (see the release assay below) ( ) . release of bound receptor-bearing rbcs by cell surface hns reflects the balance of neuraminidase activity, which would release the bonds tethering the cells, and avidity of the hns, which retain the bound cells (fig. b) . we quantified the amount of rbcs released at several time points when incubated at ph . and °c. differences between the hns was most readily observed at the earliest time point of min, when ci wt had the most release (~ %), whereas the mutant hns all had less release, reflecting either their higher receptor avidity, lower neuraminidase activity, or both. we observed two separate groups of mutants in their capability to release rbcs. a more rapidly releasing group-p l, r k, and s n-had roughly % release at min and complete release (~ %) after min. in contrast, the more slowly releasing group, consisting of h q, l f, l s, and n d, had to % release at min and to % release after min. as noted above, the higher receptor avidity of hn can override the higher-than-wt neuraminidase activity. (fig. b, yellow squares) , producing no detected progeny compared to a titer of to pfu/ml for each of the viruses bearing a single adaptive hn mutation. the growth in cv- cells reveals that while the clinical virus is severely handicapped in monolayer culture, the mutations that emerged during the rhmk culture isolation process confer growth in these monolayer cultures. while the wt ci shows negligible fitness in monolayer culture compared to the adapted viruses on day , by day infectious virus is released into the supernatant fluid of the virus to cell culture. at day however, adaptive mutations have appeared (table ) and correlate with increased production of infectious particles (fig. b, yellow squares) . in hae tissue, the wt ci- has a growth advantage over all the mutated viruses, showing that the individual culture-adaptive hn mutations modestly impair infection of hae (fig. b, orange circles) . at day after hae infection, wt ci releases . ϫ infectious particles into the hae apical surface, which is between and times more particles than released by the mutant viruses. as expected based on our previous studies ( ), growth in hae did not result in emergence of mutations (table ) . mechanisms that affect respiratory virus-cell interplay in the context of humans are finely tuned to the environment of the host ( , ) . to understand natural viral infection and the features that govern infectivity and transmissibility in humans, we have endeavored to analyze field strains in airway tissues. we have shown for human parainfluenza virus type that conclusions drawn from laboratory strains can be misleading with regard to the receptor interaction and viral fusion properties that govern entry and fitness in vivo ( , ) . analysis of clinical strains suggests that the hn-f fusion pairs of circulating hpiv- viruses maintain a balance of properties that result in an inverse correlation between fusion in cultured cells and growth in vivo; the fusion we have previously used whole-genome sequencing of field strains of hpiv- isolated from humans and grown only in airway cultures to analyze the diversity and the commonalities of circulating strains of hpiv- ( ). the sequences were used to define functional and structural properties of proteins of circulating strains in order to identify the genetic basis for properties that confer fitness in the field. while laboratoryadapted and ci airway-grown strains shared the basic properties of the hn-f fusion complex-receptor binding, fusion triggering, receptor cleavage-the balance between these properties at each step is shared across all cis we studied and different from all laboratory strains. the ci fusion pairs all possessed lower fusogenicity and decreased f activation compared to those of laboratory strains. here we carried out deep genomic screens of paired hpiv- clinical samples; one portion of each sample was sequenced directly from the patient with no culture step, while the other portion was exposed to brief culture for viral isolation in immortalized rhesus monkey kidney (rhmk) cells, a standard cell line used in clinical laboratories for virus identification. we hypothesized that even during such brief periods of culture, mutations arising in the hn-f fusion complex could be informative about features essential for growth in humans that would be stringently selected against in culture. hpiv- evolution upon exposure to culture was extraordinarily rapid and almost entirely limited to the hn protein. bioinformatic analyses of existing hn sequences revealed that hn mutations that arose during brief exposure to culture are almost uniformly found in consensus sequences from hpiv- that are either adapted to culture or exposed to culture-and not found in hpiv- sequenced directly from clinical samples. deep sequencing of newly derived hpiv- isolates and clinical samples revealed that more than one-third of hpiv- isolates had subpopulations of hn dimer interface variants that were not reflected in consensus sequences and were not present in hpiv- sequences obtained directly from clinical samples. the sequences were used to define functional and structural properties that differ from laboratory-selected circulating strains in order to identify the genetic basis for properties that may confer fitness in the field. the hn mutations that we identified in response to exposure to culture included both novel and previously observed and characterized adaptations to immortalized cells. remarkably, all the mutations map to the dimer interface and receptor binding site ii of hn. these results point to the critical role of this region in the hpiv- entry mechanism and for fitness in vivo, as we have deduced from smaller-scale and mechanistic studies ( - , ). the h residue at receptor binding site ii at the dimer interface of hpiv- hn influences hn's dimerization and its activation of f ( , ). we have shown that the presence of the glutamine at position at the dimer interface of the hpiv- hn increases hn oligomerization and thereby modulates f triggering, with this hn more actively triggering f ( , ). when the virus bearing this mutated hn was subjected to growth in human airway, it rapidly acquired a compensatory mutation, q r, which reduced fusion triggering and reduced hn dimerization. structural analysis of footprints of the dimer interfaces showed that when hn h q is present in receptor binding site ii, more of the interface residues are in contact with the opposing monomer face. however, in the hn with the q r compensatory mutation that permits growth in the airway, the dimer interface footprint shows decreased points of contact. the dimer interface footprint pointed to several residues and domains whose contact with the opposing monomer may be critical for the key functions governed by hn's receptor binding site ii. the emergence of the h q mutation after brief culture highlights both the flexibility of hn's site ii to adapt to new conditions and also the exquisite constraint of the virus's fusion complex with respect to alterations in the environment. adjacent to the h residue in the dimer interface, but not making contact in the dimer interface footprint mentioned above, is n , a residue we have previously shown to be highly conserved in all clinical strains. this residue was previously found to be an aspartic acid in laboratory-adapted strains, associated with a dramatic decrease in neuraminidase activity consistent with the overall high-receptor-contact/highfusion laboratory-adapted phenotype. in the present study, the n d mutation emerged during the very brief exposure to culture, highlighting the advantage of lowering neuraminidase activity to reduce receptor cleavage for culture conditions versus the importance of neuraminidase for human infection. within the same cluster, s was altered during culture adaptation; this is a residue that contacts the opposing monomer in the dimer interface cluster ( , ) . the precise role of this residue in the functions of the dimer region will be explored. strikingly, several mutations emerged in residues at the opposite side of the hn dimer interface from the cluster at positions to : residues , , and . the points of contact of n and r with the opposing hn monomer in the dimer interface footprint had been noted to correlate with fusion promotion and airway adaptation; however, the function of these residues had been unexplored ( ) . the selection for l f, p l, and r k in the absence of mutations in the domain from positions to suggests that the newly identified important region in the hn dimer interface may act independently of receptor binding site ii, and the functional relationships between these two sites will be explored in future studies. numerous deep sequenced viral isolates also demonstrated p l and r k hn variants in the absence of mutations in the domain from positions to . the finding that this second dimer interface domain undergoes rapid selection in the background of authentic clinical strains suggests that it may be relevant to fitness in the airway. to investigate the biological significance of these adaptations, we generated singly mutated hn molecules in the background of our well-characterized hpiv- clinical virus hn, ci- ( ), and generated recombinant viruses bearing the hn mutations on the genetic background of ci- . brief exposure to culture, through this diversity of strategies, resulted in increased fusion promotion for all the hn variants tested: h q, s n, l f, l s, n d, p l, and r k. while perhaps not unexpected based on our previous hypotheses, the absolute consistency of this finding-that through a range of specific mutations and specific functional and structural differences, the virus arrives at increased fusion-is remarkable. each individual hn mutation conferred fitness for recombinant virus infection of immortalized monolayer culture cells and for spread of virus through the culture, compared to the parent wt ci. to explore the components of hn's properties that contribute to the enhanced fusion promotion and infection properties for all the emerging hn mutants, we assessed neuraminidase activity and hn's release of receptor-bearing target cells, a measure of the balance between receptor avidity and cleavage ( ) . we have shown that clinical viruses, compared to laboratory strains, have a combination of high neuraminidase, low avidity, low f triggering, resulting in lower fusogenicity. the hn mutants that emerged here all except for hn h q had decreased neuraminidase activity. it is of interest that mutations at site ii affect neuraminidase enzymatic activity, since this site is not known to possess enzymatic activity, which resides in the bifunctional active site at t /d ( , ( ) ( ) ( ) . one mechanistic conjecture that will be explored is that alterations or interactions at one hpiv- hn site (site i or ii) modulate the function of the other site, as we have shown for another paramyxovirus, newcastle disease virus, where receptor interaction at hn site i leads to the activation of site ii ( , ) . these data may suggest an unexplored cross talk between site i and site ii that could alter neuraminidase activity allosterically. all the mutants-including hn h q-showed reduced release of rbcs compared to the clinical strain. the hn h q mutant's decreased release of rbcs in the face of increased neuraminidase activity relative to the ci wt is in agreement with our previous finding that this mutation confers increased receptor avidity ( ) that balances the receptor-cleaving activity for an overall retention of contact. interestingly, the h q mutation in the lab-adapted hn background does not have higher neuraminidase activity ( ), supporting the notion that the phenotype conferred by these sites may be modified in the context of the genetic background. collectively, the pattern is of enhanced receptor engagement and an increase in fusogenicity that confers fitness in vitro. the genetically engineered viruses bearing the individual alterations in hn show that each is fit for growth in monolayer culture compared to the wt parent and less fit for growth in hae. the mutations emerging during the brief viral isolation process thus led directly to fitness for growth in monolayer culture. interestingly, there is no single mutation or minor allele required for successful growth in culture, although all samples in this study did produce at least one nonsynonymous minor allele on either the f or hn proteins. the minor alleles were extremely biased toward mutations in the hn or f protein, with eight of ten cultures presenting minor alleles on the hn protein and four presenting a minor allele on the f protein, supporting the critical nature of the hn-f fusion complex for host adaptation ( ). intriguingly, single mutations in hn did not confer fatal growth disadvantage in the airway model, despite conferring fitness in monolayer culture. it will be of interest to determine whether an accumulation of several fusion-skewing mutations is necessary for an absolute inability to grow in airway and whether a more sensitive experimental system (i.e., infection in vivo) would be responsive to single alterations or only to a greater dimer interface disturbance. the genome sequences presented here provide a significant resource for future hpiv- genomic epidemiology and biochemical characterization, increasing the number of publicly available genomes by more than % ( , ) . we obtained full genome sequence coverage from as few as an estimated , starting copies ( ) and found that relatively uniform whole viral genome coverage can be achieved with , paired-end reads, suggesting that many samples could be sequenced in a single miseq run. whole-genome viral sequencing from clinical samples should be both technically and economically feasible, permitting investigators to avoid the use of laboratoryadapted strains of virus in immortalized cultured cell lines. the deep genome-wide screens of paired clinical samples and viral isolates allow for simple, rapid interrogation of viral evolution in the context of multiple clinical strain backgrounds. the methods pursued here allowed for discovery of novel hn residues of interest that heretofore had not been identified based on consensus sequence. deep sequencing after rapid selection pressure may allow for discovery of a greater number of novel mutations that may be removed from the population by drift or competition with other variant subpopulations after longer selection periods yet provide clues to function. other techniques that can more comprehensively profile single mutations, such as deep mutational scanning, are often limited by their use of singular backgrounds, often in the context of culture-adapted strains, and focus on singular proteins. our results are consistent with an "uncertainty principle" for virology: the act of attempting to study a clinical viral sample through commonly performed laboratory procedures, such as viral culture in cell lines, affects the authenticity of the viral biology even before the first passage. that viruses evolve uniquely in culture has been shown for hpiv- ( ) ; however, the extraordinarily rapid nature of evolution in culture, even without passage, is notable. most of the adaptive genotypes could be found as minor alleles only by deep sequencing of clinical samples and viral isolates. with the increased provision of deep coverage viral genomes obtained directly from clinical samples due to diagnostic mngs, experiments similar to the one outlined here will be critical for potentially pathogenic viruses in order to assess whether previous viral characterization studies apply to newly sequenced clinical samples ( , ) . for hpiv- , our results are consistent with the notion that use of authentic culture models that better reflect hpiv- growth in vivo is critical if we are to understand and characterize pathogenesis ( ) . the specific mutations that emerged in the cultured samples, while disturbing from the perspective of the "uncertainty" described above, provide a wealth of information about hn domains that may be critical for fitness in humans. in our focus on the correlation of emerging hn mutations to function, we did not characterize all combinations of hn mutations, and for functional studies, we paired hns with a lab-adapted more fusogenic f protein in order to readily discern differences in hn properties; ongoing studies will explore the potential for epistatic interactions in hpiv- evolution. of particular interest is the cluster of residues around p and r that were uncovered as critical in this study. the interactions of these residues with the opposing monomer, and these points of contact of this domain within the interface, were suggested based on structural data to be relevant for fusion promotion ( ) . future studies will address the function of this hn dimer interface domain in hpiv- fusion and cell entry. sample description. the term "clinical sample" is used to describe primary nasal swabs in viral transport media, while "viral isolate" or "culture isolate" is used to describe culture harvest from zero passage virus (i.e., brief exposure to culture only during viral isolation). for paired clinical sample/culture isolate sequencing, l of nasal swab viral transport medium from nine patients from to with an hpiv- cycle threshold (c t ) of Ͻ was inoculated onto primary rhesus monkey kidney (rhmk) cells (quidel diagnostics) and incubated on a roller drum at °c. the tubes were assessed for cytopathic effect daily and confirmed via hemadsorption with % guinea pig red blood cells (rbcs). positive cultures were harvested and stored at Ϫ °c. deidentified bronchial alveolar lavage (bal) fluid and nasal swab samples from patients from used for prior clinical testing were stored at Ϫ °c and subjected to mngs. in addition, culture harvests for which viral isolation was performed for prior clinical testing of bal fluid samples, nasal swabs, and sputum samples from to at the university of washington virology laboratory were also stored at Ϫ °c and subjected to mngs. culture harvests from to were performed using the same protocol detailed above for the paired sequencing experiment. use of excess deidentified clinical specimens was approved by the local university of washington institutional review board (irb) (protocol study ). sample metadata, including genbank accession numbers for consensus hpiv- genomes, are available in table s in the supplemental material and associated with ncbi bioproject . mngs and amplicon sequencing. mngs was performed as previously described ( ) . rna was extracted from l of thawed culture harvests and clinical samples using the zymo viral rna kit (zymo research) and treated with turbo dnase i (thermo fisher) for min at °c ( ) . doublestranded cdna was made using random hexamers, superscript iii reverse transcriptase (thermo fisher), and sequenase v . (thermo fisher) and cleaned using zymo- dna clean and concentrator (zymo research). mngs libraries were produced by adding one-third volume of nexteraxt (illumina) and finished with cycles of dual-indexed pcr amplification and cleaned using . ϫ ampure beads (beckman coulter) ( ) . libraries were sequenced using one -bp and two -bp runs on an illumina miseq system. libraries from the matched viral isolates were prepared twice in independent library preparations and sequencing runs to control for artifacts of library preparation. amplicon sequencing was performed to investigate linkage between mutations recovered during mngs. reverse transcription-pcr (rt-pcr) was performed using cycles of the default parameters (melting temperature [t m ], °c) in the one-step rt-pcr kit (qiagen) with pcr primers hpiv - f (f stands for forward) ( =-tacagatagggataataactgtaaa- =) and hpiv - r (r stands for reverse) ( =-gctttgctcctaagttttttatatt- =). amplicons were purified using . ϫ ampure beads, and dualindexed truseq libraries were prepared using the kapa hyperprep kit with eight cycles of pcr amplification ( ) . sequence data alignment and analysis. sequencing reads were adapter and quality trimmed using cutadapt and then aligned to the hpiv- reference sequence (nc_ ) with geneious v . . . consensus genomes were extracted for original patient samples, and culture sample sequencing reads were aligned to the sample consensus. consensus genomes were visualized and mutations and minor alleles with coverage of Ͼ ϫ and minor allele frequency (maf) of Ͼ % were called using geneious . . . the known locus of rna editing in the phosphoprotein gene was not included among the variant nonsynonymous alleles, nor were adenosine deaminase-like changes present on the same strand in p virus (affecting minor variants at r g and i v in ci- in table ). phylodynamic analysis. for phylodynamic analysis, coding nucleotide sequences from the newly generated hpiv- genomes sequenced were extracted, concatenated, and aligned using genious v . . and mafft ( ) . the resulting alignment was used to generate a bayesian skyline tree with an uncorrelated relaxed clock and hasegawa, kishino, and yano (hky) substitution model with gamma plus invariant site heterogeneity model (four gamma categories) and three codon partitioning in beast v . . with a markov chain monte carlo (mcmc) length of , , iterations ( ) . tree generation trace information and evolutionary clock rates were gathered and analyzed using tracer v . . ( ). the resulting trees were annotated and compiled using treeannotator v . . and then annotated and scaled in figtree v . . . the same phylodynamic protocol was also performed on all complete hpiv- genomes present in ncbi nt as of august . recombination detection was performed using rdp 's rdp, chimaera, maxchi, genecov, siscan, and seq algorithms with default settings ( ) . to determine whether culture-associated hn amino acid changes were present in our paired sample sequencing, we downloaded all partial and complete hpiv- hn sequences from ncbi nt as of august . metadata on whether specific sequences originated from clinical sample or culture material were gathered from the ncbi genbank sequence record and associated manuscripts in pubmed. however, it is not always possible to determine whether there was brief exposure to culture conditions prior to sequencing. to show how the background of hn protein sequence could influence the amino acid mutations selected during culture, a majority consensus amino acid sequence of hn protein from each putative clinical sample was extracted and aligned using mafft. a neighbor-joining tree was created using the genious tree building algorithm, jukes-cantor distance method, and , replicates. functional characterization of hpiv- hn. the hn mutants examined in this study were generated through site-directed mutagenesis of the previously constructed pcaggs mammalian expression vector with the ci- hn gene ( ) . plasmids were sanger sequence confirmed prior to usage. transfections with plasmids were performed in hek t cells using lipofectamine in accordance with the manufacturer's instructions. cell surface expression of each hn was measured through standard immunofluorescence assay procedures using monoclonal antibodies against hpiv- hn and used for normalization as appropriate. two monoclonal anti-hn antibodies used here (vt- g -b and vt- f -b ) were custom-made by aldevron using dna immunization. briefly, a cdna encoding hpiv- hn was cloned into expression plasmids (pegfp) ( ) . groups of laboratory rats (wistar) were immunized by intradermal application of dna-coated gold particles using a handheld device for particle bombardment (gene gun). serum samples were collected after a series of immunizations and tested in flow cytometry on hek cells transiently transfected with the above expression plasmids. antibody-producing cells were isolated and fused with mouse myeloma cells (ag ) according to standard procedures. measurement of fusion promotion. the ability of individual hns to promote fusion was measured through a ␤-galactosidase complementation assay as previously described ( , ) . hns were paired with the f protein from a lab-adapted strain ( ), transiently transfected in hek t cells, and then cell fusion was quantified. lab-adapted f partner molecules were used as previously described ( , ) , since fusion is minimal when ci hn and f are expressed in pairs, and does not allow for quantitative comparisons. measurement of neuraminidase activity. neuraminidase activity of hns were measured as previously described ( , ) . hek t cells were transiently transfected with individual hns, collected through detachment with mm edta, washed with dulbecco phosphate-buffered saline (dpbs) supplemented with g/liter d-glucose and mg/liter sodium pyruvate (gibco), then resuspended in co -independent medium (ph . ) (gibco). cells were then added to =-( -methylumbelliferyl)-␣-d-n-acetylneuraminic acid, sodium salt hydrate ( -munana) at a : ratio for a final concentration of . mm. fluorescence was measured every min for h at °c with a microplate reader (tecan). measurement of rbc release. the ability of hns to release bound rbcs was measured as described previously ( , ) . rbcs were bound to cells for min at °c and washed to remove unbound rbcs. co -independent medium (ph . ) was then added to the cells, and the cells were incubated at °c for the appropriate times before being collected and replaced for the next time point. after min, rbcs that remained bound to the cells were lysed with ack (ammonium-chloride-potassium) lysing buffer (gibco) and quantified by measurement of absorbance at nm. immortalized cell culture. cv- (african green monkey kidney) and t (human kidney epithelial) cells were grown in dulbecco's modified eagle's medium (dmem) (cellgro; mediatech) supplemented with % fetal bovine serum (fbs) and antibiotics at °c in % co . recombinant virus production and analysis. rhpiv clinical isolate (ci- )-egfp is a recombinant virus generated with a plasmid coding for the majority consensus sequence of hpiv- ci- ( , , ) obtained by deep sequencing and containing an enhanced green fluorescent protein (egfp) expression cassette between p and m viral genes. all mutants were derived from this unique sequence. recombinant viruses were generated by reverse genetics in the rhpiv -ci- -egfp-f k background using a modification of methods previously described ( ) . briefly, t cells were seeded in six-well plates. the following day, the cells were transfected using profection mammalian transfection system, calcium phosphate kit (promega) according to the manufacturer's recommendations with g of pflc plasmid encoding the full-length viral genome, ng of pcaggs-hpiv p, ng of pcaggs-hpiv l, ng of pcaggs-hpiv n, ng of pcaggs-t polymerase, ng of pcaggs-hpiv lab-adapted hn, and ng of pcaggs-hpiv lab-adapted f ( ). the following day, supernatant fluids were replaced with dmem supplemented with % fbs. two days after transfection, the cells were mechanically detached by pipetting up and down and overlaid onto cv- cells in a -cm culture dish. cultured cells were monitored by fluorescence microscopy until % of the cells were infected. the medium was replaced with dmem without fbs, and cells were incubated for up to days at °c in % co . supernatant fluid was collected, centrifuged at ϫ g for min at °c, aliquoted, and kept at Ϫ °c until use. all recombinant viruses were sequenced at this stage using the same mngs methods detailed above on l of supernatant fluid to confirm the correct ci backbone and hn mutation sequences before use in experiments. viral propagation in human airway epithelia. the hae epiairway air- system (mattek corporation) consists of normal human-derived tracheo/bronchial epithelial cells that have been cultured to form a pseudostratified, highly differentiated mucociliary epithelium closely resembling that of epithelial tissue in vivo. upon receipt from the manufacturer, human airway epithelium (hae) cultures were transferred to six-well plates (containing ml medium per well) with the apical surface remaining exposed to air and incubated at °c in % co . hae cultures were infected with , pfu of recombinant clinical isolate egfp ( ) (referred to as ci- -egfp-f k ) at the apical surface for min at °c. the wild type (wt) ci- -egfp-f k or ci- -egfp-f k harboring single mutations in hn (i.e., p l, r k, h q, s n, or l f) was used. at min, the medium containing the inoculum was removed, and cultures were placed at °c and fed each day with ml medium via the basolateral surface. viruses were harvested by adding l of ϫ phosphate-buffered saline (pbs) with calcium and magnesium chloride (mattek corporation) per well to the hae cultures' apical surface and equilibrating for min at °c. the suspension was then collected, and viral titers were determined as previously described ( ) . this viral collection was performed sequentially with the same wells of cells on each indicated day postinfection. viral propagation in cv- . cv- cultures were seeded in -well plates. seventy percent confluent cells were infected with , pfu of recombinant ci- -egfp-f k (wt or harboring single mutations in hn [i.e., p l, r k, h q, s n, l f]) at the apical surface for min at °c. after min, the medium containing the inoculum was replaced with ml of dmem supplemented with % fbs. the supernatant fluid was then collected, aliquoted, and replaced with the same volume of dmem supplemented with % fbs, and viral titers were determined as previously described ( ) . this viral collection was performed sequentially with the same wells of cells on each indicated day postinfection. supplemental material for this article may be found at https://doi.org/ . /mbio . - . table s , pdf file, . mb. global burden of acute lower respiratory infections due to respiratory syncytial virus in young children: a systematic review and meta-analysis control of an outbreak of human parainfluenza virus in hematopoietic stem cell transplant recipients epidemiology and clinical presentation of the four human parainfluenza virus types features of circulating parainfluenza virus required for growth in human airway circulating clinical strains of human parainfluenza virus reveal viral entry requirements for in vivo infection interaction between the hemagglutinin-neuraminidase and fusion glycoproteins of human parainfluenza virus type iii regulates viral growth in vivo adaptation of human parainfluenza virus to airway epithelium reveals fusion properties required for growth in host tissue unity in diversity: shared mechanism of entry among paramyxoviruses triggering of human parainfluenza virus fusion protein (f) by the hemagglutininneuraminidase (hn) protein: an hn mutation diminishes the rate of f activation and fusion mechanism of fusion triggering by human parainfluenza virus type iii: communication between viral glycoproteins during entry regulation of paramyxovirus fusion activation: the hemagglutinin-neuraminidase protein stabilizes the fusion protein in a pretriggered state the hemagglutininneuraminidase of human parainfluenza virus type : role of the neuraminidase in the viral life cycle a structural explanation for the low effectiveness of the seasonal influenza h n vaccine effects of egg-adaptation on receptor-binding and antigenic properties of recent influenza a (h n ) vaccine viruses cell culture adaptation of hepatitis c virus and in vivo viability of an adapted variant sequential mutations associated with adaptation of human cytomegalovirus to growth in cell culture antigenic drift of the pandemic a(h n ) influenza virus in a ferret model clinical sequencing uncovers origins and evolution of lassa virus quantification of bk virus standards by quantitative real-time pcr and droplet digital pcr is confounded by multiple virus populations in the who bkv international standard copy number heterogeneity of jc virus standards cooperating h n influenza virus variants are not detectable in primary clinical samples prolonged shedding of human coronavirus in hematopoietic cell transplant recipients: risk factors and viral genome evolution global epidemiology of non-influenza rna respiratory viruses: data gaps and a growing need for surveillance a decade of rna virus metagenomics is (not) enough measurements of intrahost viral diversity are extremely sensitive to systematic errors in variant calling a second receptor 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virus hemagglutinin-neuraminidase receptor binding: effect of receptor avidity and steric hindrance at the inhibitor binding sites paramyxovirus receptor-binding molecules: engagement of one site on the hemagglutinin-neuraminidase protein modulates activity at the second site second sialic acid binding site in newcastle disease virus hemagglutinin-neuraminidase: implications for fusion database resources of the national center for biotechnology information clinical metagenomic identification of balamuthia mandrillaris encephalitis and assembly of the draft genome: the continuing case for reference genome sequencing rapid metagenomic identification of viral pathogens in clinical samples by real-time nanopore sequencing analysis rapid metagenomic next-generation sequencing during an investigation of hospital-acquired human parainfluenza virus infections a metagenomic analysis of pandemic influenza a ( h n ) infection in patients from north america rule-out outbreak: -hour metagenomic next-generation sequencing for characterizing respiratory virus source for infection prevention myeloablation-associated deletion of orf in a human coronavirus e infection mafft: a novel method for rapid multiple sequence alignment based on fast fourier transform bayesian phylogenetics with beauti and the beast . tracer v mechanism of interference mediated by human parainfluenza virus type infection infection of ciliated cells by human parainfluenza virus type in an in vitro model of human airway epithelium human parainfluenza virus infection of the airway epithelium: the viral hemagglutinin-neuraminidase regulates fusion protein activation and modulates infectivity we thank the patients from western washington state and staff of the university of washington virology laboratory who contributed to the project. this work was supported by nih/national institute of allergy and infectious diseases (niaid) r ai and r ai to a.m. r.c.s. received support from the mary gates endowment for students. key: cord- -v uc ijw authors: girardi, erika; chane-woon-ming, béatrice; messmer, mélanie; kaukinen, pasi; pfeffer, sébastien title: identification of rnase l-dependent, ′-end-modified, viral small rnas in sindbis virus-infected mammalian cells date: - - journal: mbio doi: . /mbio. - sha: doc_id: cord_uid: v uc ijw small rnas play a critical role in host-pathogen interaction. indeed, small rna-mediated silencing or rna interference (rnai) is one of the earliest forms of antiviral immunity. although it represents the main defense system against viruses in many organisms, the antiviral role of rnai has not been clearly proven in higher vertebrates. however, it is well established that their response to viral infection relies on the recognition of viral rnas by host pattern recognition receptors (prrs) to trigger activation of the interferon pathway. in the present work, we report the existence of a novel small noncoding rna population produced in mammalian cells upon rna virus infection. using sindbis virus (sinv) as a prototypic arbovirus model, we profiled the small rna population of infected cells in both human and african green monkey cell lines. here, we provide evidence for the presence of discrete small rnas of viral origin that are not associated with the rna-induced silencing complex (risc), that are highly expressed and detected by northern blot analysis, and that accumulate as - to -nucleotide (nt) species during infection. we report that the cellular antiviral endoribonuclease rnase l cleaves the viral genome, producing in turn the small rnas. surprisingly, we uncovered the presence of a modification on the ′-end nucleotide of sinv-derived viral small rnas (svsrnas) that might be at the origin of their stability. altogether, our findings show that stable modified small viral rnas could represent a novel way to modulate host-virus interaction upon sinv infection. continuous arms race with their hosts, the outcome of which might ultimately result in either viral persistence, clearance of the viral infection, or death of the infected cells. the arsenal of antiviral defenses and viral counter defense mechanisms is vast and very diverse among the different phyla of life. plants, insects, nematodes, and fungi mostly (if not exclusively) rely upon an innate immune response, whereas mammals have evolved an adaptive response in addition to this first line of defense. in the former organisms, the antiviral component of the innate immune response is mainly based on a highly conserved process commonly referred to as rna silencing, or rna interference (rnai). in this phenomenon, long double-stranded rnas of viral origin are cleaved by the rnase dicer into small interfering rnas (sirnas), which are then assembled into effector complexes that contain a member of the argonaute (ago) family; this results in targeted degradation of viral messenger or genomic rnas ( ) . in mammals, another type of innate immunity has evolved to control viruses ( ) . the initial recognition of nonself viral nucleic acids by extra-and intracellular sensors triggers the activation of type i interferons (ifns) ( ) . this leads in turn to the upregulation of numerous ifn-stimulated genes (isgs) ( ) , which either directly trigger a signaling cascade directed against the viral infection or mobilize other cells of the immune system. isgs encode proteins involved in apoptosis induction, protein synthesis blocking, or in the regulation of mrna editing or rna degradation ( ) . one key factor involved in ifn type i-mediated host defense is rnase l, a latent cytoplasmic endoribonuclease that is activated by =, =-oligoadenylates in response to double-stranded rna (dsrna) sensing and that cleaves both viral and cellular rnas ( ) . although extensive deep sequencing analyses have been performed to identify sirnas in mammalian cells infected with several rna viruses, there is so far no good evidence for a possible antiviral role of rnai in mammalian somatic cells ( ) . nevertheless, another class of small noncoding rnas, micrornas (mirnas), has been shown to play an important role during viral infection in mammals. mirnas are evolutionarily conserved small rnas derived from large primary transcripts, which are sequentially processed by the respective nuclear and cytoplasmic rnase iii enzymes, drosha and dicer, to generate mature single-stranded~ -or -nucleotide (nt) rnas ( , ) . similar to sirnas, they are incorporated into an effector ago-containing rna-induced silencing complex (risc), whereby they mediate posttranscriptional regulation of target mrnas via partially complementary sites ( ) . on one hand, mirnas of cellular origin can directly or indirectly regulate viral infections ( , ) . on the other hand, some viruses have evolved the capacity to encode their own mirnas, which represent an ideal tool to stealthily modulate the cellular environment. to date, viral mirnas have been almost exclusively identified in the genomes of dna viruses, mostly herpesviruses ( ) ( ) ( ) ( ) ( ) , with the notable exception of bovine leukemia virus, a retrovirus with an rna genome ( ) . it has been commonly assumed that cytoplasmic rna viruses cannot encode mirnas, not only because they do not have access to the nuclear biogenesis machinery but also because their genomic integrity would be destabilized by mirna processing ( ) . nevertheless, it has recently been demonstrated that the insertion of a cellular mirna precursor into the =-end nontranslated region (ntr) of sindbis virus (sinv) genomic rna leads to its cytoplasmic processing without affecting the viral replication ( , ) . the arthropod-borne sinv is a small, enveloped, positive, single-stranded rna virus and is the prototype for the alphavirus genus. alphaviruses represent a group of widely distributed human and animal pathogens, which pose a serious public health threat ( ) . some of them induce febrile and arthritogenic diseases, while others can cause highly debilitating diseases, such as encephalitis. the sinv genomic rna is capped and polyadenylated and is infectious as naked rna. upon entry into the cytoplasm by endocytosis, the host translational machinery recognizes the genomic rna, and four nonstructural proteins are produced ( ) . their expression is sufficient for the establishment of replication complexes and the synthesis of a complementary antigenomic negative rna strand. the antigenome is necessary for the replication of the viral genome and the production of a subgenomic rna that ensures the translation of the structural proteins during the late phase of infection ( , ) . to date, sinv has not been shown to give rise to any small rna species in mammalian cells. as such, we decided to investigate the small rna profile of sinv-infected human and african green monkey cell lines. using small rna cloning and deep sequencing techniques, we provide evidence that the sinv genome is a source of small rnas in infected mammalian cells. some of these small rnas accumulate to levels detected by northern blot analysis. we set out to identify the factors involved in their biogenesis and show that they do not depend on the rna silencing machinery, but rather, that they are downstream products of the cytoplasmic ifn-induced endoribonuclease rnase l. finally, we also present experimental evidence that these viral small rnas are modified at their = extremity. altogether, our results indicate that the sinv-derived small rnas could represent key markers of the host defense mechanism. in order to investigate the presence and nature of virusderived small rnas in mammalian cells, we used a small rna cloning and deep sequencing approach. we generated and analyzed small rna libraries from both hek and vero cells infected with sinv or not infected with sinv. total rna was isolated from uninfected cells or cells infected with sinv at a multiplicity of infection (moi) of . plaque forming units (pfu) per cell h postinfection (hpi). using these conditions, we could detect cells with a high viral load without observing any apparent cell death as assessed by propidium iodide staining (see fig. s in the supplemental material). we also confirmed that until hpi, cells were still actively proliferating (data not shown). high-throughput sequencing yielded , and , , reads that mapped to the sinv genome in infected hek and vero cells, respectively (fig. a) ; these two numbers of reads represented a percentage of . and . %, respectively, of total mapped small rnas in hek and vero cells. the greater fraction of viral reads in vero cells is consistent with the fact that these cells are known to be more permissive to viral infections due to a defect in interferon production ( ) . the distribution of the reads on the viral genomic (positive) and antigenomic (negative) strands is indicated in fig. a . interestingly, the vast majority of the reads ( to %) originate from the genomic strand of the virus. nonetheless, we identified limited sequencing reads that were derived from the negative strand, albeit at extremely low levels in both libraries (fig. a) . their genomic distribution showed a peak at the = end of the antigenome (see fig. s a in the supplemental material). this specific region corresponds to the viral promoter, necessary for the replication of the viral genome and is known to fold into a conserved -nt stemloop structure ( ) . the length distribution of the antigenomic small rna reads peaked at a size of nt (fig. s b) , which corresponds to the = arm of the stem-loop structure (fig. s c ). although we could validate the presence of the -nt-long rna by northern blot analysis, we were unable to detect the accumulation of the small -nt rna as a discrete species (fig. s d) . the size distribution of the main population of viral reads mapping on the positive strand of the virus was broad with no defined peak at a specific length (fig. b) . however, a closer inspection revealed that the overall size and genomic distribution of the viral reads were highly similar in both hek and vero cells (fig. c) . moreover, a greater number of reads were found between nucleotides and of the viral genome, corresponding to the abundantly expressed subgenomic rna. we selected candidates among the most abundant peaks obtained in both cell types for validation by northern blot analysis (see fig. s a in the supplemental material). we could detect several sinv-derived viral small rnas (svsrnas) of to nt in length that were derived from all along the viral genome (fig. s b ). among them, svsrna- , - , and - were detected in both hek and vero cells (fig. s c) . the presence of abundant larger bands accumulating between and nt was also evidenced. of note, the expression of both larger and smaller viral rnas increased proportionally to the accumulation of the viral genome during a time course of infection ( fig. d and e) . the identification of sinv-derived small rnas prompted us to analyze more closely their origin and biochemical properties. the production of svsrnas does not rely on the mirna biogenesis machinery. the identification of small rnas from rna viruses could be seen either as a result of a productive mechanism for the pathogen (as for virus-encoded mirnas) or as the consequence of a host response to control the infection by degrading viral rnas. sinv expresses four enzymes that play key functions during the viral replicative cycle. however, none of these enzymes is known to possess an endoribonuclease activity ( ) that could enable the processing of small rnas from a viral rna precursor. therefore, cellular enzymes have to be involved in the biogenesis of sinv srnas. in order to identify them, we decided to knock down cytoplasmic enzymes with a known rnase activity. we first focused on proteins important for the production of small rnas, such as mirnas. hence, we transfected sirnas against drosha, dicer, or ago followed by an infection with sinv and an analysis of viral small rna accumulation. although drosha is known to be mostly nuclear, we also took it into consideration because it was recently shown to relocalize to the cytoplasm upon sinv infection ( ) . although we were able to downregulate very efficiently drosha, dicer, and ago (see fig. s a to c in the supplemental material), the knockdown of these factors neither affected the viral load nor the accumulation of svsrna- , - and - ( fig. s d to g). we also examined whether we could detect an sirna signature among all viral reads by looking for small rnas that could perfectly base pair on or nt and present a -nt overhang in =. a very limited number of such duplexes could be found, but they were not more represented than larger duplexes presenting with = or = overhangs (data not shown). it has been reported that small rnas, which are not generated by dicer or drosha cleavage, could nevertheless be assembled into argonaute proteins (e.g., trna fragments [ ] ). we therefore immunoprecipitated argonaute proteins followed by northern blot analysis to see whether svsrnas could be detected within the risc. however, although we could detect the accumulation of the cellular mirna mir- , using both an antibody specific for ago (fig. s h , left panel) and an antibody able to recognize all four ago proteins (fig. s h , right panel), we were unable to detect svsrnas within immunoprecipitated risc. rnase l is involved in the production of svsrnas. we then hypothesized that an endoribonuclease involved in the antiviral response could be implicated in svsrna production. rnase l is a ubiquitous, cytoplasmic, interferon-induced endonuclease that plays a key role in the cellular response to viral infections. it cleaves single-stranded rna regions after the dinucleotides ua/uu, thereby preventing virus accumulation ( ) . as such, we knocked down rnase l using sirnas, before infecting the cells with sinv. as expected, rnase l knockdown (kd) resulted in the upregulation of the sinv genome by approximately . -fold ( fig. a and b) . interestingly, it also resulted in a dramatic reduction of the viral small rnas svsrna- , - , and - and their precursors (fig. c) . in order to extend this observation and assess the role of rnase l in svsrna generation, we also produced small rna libraries from hek cells treated with a control or an rnase l-specific sirna prior to sinv infection. in accordance with the striking effect seen on the three specific svsrnas, reduction in rnase l protein level decreased the entire population of clonable viral sequences from . to . % (see fig. s a in the supplemental material). however, rnase l knockdown affected neither the size distribution of viral reads nor their genomic distribution ( fig. s b and c). given the cleavage properties of rnase l (see next section), it was unlikely that the identified small rnas were direct products of this enzyme. we therefore looked for other factors implicated in rna degradation that could affect svsrna accumulation. we used rnai to downregulate the expression of the major cytoplasmic =- = exoribonuclease xrn (fig. d ). as expected, the capped viral genome was not affected by downregulation of xrn (fig. e) . however, the levels of svsrna- , - , and - and their precursors were stabilized (fig. f) . we then looked at the effect of combining knockdown of these factors (see fig. s in the supplemental material). we found that the double knockdown of both rnase l and dicer did not affect the accumulation of svsrna- compared to the single rnase l knockdown, which confirms that dicer does not play a role (fig. s a) . on the contrary, the accumulation of svsrna- upon double knockdown of both rnase l and xrn increased compared to the single rnase l knockdown (fig. s a ). this finding suggests that both rnase l and xrn can act upon viral rnas and have opposite activities in terms of generation and stabilization of the svsrnas. xrn might act upstream of rnase l in degrading, for example, uncapped viral rnas that would otherwise be a substrate for rnase l; alternatively, it could directly act upon the rnase l products. the former hypothesis is more likely though because xrn has a preference for = phosphorylated rnas, whereas rnase l leaves a = oh after cleavage. we also performed a high-molecular-weight northern blot analysis to look at the effects of the various knockdowns on the accumulation of the genomic and subgenomic viral rnas. the results confirm the quantitative pcr data, in that only the rnase l kd has an impact on the accumulation of both genomic and subgenomic rnas (fig. s b) . we also tested several cytoplasmic =- = exoribonucleases, including dis l, dis l , and rrp (also known as exosc , a core subunit of the exosome); knockdown of these factors had no effect on svsrna accumulation (data not shown). this indicated that at least the main cytoplasmic =- = exoribonucleases do not seem to be implicated in the production of the viral small rnas. the = extremity of svsrna- is modified. rnase l processing produces a typical signature: the enzyme leaves a = hydroxyl (oh) and a = phosphate (p) after cleavage ( ) . however, these features are not compatible with our small rna cloning protocol ( ) , which strictly relies on the presence of a = p and a = oh on the small rna extremities. this prompted us to characterize the extremities of svsrnas to better understand their biogenesis. we initially decided to restrict our analysis to svsrna- because it accumulated as a major form of nt. the assay used for the analysis of rna = ends involved digestion with the terminator exonuclease, a processive =- = exonuclease that specifically targets and degrades rnas with a = monophosphate, but not rnas with = triphosphates, = oh, or a = cap ( ) . for a control, the terminator reaction was carried out on both a = oh and a = p oligoribonucleotide with the same sequence as svsrna- . terminator treatment did not result in the degradation of the = oh synthetic oligoribonucleotide, whereas the = p rna oligonucleotide was almost completely degraded. accordingly, svsrna- is prone to degradation by the terminator exonuclease, which is consistent with the presence of a monophosphate at the = end, while the accumulation of the precursor bands was less affected by the treatment (fig. a) . next, we performed sodium periodate (naio ) treatment followed by ␤-elimination to specifically examine the = end of svsrna- . the presence of hydroxyl groups in positions = and (b and e) sinv genomic rna was quantified by rt-qpcr, before and after rnase l or xrn kd. the relative quantification is normalized to gapdh levels and is represented on a logarithmic scale as a mean of three replicates. (c and f) northern blot analysis on total rna from infected hek cells before and after rnase l or xrn kd. u was used as a normalizer. = on the last ribose is needed for the periodate reaction. ␤-elimination of the oxidized rna results in an rna product one or two nucleotides shorter. on the contrary, the presence of modifications on the ribose of the = terminal residue renders the rna insensitive to periodate oxidation and impairs the chain scission ( ) . for a control for the reaction, we measured its effect on the unmodified cellular mirna mir- . as expected, the electrophoretic mobility of the mirna strongly changed after treatment (fig. b) . in contrast, svsrna- mobility was found to be insensitive to periodate oxidation and ␤-elimination (fig. b) . this indicated the presence of a modification on the terminal ribose, most likely in the = position, which is perfectly compatible with our cloning protocol. interestingly, it also seems that the intermediate rna products are insensitive to periodate oxidation and are consequently modified (see fig. s in the supplemental material). moreover, we were also able to assess the existence of a modified = end for other svsrnas by northern blotting (fig. s ) . in order to gain more insight into the nature of this modification, we treated our samples with calf intestinal phosphatase (cip) (fig. c, lanes , , , , , , , and ) prior to ␤-elimination (fig. c, lanes , , , and ) . the double treatment strongly affected the mobility of a control oligoribonucleotide with a = p (fig. c, lane ) , while the mobility of the = oh oligonucleotide was affected independently of the cip treatment (fig. c, lanes and ) . of note, the combined reactions changed neither the mobility of a control oligoribonucleotide bearing a cip-insensitive modification (methyl group in position = of the last ribose) (fig. c, lane ) nor the mobility of svsrna- (fig. c, lane ) . taken together, these results show that svsrna- is modified at its = extremity but that the modification is not due to a monophosphate group caused by a direct rnase l cut. viral genomic rna is modified on a . the presence of posttranscriptional modifications, such as =-o-methylation, is a characteristic of several cellular rnas in mammals, such as rrnas ( ) , snrnas ( ) , and piwi-interacting rnas (pirnas) ( , ) . recently, it has also been reported for several viruses that their genomic rna can be =-o-methylated at specific positions ( , ) . we hypothesized that the modification on the small rna could be derived from a modification at the level of the genomic rna, which is reasonable given the fact that svsrna- precursor rnas also appear to be modified (see fig. s in the supplemental material). we thus looked for modifications in the genomic region spanning svsrna- using the rtl-p (reverse transcription at low deoxyribonucleoside triphosphate concentrations followed by polymerase chain reaction) technique ( ) : the tendency of the reverse transcriptase to pause immediately before a =-omethylated nucleotide is exploited to establish the efficiency of retrotranscription by semiquantitative pcr, using differential deoxynucleoside triphosphate (dntp) concentrations. as shown in fig. a , we designed a reverse transcription primer specific for the viral genome, as well as primers for semiquantitative pcr in the region surrounding svsrna- (shown in red): one reverse primer (r) and three forward primers, upstream (f u ) or downstream of the putative modification (f d and f d ). after retrotranscription with low and high dntp concentrations, we performed multiplex pcr using primers f u /f d (in the svsrna- region) or primers f d /f d (in the control region) and then compared the signal intensity ratios of pcr products amplified with different numbers of pcr cycles (fig. b ). our data indicated that only the f u /f d ratio changed depending on the rt conditions (fig. b, top panel) , suggesting that at a low dntp concentration, the reverse transcriptase paused in the svsrna- region, most probably due to the presence of a modification on the viral genome in the region of interest. to further strengthen this observation and to identify the precise position of the nucleotide modification, we performed primer extension assays on rna isolated from either sinv-infected hek or vero cells (fig. c ). =-o-methylation can cause reverse transcriptase to pause one nucleotide before and/or at the o-methylated nucleotide. indeed, we identified two bands (rt stops) that correspond to nucleotides g and a , the latter corresponding precisely to the = end of the svsrna- sequence (fig. c) . these data imply that the =-end modification of svsrna- was likely already present on the viral genome and might be a =-o-methylation. =-end modification is a hallmark of svsrnas. taken together, our observations indicate that the sinv genomic rna is modified, and are therefore suggestive of a potential modification of svsrnas. we then decided to investigate to what extent reads mapping to the sinv genome were modified at the last nucleotide position. small rna libraries were prepared from noninfected or sinv-infected hek cells using total rna treated (or not) with naio . this treatment specifically impairs the = adapter ligation to rna species with unmodified = ends ( = and = oh), while rnas bearing a modification at one of these positions are resistant to oxidation, and can therefore be ligated. overnight ligation of the = adapter at °c was performed in order to enhance the cloning of =-o modified small rnas ( ) . after deep sequencing of these libraries, we mapped the total number of reads against both the human and sinv genome (table ). as expected, in both noninfected and infected cells, the naio treatment drastically reduced the number of clonable mirna sequences (see also fig. s a in the supplemental material). in contrast, modified small rnas (such as trna fragments) were preferentially cloned over mirnas upon naio treatment (fig. s b) . strikingly, almost % of the viral reads were kept after oxidation ( table ). the oxidation treatment did not dramatically change the genomic distribution of the viral reads compared to the nontreated sample (fig. a) . however, deep sequencing revealed some new peaks (fig. a, arrows) that we could also validate by ␤-elimination and northern blot analysis (see svsrna- and - in fig. s ) . the size distribution of the viral reads remained unchanged before and after naio treatment, indicating that sinv-derived modified rnas belonged to each length (fig. b) . finally, we analyzed the nucleotide composition of the = end of sinv small rnas and observed that we had enrichment in adenosine (a) at the = end after oxidation, which was not clearly discernible in the nontreated sample (fig. c ). this is in line with the specific =-omethylation occurring on internal adenosines recently identified on the genome of dengue virus ( ) . mammals have evolved a very sophisticated innate immune response to ward off pathogens that is based on the sensing of pathogen-associated molecular patterns (pamps) by dedicated pattern recognition receptors (prrs). when it comes to viral infection, one of the most potent pamps is dsrna. the recognition of dsrna by prrs triggers a signaling cascade that ultimately leads to the activation of interferon-sensitive genes (isgs) ( ) . rna silencing is another type of antiviral innate immunity, which, as of now, has mostly been demonstrated in plant and insect organisms ( ). nonetheless, a role for rna silencing as a host defense system against mammalian viruses could reasonably be considered. indeed, dicer, the key enzyme for dsrna cleavage is conserved in higher vertebrates, and although its main substrate in vivo is pre-mirna molecules, it is able to process long dsrna into short rnas in vitro ( ) . however, the question as to which other types of dsrna molecules mammalian dicer can cleave in vivo has been the matter of intense research, and especially so when considering rnas of viral origin. here, we wanted to make our contribution to the field by studying the prototypical arbovirus, sindbis virus. this type of virus can infect both insects and mammals, and as such is a useful tool to study the conservation of antiviral mechanisms in both phyla. intriguingly, it has been shown for some members of the alphavirus genus that their genomic rna can be processed into both sirnas and pirnas in insect somatic cells ( , ) . in this study, we generated small rna libraries from two different mammalian cell lines infected with sinv. although the percentage of sequences mapping to the viral genome was significant (~ . to~ % in hek and vero cells, respectively), we were unable to detect either sirna or pirna signatures. on the contrary, the viral reads displayed a highly biased strand distribution. although antigenomic (negative) strand-derived sequences represented a very small percentage of the total viral reads, they showed properties distinct from the genomic (positive) strand reads: their size distribution peaked at nt, and their localization on the viral genome is restricted to the promoters required for the synthesis of both the genomic and subgenomic rna. although the cloned -nt sequences were not visible by northern blot analysis, we could detect the accumulation of an~ -nt product that in our knowledge has never been observed before. we hypothesize that these rna species could be critical for viral replication, similarly to what has been previously reported for influenza virus ( ) . consistent with the relative abundance of the positive strand compared to the negative strand, the majority of reads were derived from the plus-strand rna and more specifically from the region corresponding to the subgenomic rna sequence. we could detect several hot spots of small rna accumulation, indicating that discrete sinv-derived viral small rna species (svsr-nas) could be produced in infected mammalian cells. these svs-rnas do not display any peak in size distribution and do not map to any alphavirus conserved regions. nonetheless, we noticed that they originate from the same viral genomic positions in both infected african green monkey and human cells. in order to confirm that this was not solely due to a bias in the small rna cloning protocol, we performed northern blot analyses of several small rna candidates. for some of the candidates, we were able to detect discrete bands, which increased in intensity throughout the time course of infection. we therefore focused on the characterization of the positivestrand-derived small rnas. one hypothesis regarding their identity could be that some of these svsrnas are in fact virus-encoded mirnas. using bioinformatics tools such as mirdeep ( , ) and miranalyzer ( , ) , we excluded the possibility that putative mirna precursor structures were present in the viral genome. nonetheless, we tested whether the mirna machinery was required for the production of viral small rnas by knocking down drosha, dicer, and ago . the downregulation of these factors affected neither sinv replication nor accumulation of the three most abundant identified svsrnas, indicating that their production must involve another rnase. we discovered that the rnase l endonuclease is implicated in svsrna genesis. indeed, its downregulation causes a dramatic reduction in the accumulation of sindbis viral small rnas detected by northern blot analysis; this is accompanied by an upregulation of the viral genomic rna. in order to extend this observation, we also generated small rna libraries from hek cells treated with sirnas against rnase l and infected with sinv. we found that, under these conditions, the global population of sinv srnas was downregulated. rnase l is an endoribonuclease that is present in the cell in the form of an inactive monomer. upon type i interferon induction, and more specifically =- = oligoadenylate synthetase activation ( ) , it can dimerize and then act upon its viral rna targets. rnase l is known to play an antiviral role against viruses such as west nile virus ( ), hepatitis c virus ( ) , and sinv ( ) . we also looked at the involvement of exoribonucleases in the production of svsrnas and found that, indeed, the accumulation of these small rnas is counteracted by the cellular =- = exonuclease xrn . this suggests that svsrnas represent stable viral products of cellular decay enzymes. we therefore hypothesize that rnase l is the enzyme primarily involved in the generation of small rnas and that the further processing operated by other uncharacterized enzymes could give rise to the final -to -nt-long viral rnas. it also remains possible that some of the fragments we detected are direct products of rnase l but are further modified by kinases and dephosphorylases, which would allow their cloning. the fact that rna fragments generated by rnase l are detected implies that some of them must be stabilized. indeed, we discovered that the viral small rna candidates appear to be modified on the = proximal nucleotide. we could extend this peculiarity to the global small rna population by cloning and sequencing small rnas after an oxidation treatment designed to prevent ligation of nonmodified rnas. our data indicate that this modification is most likely a =-o-methylation and seems to occur at the level of the viral genomic rna, preferentially on adenosines. thanks to a double approach of rtl-p and primer extension, we were able to map such a modification on position a of the sinv viral genome. moreover, our sequencing data indicate that there are most likely many others given the enrichment in a residues at the = end of small rnas cloned after oxidative treatment. the main protective mechanism against =- = degradation is a =-o-methylation on the = proximal ribose, which prevents degradation by exoribonucleases and thus helps to stabilize small rnas such as mirnas and sirnas in plants, pirnas in animals, and sirnas in drosophila melanogaster ( ) . this suggests that rna modification could have evolved as a response to protect the anti-pathogen small rnas during infections. posttranscriptional rna modifications represent an important but not yet fully understood system to regulate transcripts. cellular rnas contain many distinct posttranscriptional modifications at thousands of sites. in the same way, rna viruses take advantage of nucleoside modifications on the base or on the ribose of their genomic rna ( , ) as a host mimicry strategy to abrogate innate immune signaling ( ) . the enzyme responsible for the methylation of sinv rna remains unknown. however, it has been reported that the alphavirus nsp protein contains a putative methyltransferase (mt)-like domain, similar to dengue virus ns mtase ( ) . although the activity of this domain has not been verified yet, nsp -mtase mutant viruses exhibit a strong phenotype: negative-strand synthesis is continuous, and unstable viral replication complexes are produced. surprisingly, rnase l-deficient cells infected with sinv display the same phenotype, suggesting that the two proteins function in similar events ( ) . for all these reasons, we propose that rnase l-dependent, =-modified small rnas from sinv could be involved as intermediates in this mechanism. altogether, our findings show a link between the host degradation pathway and stabilization of modified viral small rnas. clearly, not all the small rnas identified by our deep sequencing approach correspond to rnase l downstream products. nonetheless, the fact that at least some of these small rnas are protected and detected might implicate them in signaling events, maybe upstream of the infection front, which might prove difficult to assess in cell culture. we hypothesized that by removing rnase l we might uncover signatures corresponding to other types of small rnas, such as sirnas or pirnas, but it did not turn out to be the case. this could be due to the fact that other enzymes might also be involved in the production of rna fragments and is an indication that it will make the detection of antiviral rnai complicated in differentiated mammalian cells. an alternate hypothesis could also be that sinv encodes an rna silencing suppressor that blocks dicer activity but has no effect on rnase l. finally, it is also possible that the antiviral rnai response might be detected only in specific subtypes of cells in mammals, which would be different from the differentiated somatic cells we used for our study. further investigation will be needed to answer these questions and to decipher the roles played by this type of virusderived small rnas, as well as their potential implication in the design of new antiviral therapeutic strategies. plasmid carrying wild-type (wt) and green fluorescent protein (gfp)-sinv genomic sequence (kindly provided by m. c. saleh) was linearized with xhoi and used as a substrate for in vitro transcription using mmessage mmachine capped rna transcription kit (ambion) following the manufacturer's instructions. sindbis viral stock was prepared in bhk hamster kidney cells, and titers were measured by plaque assay. hek and vero cells were maintained in dulbecco's modified eagle medium (dmem) (gibco) supplemented with % fetal bovine serum (fbs) (clontech) in a humidified atmosphere of % co at °c. cells were infected with sinv at an moi of . . samples were harvested at , , , , , , and h postinfection (hpi). rnai-mediated protein depletion. for sirna transfection, . ϫ hek cells were seeded in -well plates. transfections were carried out for h using nm on-target plus smart pool sirnas (dharmacon) for one-step depletion (human rnase l and xrn ) or nm on-target plus smart pool sirnas (dharmacon) for two-step depletion (human drosha, dicer, and ago ) with lipofectamine (invitrogen) following the manufacturer's instructions. the on-target plus nontargeting pool sirnas (dharmacon) was used as a negative control. cells were infected with sinv and harvested after h for rna and protein analysis. small rna cloning and sequencing. rna was extracted from noninfected and sinv-infected hek and vero cells hpi. small rna cloning was conducted with or g of total rna as previously described ( , ) . small rna libraries from sinv-infected hek cells treated with control or rnase l-specific sirnas were generated following the same protocol with some modifications. mainly, small rnas were ligated using degenerate = and = adapters to limit biases in the ligation step ( ) . this procedure was also used to prepare small rna libraries after naio treatment of g of total rna, as previously described ( ) , except that the = ligation was performed at °c overnight, in % polyethylene glycol (peg ) to favor the cloning of modified small rnas. sequencing was performed at the igbmc microarray and sequencing platform, illkirch, france, using different illumina instruments (genome analyzer iix, hiseq , and hiseq ) with a read length of or bp. a detailed description of the deep sequencing data analysis is available in text s in the supplemental material. microarray data accession number. the data discussed in this publication have been deposited in ncbi's gene expression omnibus ( ) and are accessible through geo series accession number gse (http: //www.ncbi.nlm.nih.gov/geo/query/acc.cgi?accϭgse ). supplemental material for this article may be found at http://mbio.asm.org /lookup/suppl/doi: . /mbio. - /-/dcsupplemental. text s , doc file, . mb. figure s , tif file, mb. figure s , tif file, . mb. figure s , tif file, . mb. figure s , tif file, . mb. figure s , tif file, . mb. figure s , tif file, . mb. figure s , tif file, . mb. figure s , tif file, . mb. figure s , tif file, . mb. antiviral immunity directed by small rnas innate immune recognition of viral infection pathogen recognition and innate immunity interferons at age : past, current and future impact on biomedicine identification of genes differentially regulated by interferon alpha, beta, or gamma using oligonucleotide arrays viral encounters with =, =-oligoadenylate synthetase and rnase l during the interferon antiviral response six rna viruses and forty-one hosts: viral small rnas and modulation of small rna repertoires in vertebrate and invertebrate systems a cellular function for the rna-interference enzyme dicer in the maturation of the let- small temporal rna the nuclear rnase iii drosha initiates mi-crorna processing the widespread regulation of microrna biogenesis, function and decay interferon modulation of cellular micrornas as an antiviral mechanism a cellular microrna mediates antiviral defense in human cells identification of virusencoded micrornas identification of micrornas of the herpesvirus family kaposi's sarcoma-associated herpesvirus expresses an array of viral mi-crornas in latently infected cells cloning and identification of a microrna cluster within the latency-associated region of kaposi's sarcoma-associated herpesvirus small rna deep sequencing identifies micrornas and other small noncoding rnas from human herpesvirus b rna virus microrna that mimics a b-cell oncomir is rna interference involved in intrinsic antiviral immunity in mammals? noncanonical cytoplasmic processing of viral micrornas evidence for a cytoplasmic microprocessor of pri-mirnas fields virology evasion of the innate immune response: the old world alphavirus nsp protein induces rapid degradation of rpb , a catalytic subunit of rna polymerase ii the alphaviruses: gene expression, replication, and evolution a structural and functional perspective of alphavirus replication and assembly defectiveness of interferon production and of rubella virus interference in a line of african green monkey kidney cells (vero) extensive terminal and asymmetric processing of small rnas from rrnas, snor-nas, snrnas, and trnas interferon action: rna cleavage pattern of a ( =- =)oligoadenylate dependent endonuclease identification of virally encoded micrornas human rna methyltransferase bcdin d regulates microrna processing use of specific chemical reagents for detection of modified nucleotides in rna small nucleolar rnas direct sitespecific synthesis of pseudouridine in ribosomal rna biogenesis of small nuclear rnps mouse piwi-interacting rnas are =-omethylated at their = termini the = termini of mouse piwi-interacting rnas are =-o-methylated =-o methylation of internal adenosine by flavivirus ns methyltransferase biochemical and structural insights into the mechanisms of sars coronavirus rna ribose =-omethylation by nsp /nsp protein complex rtl-p: a sensitive approach for detecting sites of =-o-methylation in rna molecules optimization of enzymatic reaction conditions for generating representative pools of cdna from small rna genetic analysis of resistance to viral infection dicing and slicing: the core machinery of the rna interference pathway production of virus-derived ping-pong-dependent pirna-like small rnas in the mosquito soma arbovirus-derived pirnas exhibit a ping-pong signature in mosquito cells influenza a virus expresses high levels of an unusual class of small viral leader rnas in infected cells discovering micrornas from deep sequencing data using mirdeep . mirdeep accurately identifies known and hundreds of novel microrna genes in seven animal clades miranalyzer: a microrna detection and analysis tool for nextgeneration sequencing experiments miranalyzer: an update on the detection and analysis of micrornas in highthroughput sequencing experiments rnase l plays a role in the antiviral response to west nile virus rnase l releases a small rna from hcv rna that refolds into a potent pamp alphavirus minus-strand synthesis and persistence in mouse embryo fibroblasts derived from mice lacking rnase l and protein kinase r regulation of small rna stability: methylation and beyond =-o methylation of the viral mrna cap evades host restriction by ifit family members overcoming the innate immune response to small interfering rna role for conserved residues of sindbis virus nonstructural protein methyltransferase-like domain in regulation of minus-strand synthesis and development of cytopathic infection reducing ligation bias of small rnas in libraries for next generation sequencing molecular characterization of geminivirus-derived small rnas in different plant species gene expression omnibus: ncbi gene expression and hybridization array data repository mfold web server for nucleic acid folding and hybridization prediction we are grateful to m. c. saleh for the gift of the plasmids for sinv preparation and g. meister for the anti-ago antibody. we thank frédéric gros for technical help with the fluorescence-activated cell sorting (facs) analyses. we acknowledge valérie cognat and the bioimage fa-cility (ibmp, strasbourg, france) for help with the initial analysis of the deep sequencing data. we also thank the igbmc microarray and sequencing platform, member of the france genomique program, for the sequencing of our libraries. we thank all members of the pfeffer laboratory, especially lee tuddenham for critically reading the manuscript and gabrielle haas for fruitful discussions. key: cord- - vsdqhi authors: burgess, stacey l.; buonomo, erica; carey, maureen; cowardin, carrie; naylor, caitlin; noor, zannatun; wills-karp, marsha; petri, william a. title: bone marrow dendritic cells from mice with an altered microbiota provide interleukin a-dependent protection against entamoeba histolytica colitis date: - - journal: mbio doi: . /mbio. - sha: doc_id: cord_uid: vsdqhi there is an emerging paradigm that the human microbiome is central to many aspects of health and may have a role in preventing enteric infection. entamoeba histolytica is a major cause of amebic diarrhea in developing countries. it colonizes the colon lumen in close proximity to the gut microbiota. interestingly, not all individuals are equally susceptible to e. histolytica infection. therefore, as the microbiota is highly variable within individuals, we sought to determine if a component of the microbiota could regulate susceptibility to infection. in studies utilizing a murine model, we demonstrated that colonization of the gut with the commensal clostridia-related bacteria known as segmented filamentous bacteria (sfb) is protective during e. histolytica infection. sfb colonization in this model was associated with elevated cecal levels of interleukin a (il- a), dendritic cells, and neutrophils. bone marrow-derived dendritic cells (bmdcs) from sfb-colonized mice had higher levels of il- production in response to stimulation with trophozoites. adoptive transfer of bmdcs from an sfb(+) to an sfb(−) mouse was sufficient to provide protection against e. histolytica. il- a induction during bmdc transfer was necessary for this protection. this work demonstrates that intestinal colonization with a specific commensal bacterium can provide protection during amebiasis in a murine model. most importantly, this work demonstrates that the microbiome can mediate protection against an enteric infection via extraintestinal effects on bone marrow-derived dendritic cells. port neutrophil infiltration in inflammatory-disease models ( ) ( ) ( ) ( ) . neutrophils play a central role in cell-mediated clearance of parasites and may be important in clearance of e. histolytica ( ) . iga was also implicated in protection against e. histolytica infection in a childhood cohort and thus may be involved in immunity against the parasite ( ) . given that il- a and downstream mediators are important in immunity to e. histolytica, components of the intestinal microbiota that elicit exogenous il- a in the absence of intestinal pathology might prove to be protective during amebiasis. segmented filamentous bacteria (sfb), or "candidatus savagella," are genetically and morphologically unique members of the clostridia. they represent an uncultivable component of the mammalian intestinal microbiota, reported to colonize both humans and mice ( ) ( ) ( ) . it has been demonstrated that sfb colonization induces a potent th helper response in the intestine that is dependent on intestinal dendritic cells ( , ) . sfb colonization is associated with higher intestinal levels of both il- a and the damage-associated molecular pattern molecule and antimicrobial peptide serum amyloid a (saa) ( ) . sfb infection influences many models of intestinal and extraintestinal inflammatory disease, suggesting that it may have a systemic influence on the immune response ( ) ( ) ( ) . indeed, a growing body of literature suggests that intestinal colonization with commensal microorganisms can have extraintestinal effects on dendritic-cell precursors that influence susceptibility to pathogens ( ) ( ) ( ) . it is also quite possible that mediators induced by the intestinal microbiota in the serum might influence the bone marrow in such a way as to prime dcs to provide protection against, or exacerbate, enteropathogen infections ( , ) . bone marrow dendritic cells (bmdcs) are known to be able to recapitulate effector functions of some in vivo antigen-presentingcell populations, and lipopolysaccharide (lps)-matured bmdcs have been effectively utilized in adoptive transfer experiments to examine the influence of dendritic cells in infection models ( ) ( ) ( ) . thus, to begin to examine the influence of changes in the microbiota on resistance to amebic infection, we utilized a murine model and bmdcs to explore what influence colonization with sfb had on intestinal infection with e. histolytica. here we dem-onstrate that colonization with sfb provided protection against e. histolytica colitis and that bone marrow dendritic cells (bm-dcs) derived from sfb-colonized mice were able to recapitulate protection in mice that had not been colonized with the bacteria. protection mediated by bmdcs from sfb ϩ mice was il- a dependent. these data suggest that intestinal colonization with a commensal bacterium can alter bone marrow in such a way as to provide protection against parasite infection. cohousing charles river (sfb ؉ ) and jackson mice (sfb ؊ ) protected jackson cba mice from e. histolytica infection. to test if alteration if the intestinal microbiota could alter susceptibility to e. histolytica infection, mice from two different animal vendors, charles river and jackson laboratories, were cohoused ( , ) . the sfb status of mice after weeks of cohousing was measured via qpcr and gram stains. the cohoused mice were then challenged with e. histolytica trophozoites ( ϫ ) via intracecal injection. seven days later, e. histolytica and sfb burden were measured by qpcr. cohousing mice transferred sfb (fig. a) , and potentially many other bacteria, and provided protection against e. histolytica infection (fig. b) . segmented filamentous bacteria specifically protect against e. histolytica and induce increased il- a, il- , neutrophils, and dendritic cells in the intestine and saa in the blood. as cohousing of mice both transferred sfb and was protective against e. histolytica infection, we sought to determine if sfb specifically provided protection against the ameba. we directly colonized cba/j mice from jackson laboratories with sfb by orogastric gavage with sfb-monoassociated feces resuspended in phosphate-buffered saline (pbs), provided by the yakult central institute for microbiological studies, week prior to e. histolytica infection. mice became colonized with sfb following gavage ( fig. a) and were protected from infection with the ameba (fig. b) . additionally, there was increased il- a and il- expression before ( onized mice after e. histolytica infection. we also observed increased saa in serum in sfb-colonized mice after ameba infection (fig. g ). bmdcs derived from sfb-colonized mice have an increased capacity to produce il- that is partially recapitulated with saa treatment. il- is a key cytokine in the generation and maintenance of il- a-producing cells ( ) . saa can directly induce il- from both dcs and macrophages ( , ) and is known to upregulate an epigenetic mediator, jmjd ( ) , that specifically increases il- production in a macrophage cell line. thus, as sfb increased frequency of intestinal dcs, il- , il- a expression, and circulating serum saa, we examined the capacity of bone marrow dcs from sfb-infected mice to produce il- in response to trophozoites. bmdcs were generated from sfb-free and sfb-infected mice and treated with trophozoites for h. bmdcs and trophozoites were cocultured in rpmi with % fetal bovine serum (fbs) under aerobic conditions rather than in ameba culture medium under anaerobic conditions, as both dcs and trophozoites were viable after h in these media; however, some cell death had occurred, as observed via trypan blue. cytokines in the supernatants were then determined by enzyme-linked immunosorbent assay (elisa) (il- ; r&d systems). bmdcs derived from mice that were colonized with sfb produced significantly more il- in response to trophozoites (fig. a) . these data suggested that sfb altered bone marrow cells in such a way as to favor increased il- production. we hypothesized that saa induced in the serum by intestinal sfb colonization could influence bmdcs to produce increased il- . therefore, we wanted to test if the presence of saa during the differentiation of sfb-free bmdcs might alter them in such a way that they also would produce more il- . thus, saa ( g/l) was added for the first days of bone marrow dc culture. addition of saa to bmdc culture from sfb-free mice partially recapitulated the increased il- seen in bmdcs derived from sfb ϩ mice (fig. b ). this suggested that saa might be at least partially responsible for alteration of bone marrow dcs in this model. bmdcs from sfb ؉ but not sfb ؊ mice migrated to the intestine. to test if the increase in dcs in the gut of sfb ϩ mice was due to a direct effect on the bone marrow, bmdcs from sfbcolonized and sfb-free mice were adoptively transferred to sfb Ϫ mice. for in vivo transfers, day bmdc from sfb Ϫ and sfb ϩ mice were matured with lps ( g/ml), and h later, cells were stained with carboxyfluorescein succinimidyl ester (cfse; nm), and ϫ cells were administered via intraperitoneal injection. cfse ϩ bmdc were detected in the lamina propria but only from sfb ϩ mice (fig. ) . the ability of sfb ϩ bmdcs to home to the gut may help explain the increased numbers of dcs seen in the lamina propria of sfb-colonized mice during e. histolytica infection (fig. e) . these results were consistent with other studies that have shown that adoptive transfer of lps-matured bmdcs results in dc trafficking to the intestine ( , , ) . adoptive transfer of bmdcs from sfb colonized mice was sufficient to provide protection against infection in an il- adependent manner. we hypothesized that bmdcs from sfbcolonized mice may provide protection against amebiasis and that the mechanism of this protection might be via downstream induction of il- a. to explore this idea, we adoptively transferred bmdcs ( ϫ cells) from sfb Ϫ or sfb ϩ mice to sfb Ϫ mice prior to e. histolytica infection. indeed, we found that bmdcs from sfb ϩ mice were sufficient both to confer protection (fig. a) and to recapitulate the increase in neutrophils (fig. b ) observed with sfb colonization alone. blockade of il- a during transfer abrogated protection (fig. c ) and neutrophil influx (fig. d ) and led to a decrease in iga induction (fig. e ). we concluded that il- a and downstream innate and perhaps adaptive immune responses underlie the protection observed during adoptive transfer of bmdcs from sfb ϩ mice. we have demonstrated that alteration of the microbiota via introduction of the commensal bacteria sfb impacts susceptibility to amebic infection in a murine model, likely via its impact on the mucosal immune system ( ) . there was an increase in il- a and il- expression in the intestines of sfb-colonized mice prior to and following amebic infection and a relative increase in neu- trophils after amebic infection. our laboratory and others have shown that these effectors may provide protection against e. histolytica ( ) . we also observed increased cd c ϩ mhcii ϩ cells in the intestines of mice, suggesting that dendritic cells induced by sfb may also help mediate protection against infection. this is not surprising given that goto et al. recently demonstrated that major histocompatibility complex class ii (mhc-ii)-dependent presentation of sfb antigens by intestinal dcs is crucial for th cell induction ( ) . interestingly, protection in our model appeared to involve not only the mucosal immune response in the gut but also the bone marrow. resistance to amebic colitis was adoptively transferred to sfb-free mice with bone marrow-derived dendritic cells (bm-dcs) from sfb-colonized mice. this suggested that sfb alteration of bone marrow cells might underlie protection against amebic colitis. bmdcs derived from sfb colonized mice produced more il- in response to e. histolytica trophozoites than bmdcs from mice that were not colonized with the bacteria. however, this response was not specific to e. histolytica, as increased il- production from sfb ϩ bmdcs was also seen with lps and saa treatment (data not shown). this suggests that sfb colonization of the intestine has conditioned bone marrow cells to be more responsive to stimulation with pathogen-associated molecular patterns (pamps) or damage-associated molecular patterns (damps), such as saa, or, alternatively, that there were shifts in the populations of dendritic-cell precursors present in the bone marrow prior to bmdc culture in sfb-colonized mice. therefore, in this model of sfb-induced alteration of the microbiome, it is not yet clear how sfb altered bone marrow cells. future studies will be required to examine the possibility of shifts in dendritic-cell precursors or changes in responsiveness to damps and pamps. in this context, intestinal inflammation or colonization with commensal organisms has been shown to alter populations of bone marrow progenitor cells ( , , ) . however, exactly how cross talk occurs between the intestine and bone marrow is not well understood. recent work has shown that gut microbiota metabolism of dietary fiber can increase the concentration of circulating short-chain fatty acids (scfas) and that treatment of mice with the scfa propionate led to alterations in bone marrow hematopoiesis ( ) . a similar mechanism might underlie protection in our model of e. histolytica/sfb coinfection. however, sfb-mediated alteration of the metabolome has not been explored. sfb do, however, have an intimate association with the gut intestinal mucosa, likely due to their limited metabolic capability ( , ) . these bacteria are thus known to alter host gene expression in the intestine ( ) . saa has been shown to be upregulated in the intestines of mice colonized with sfb ( ) , and we have demonstrated that saa is also increased in the serum of e. histolytica/sfb-infected mice. saa is known to be produced by many cell types, including epithelial cells ( ), and sfb tightly associate with the intestinal mucosa ( ). saa-dependent induction of jmjd -mediated epigenetic regulation of inflammatory cytokine gene expression has also been described ( ) , including il- , in saa-stimulated macrophages. it is thus not hard to imagine that circulating saa might act on bone marrow cells to alter il- induction via epigenetic mechanisms ( ) . indeed, we cultured bmdcs from sfb Ϫ mice in the presence of saa for the first days of culture and then removed the mediator, and the resulting differentiated bmdcs produced significantly more il- than bmdcs from sfb Ϫ mice alone. however, this did not fully recapitulate the increase in il- production seen in bmdcs from sfb-colonized mice. this study is not conclusive, however, and multiple mechanisms, perhaps including alteration of metabolic by-products, may underlie the increased il- production seen in bmdcs from sfbcolonized mice. we propose a model in which intestinal colonization with sfb leads to induction of systemically circulating me-diators, perhaps including saa, that alter bone marrow dendriticcell precursors so that they provide protection against e. histolytica via induction of downstream effectors, which may include il- a, neutrophils, and iga (fig. ) . in conclusion, we have demonstrated that colonization with a specific component of the intestinal microbiota, sfb, provided protection against infection with the protozoan pathogen e. histolytica and that bmdcs and downstream il- a induction recapitulated this protection. this work therefore suggests that alteration of the microbiome can mediate resistance to intestinal infection via extraintestinal effects on bone marrow cells. future studies will be required to describe and understand the underlying mechanisms of these microbiome-induced changes in bone marrow dendritic-cell precursors. male -week-old cba/j mice (jackson laboratories, charles river) were housed in a specific-pathogen-free facility in micro-isolator cages and provided autoclaved food (lab diet ) and water ad libitum. all procedures were approved by the institutional animal care and use committee of the university of virginia. e. histolytica culture and intracecal injection. animal-passaged hm :imss e. histolytica trophozoites were cultured from cecal contents of infected mice in complete trypsin-yeast-iron (tyi- ) medium supplemented with diamond vitamin mix (jrh biosciences), u/ml of penicillin and streptomycin, and bovine serum (sigma-aldrich). prior to injection, trophozoites were grown to log phase, and ϫ parasites were suspended in l culture medium and injected intracecally ( ) . sfb colonization. mice were colonized with sfb by cohousing for weeks with sfb ϩ age-matched mice or directly colonized with sfb by orogastric gavage with sfb-monoassociated feces (a kind gift from the yakult central institute for microbiological studies) week prior to e. histolytica infection. sfb-monoassociated feces were independently confirmed to be associated with sfb only, with no presence of other bacteria or fungal contamination, by both y. umesaki and us via qpcr, sanger sequencing, and culture ( ) . quantitative real-time rt-pcr. sfb and e. histolytica colonization were measured by real-time pcr. for sfb colonization, qpcr with sybr green was performed, and data were normalized to expression of a conserved eubacteria s rna gene (eub). primer sequences are as follows: eub forward, =-actcctacgggaggcagcagt- =; eub reverse, =-attaccgcggctgctggc- =; sfb forward, =-gacgctgaggcat gagagcat- =; sfb reverse, =-gacggcacggattgttattca- =. a -carboxyfluorescein (fam)-labeled probe ( ), a standard curve prepared from trophozoites, and quantitative pcr (qpcr) were utilized for e. histolytica quantification; the primer and probe sequences are as follows: eh-f, aac agt aat agt ttc ttt ggt tag taa aa; eh-r, ctt aga atg tca ttt ctc aat tca t; eh-yyt, att agt aca aaa tgg cca att cat tca-dark quencher; il- a f, =-actacctcaa ccgttccacg- =; il- a r, =-agaattcatgtggtggtccag- =. cytokines were measured via qpcr with sybr green, and data were normalized to expression of s : il- p f, =-gacccacaaggactcaa gga- =; il- p r, =-catggggctatcagggagta- =; s f, =-t ggtgtctgccacatctttgcatc- =; s r, =-agtcactcggcag atggtttcctt- =. melting temperatures were °c for eub, sfb, il- a, and il- and for e. histolytica ( ) . primers and probes were purchased from integrated dna technologies, coralville, ia, usa. serum saa was measured by elisa (ab ; abcam, cambridge, england). bone marrow-derived-dendritic-cell (bmdc) culture. bone marrow cells were harvested from weeks old male cba/j mice from jackson laboratories that had been treated by gavage with pbs or sfb the week prior. cells were cultured in rpmi with % fbs supplemented on days and with granulocyte-macrophage colony-stimulating factor (gm-csf) ( ng/ml; peprotech) and harvested on day . for in vitro experiments, . ϫ cells were plated per well of a flat-bottom -well dish and treated with trophozoites for h. cytokines in the supernatants were determined by elisa (il- ; r&d systems). dc transfer. for in vivo transfers, day bmdcs were stimulated with lps ( g/ml); h later, cells were washed in pbs, and ϫ cells were administered intraperitoneally. for cfse experiments, cells were stained with cfse in pbs for min ( nm) and then washed twice in pbs prior to adoptive transfer. il- a blockade. four-week-old cba/j mice were treated with g rat anti-mouse il- a monoclonal antibody (mab) (clone m ; amgen) or rat igg a isotype control (clone a ; bioxcell) at days before and on the day of bmdc transfer ( , intraperitoneally from mice receiving sfb gavage or those not receiving sfb gavage). mice were challenged intracecally with ϫ trophozoites days after bmdc transfer. in vitro culture and flow cytometry of intestinal cells. for intracellular staining, intestinal tissue was digested in liberase tl ( . mg/ml roche) and dnase ( . mg/ml; sigma) for min at °c and processed into a single-cell suspension. a total of ϫ cells were incubated with cd /cd mab (bd biosciences) to block fc binding, followed by staining with cd v-bv , mhcii-fitc, tcr-beta-apc, b -apc, cd . -apc, ly g-pe, ly c-percp cy . , or cd b-pacific blue and run on an lsr fortessa cell analyzer (bd biosciences). the data were analyzed with flowjo (tree star inc.). statistical analysis. an analysis of variance (anova) followed by the tukey-kramer test was used for analysis of differences among multiple groups. student's t test was used for comparisons between two groups. p values of less than . were considered significant. results of representative experiments are shown; all experiments were replicated to times, with to individuals per group. supplemental material for this article may be found at http://mbio.asm.org /lookup/suppl/doi: . /mbio. - /-/dcsupplemental. figure s , eps file, . mb. who/paho informal consultation on intestinal protozoal infections. world health organization update on human infections caused by intestinal protozoa contribution of enteric infection, altered intestinal barrier function, and maternal malnutrition to infant malnutrition in bangladesh burden and aetiology of diarrhoeal disease in infants and young children in developing countries (the global enteric multicenter study, gems): a prospective, case-control study the dynamic interdependence of amebiasis, innate immunity, and undernutrition. semin. immunopathol entamoeba histolytica-associated diarrheal illness is negatively associated with the growth of preschool children: evidence from a prospective study interactions between parasites and microbial communities in the human gut cd ϩ and cd ϩ t cell-and il- -mediated protection against entamoeba histolytica induced by a recombinant vaccine th cells upregulate polymeric ig receptor and intestinal iga and contribute to intestinal homeostasis th cytokines and the gut mucosal barrier regulation of airway muc ac expression by il- beta and il- a; the nf-kappab paradigm cxcr and cxcl regulate the il- /g-csf axis and neutrophil homeostasis in mice th cells: biology, pathogenesis of autoimmune and inflammatory diseases, and therapeutic strategies resistance to intestinal entamoeba histolytica infection is conferred by innate immunity and gr- ϩ cells amebiasis and mucosal iga antibody against the entamoeba histolytica adherence lectin in bangladeshi children singlecell sequencing provides clues about the host interactions of segmented filamentous bacteria (sfb) comparative analysis of the distribution of segmented filamentous bacteria in humans, mice and chickens habitat, succession, attachment, and morphology of segmented, filamentous microbes indigenous to the murine gastrointestinal tract segmented filamentous bacteria antigens presented by intestinal dendritic cells drive mucosal th cell differentiation induction of intestinal th cells by segmented filamentous bacteria naturally transmitted segmented filamentous bacteria segregate with diabetes protection in nonobese diabetic mice gut-residing segmented filamentous bacteria drive autoimmune arthritis via t helper cells proinflammatory t-cell responses to gut microbiota promote experimental autoimmune encephalomyelitis gut microbiota promote hematopoiesis to control bacterial infection commensal bacteria-derived signals regulate basophil hematopoiesis and allergic inflammation dysregulated hematopoietic stem and progenitor cell activity promotes interleukin- -driven chronic intestinal inflammation the intestinal metabolome: an intersection between microbiota and host modulation of dendritic cell differentiation in the bone marrow mediates sustained immunosuppression after polymicrobial sepsis distinct roles for il- and il- p in mediating protection against leishmania donovani and the expansion of il- ϩ cd ϩ t cells evasion by stealth: inefficient immune activation underlies poor t cell response and severe disease in sars-cov-infected mice variation in the gut microbiota of laboratory mice is related to both genetic and environmental factors temporal stability of the mouse gut microbiota in relation to innate and adaptive immunity jmjd -mediated epigenetic regulation of inflammatory cytokine gene expression in serum amyloid a-stimulated macrophages serum amyloid a activates the nlrp inflammasome and promotes th allergic asthma in mice dendritic cell immunization route determines integrin expression and lymphoid and nonlymphoid tissue distribution of cd t cells human nutrition, the gut microbiome and the immune system segmented filamentous bacteria antigens presented by intestinal dendritic cells drive mucosal th cell differentiation gut microbiota promote hematopoiesis to control bacterial infection gut microbiota metabolism of dietary fiber influences allergic airway disease and hematopoiesis the genome of th cell-inducing segmented filamentous bacteria reveals extensive auxotrophy and adaptations to the intestinal environment the lifestyle of the segmented filamentous bacterium: a non-culturable gut-associated immunostimulating microbe inferred by whole-genome sequencing the mouse model of amebic colitis reveals mouse strain susceptibility to infection and exacerbation of disease by cd ϩ t cells segmented filamentous bacteria are indigenous intestinal bacteria that activate intraepithelial lymphocytes and induce mhc class ii molecules and fucosyl asialo gm glycolipids on the small intestinal epithelial cells in the ex-germ-free mouse realtime-pcr assay for diagnosis of entamoeba histolytica infection this work was supported by nih grant r ai - to w.p. we thank yoshinori umesaki, from the yakult central institute for microbial research (tokyo, japan) for supplying the sfb-monocolonized feces. we also gratefully acknowledge the gift of anti-il- a monoclonal antibodies from alison budelsky at amgen incorporated (seattle, wa) and florent ginhoux, brian kelsall, and tom braciale for helpful discussions.s.b., w.p., and m.w.-k. contributed to the design, analysis and interpretation of the data. s.b., e.b., m.c., c.c., c.n., and z.n. conducted experiments and critiqued the work. s.b. and w.p. wrote the manuscript. key: cord- -p ydzjre authors: goodman, danielle e.; pretto, carla d.; krepostman, tomas a.; carnahan, kelly e.; spindler, katherine r. title: enhanced replication of mouse adenovirus type following virus-induced degradation of protein kinase r (pkr) date: - - journal: mbio doi: . /mbio. - sha: doc_id: cord_uid: p ydzjre protein kinase r (pkr) plays a major role in activating host immunity during infection by sensing double-stranded rna (dsrna) produced by viruses. once activated by dsrna, pkr phosphorylates the translation factor eukaryotic initiation factor α (eif α), halting cellular translation. many viruses have methods of inhibiting pkr activation or its downstream effects, circumventing protein synthesis shutdown. these include sequestering dsrna or producing proteins that bind to and inhibit pkr activation. here we describe our finding that in multiple cell types, pkr was depleted during mouse adenovirus type (mav- ) infection. mav- did not appear to be targeting pkr at the transcriptional or translational level, because total pkr mrna levels and levels of pkr mrna bound to polysomes were unchanged or increased during mav- infection. however, inhibiting the proteasome reduced the pkr depletion seen in mav- -infected cells, whereas inhibiting the lysosome had no effect. this suggests that proteasomal degradation alone is responsible for pkr degradation during mav- infection. time course experiments indicated that the degradation occurs early after infection. infecting cells with uv-inactivated virus prevented pkr degradation, whereas inhibiting viral dna replication did not. together, these results suggest that an early viral gene is responsible. degradation of pkr is a rare mechanism to oppose pkr activity, and it has been described in only six rna viruses. to our knowledge, this is the first example of a dna virus counteracting pkr by degrading it. causing inhibition of protein synthesis and reduced viral replication ( ) ( ) ( ) ( ) . many viruses encode gene products that block pkr activation or inhibit its ability to phosphorylate eif ␣ ( ) . a common mechanism is that of producing a viral protein that binds and sequesters dsrna, blocking its interaction with pkr. examples of this are vaccinia virus e l ( ) ( ) ( ) , influenza virus ns ( , ) , and ebola virus protein vp ( ) . other viruses produce proteins or rnas that bind directly to pkr to inhibit its activation, such as herpes simplex virus us ( , ) , hiv- tat protein ( , ) or trans-activation response element (tar) rna ( ) , and human adenovirus (had) virusassociated (va) rnas ( , ( ) ( ) ( ) . degradation of pkr by viruses is a less extensively documented method of regulating pkr. to date, pkr degradation has been reported in six rna viruses: toscana virus (tosv) ( ) , rift valley fever virus (rvfv) ( ) ( ) ( ) , poliovirus ( , ) , foot-andmouth disease virus (fmdv) ( , ) , encephalomyocarditis virus (emcv [strain mengovirus]) ( , ) , and enterovirus ( ) . rvfv and tosv both degrade pkr via proteasomal mechanisms involving a viral nonstructural protein (nss) ( , , ) . rvfv nss recruits a scf (skp -cul -f-box) fbxw e ubiquitin ligase to ubiquitinate pkr and target it to the proteasome, though pkr ubiquitination could not be demonstrated ( , ) . the mechanism for pkr proteasomal degradation by nss has not been described for tosv ( ) . fmdv uses the other major cellular protein degradation pathway, the lysosome, to degrade pkr during infection ( ) . though the mechanism is unclear, expression of major fmdv protease c pro is required for pkr degradation by the lysosome. however, c pro does not interact with pkr, and its protease activity is not required for pkr degradation. enterovirus a c pro causes pkr degradation by direct interaction ( ) . the mechanism of pkr depletion by poliovirus is unclear, though gene expression is required and the major poliovirus proteases ( a and c) are not directly involved ( ) . the mechanism by which mengovirus depletes pkr during infection is unknown ( , ) . adenoviruses are species specific, making the study of had pathogenesis difficult in an animal model. mav- represents a useful alternative to study adenovirus pathogenesis ( ) ( ) ( ) ( ) ( ) . mav- has molecular, genetic, and pathogenic similarities to and differences from had. their genomic structures are similar at a gross level, and both contain early genes involved in pathogenesis and immune evasion. pathogenically, their tropisms differ, with had infecting epithelial cells, leading to upper respiratory and gi tract infections and to conjunctivitis, while mav- infects endothelial cells and monocytes, causing encephalitis and myocarditis. we and others have been investigating the adaptive and innate immune responses to mav- . human adenovirus va rnas bind pkr as a monomer, preventing its transautophosphorylation ( ) . however, mav- does not produce va rnas ( ) , and whether mav- induces pkr activation is not known. in our studies of mav- pathogenesis and the innate response, we discovered that during mav- infection, pkr was depleted from cells as early as h postinfection (hpi). total pkr mrna levels and levels of pkr mrna bound to polysomes were unchanged or increased during mav- infection, suggesting that mav- did not appear to be targeting pkr at a transcriptional or translational level. however, inhibiting the proteasome blocked the pkr depletion seen in mav- -infected cells, indicating that proteasomal degradation is responsible for pkr depletion during mav- infection. we report results indicating that an early viral gene is likely responsible for mediating pkr degradation. to our knowledge, this is the first example of a dna virus counteracting pkr by degrading it. viral dna yield is increased in pkr ؊/؊ mouse embryonic fibroblasts. while pkr is an important part of the innate immune response, pkr Ϫ/Ϫ cells in culture are not always more susceptible to viral infection than wild-type (wt) cells ( ) ( ) ( ) . pkr Ϫ/Ϫ mouse embryonic fibroblasts (mefs) show increased viral yields compared to wild-type mefs when infected with vesicular stomatitis virus and influenza a virus ( , ) , but there is no change in viral yield during vaccinia virus infection compared to the results seen with wild-type cells ( ) . however, it was later discovered that the pkr Ϫ/Ϫ mef lines used were not complete pkr knockouts ( ) . there are two categories of pkr Ϫ/Ϫ mefs derived from knockout mice: n-pkr Ϫ/Ϫ mefs and c-pkr Ϫ/Ϫ mefs ( ). the pkr Ϫ/Ϫ mefs derived from mice created in the weissmann laboratory ( ) are designated n-pkr Ϫ/Ϫ mefs, because the c-terminal fragment of pkr is still expressed and can be detected by immunoblotting when there is ifn induction ( ) . the fragment has the kinase catalytic activity of pkr, but it does not bind dsrna ( ) . the pkr Ϫ/Ϫ mefs derived from mice created in the bell laboratory ( ) are designated c-pkr Ϫ/Ϫ mefs, because the n-terminal fragment of pkr is still expressed and can be detected by immunoblotting with specific pkr antibodies ( ) . the fragment is catalytically inactive, but it can still bind dsrna ( ) . susceptibility of these pkr Ϫ/Ϫ mefs to specific viruses may be dependent on the pkr mutation and the mechanism used by each virus to circumvent pkr. to determine whether pkr plays an important role during mav- infection, we tested the susceptibility of both pkr Ϫ/Ϫ mef lines to mav- infection. we infected wild-type mefs, n-pkr Ϫ/Ϫ mefs, and c-pkr Ϫ/Ϫ mefs with mav- at a multiplicity of infection (moi) of pfu/cell and collected cell pellets at and hpi. dna was purified from the cell pellets and analyzed for mav- genome copies by quantitative pcr (qpcr). n-pkr Ϫ/Ϫ mefs produced a significantly higher viral dna yield than wild-type mefs at hpi, and both pkr mutant mef lines had a significantly higher viral dna yield than wild-type mefs at hpi (fig. ). although we have not confirmed the production of truncated pkr proteins in the cells in our laboratory, the results shown in fig. mouse pkr is depleted during mav- infection. to determine whether mav- affects pkr during infection, we infected several cell types and analyzed pkr protein expression. we infected immortalized c bl/ mefs, c bl/ primary peritoneal macrophages, and cmt cells (mouse rectal carcinoma cells) with mav- at an moi of and collected cell lysates , , and hpi. we analyzed cell lysates for the presence of pkr by immunoblotting using a polyclonal antibody that detects mouse pkr. we probed blots with antibodies to actin as a loading control. to our surprise, in c bl/ mefs, pkr was almost completely depleted from lysates at hpi and remained depleted through hpi ( fig. a and b) . pkr was also depleted compared to the levels seen with mock infection at and hpi in c bl/ mefs infected at mois of and (see fig. s in the supplemental material). we also observed depletion of pkr in other cell types. in cmt cells, pkr was nearly undetectable at hpi ( fig. a) . pkr levels were decreased in c bl/ primary peritoneal macrophages at hpi compared to mock-infected lysates, and pkr was absent in infected lysates at hpi ( fig. a) . this indicates that mav- causes pkr depletion during infection. error bars represent standard errors of the means (sem). *, p Յ . ; ***, p Յ . ; ****, p Յ . . to determine whether the kinase activity of pkr is important for the depletion, we assayed infection of mefs expressing a mutant form of mouse pkr with a point mutation in the kinase domain (k r) ( ) . these cells, designated k r simian virus (sv ) mefs, showed an even higher rate of depletion of pkr than the wt sv mefs (fig. c ). at hpi, % of pkr remained in k r sv mefs compared to % in the wt sv mefs. the fraction remaining at hpi in k r sv mefs was % compared to % in wt sv mefs (fig. c ). this indicates that the pkr kinase does not have to be functional to be depleted during mav- infection. also, comparing the pkr immunoblot bands in the mock-infected wt sv mefs and mutant k r sv mefs suggests that the upper band of the pkr doublet usually seen in wild-type cells is a phospho-pkr band, because only the lower band of the pkr doublet is seen in kinase-dead mutant k r sv mefs. the data in fig. a and c thus indicate that both pkr and phospho-pkr are depleted during mav- infection. mav- does not cause pkr depletion by reducing steady-state levels of pkr mrna. to determine the mechanism of pkr depletion, we first assayed whether the reduction in the level of pkr protein during mav- infection was due to reduced pkr mrna steady-state levels. we mock-infected or infected c bl/ mefs and primary peritoneal macrophages at an moi of and collected cell lysates at , , and hpi. we synthesized cdna from rna purified from these cell lysates and assayed for pkr mrna by qpcr. the pkr mrna levels in c bl/ mefs were similar between mockinfected and infected lysates at hpi (fig. a) , a time point at which the pkr protein levels were already greatly reduced in the infected lysates compared to mock-infected lysates (fig. ) . although the pkr mrna levels were depleted % at hpi and % at hpi in mav- -infected lysates compared to mock-infected lysates, this does not correlate to the % and % reductions, respectively, in pkr protein levels seen at those time points (fig. b ). pkr mrna levels in c bl/ primary peritoneal macrophages in the infected lysates were to times higher than the levels in the mock-infected lysates at all three time points assayed (fig. b) , even though the pkr protein was almost completely depleted in infected lysates at hpi (fig. ) . this represents evidence that mav- was not causing pkr protein depletion by reducing pkr steady-state mrna levels during infection. mav- infection effects on pkr translation. because mav- did not cause reductions in the pkr mrna steady-state levels, we determined whether mav- causes pkr depletion by reducing translation of its mrna. we first assayed total pkr mrna bound to ribosomes during infection. c bl/ mefs were mock infected or infected at an moi of , and lysates were collected at hpi in the presence of cycloheximide to keep the mrna bound to the ribosomes ( ) . lysates were centrifuged through % sucrose to pellet ribosomes, and rna was purified from the pellets. the purified rnas were used to generate cdna, which we assayed for pkr mrna by qpcr. as a control for pelleting of ribosomes, we assayed the pellets and sucrose cushion supernatants by immunoblotting with antibodies to ribosomal protein rpl . we confirmed that rpl was present only in the pellets and not in the supernatants (fig. s ). there was no significant difference between the amount of pkr mrna in the ribosome pellet of mock-infected lysates and the amount in that of mav- -infected lysates (fig. a) . to confirm the results seen in total mrna bound to ribosomes, we also centrifuged cell extracts on sucrose gradients to generate polysome profiles. this enabled us to analyze levels of pkr mrna associated with actively translating ribosomes during infection. c bl/ mefs were mock infected or infected at an moi of , and lysates were collected at hpi in the presence of cycloheximide, as described above. the levels of rna content for mock-infected and infected lysates were estimated by nanodrop spectrophotometry, and equivalent optical density (od) amounts of rna were centrifuged on % to % sucrose gradients to sediment s and s ribosomal subunits, s ribosomes (monosomes), and polyribosomes (polysomes). a typical polysome profile was obtained (fig. b) . rna was purified from fractions containing monosomes mav- degrades pkr during infection ® and polysomes and then used to generate cdna, which we assayed for pkr mrna by qpcr (fig. c) . as a control, gapdh mrna was measured by qpcr, and pkr mrna levels in each fraction were normalized to the gapdh mrna content. when the data representing the percentages of pkr mrna bound to ribosomes were pooled into monosome and polysome fractions and analyzed (fig. d ), . % and . % were bound to polysomes (fractions to ) for mock-infected and infected samples, respectively, compared to . % and . % bound to monosomes (fractions to ). we performed two additional polysome gradient analyses. the pooled data from all three analyses ( fig. e ) were similar to the data shown in fig. d ; i.e., . % and . % of pkr mrna were bound to mock-infected and infected polysomes, respectively, compared to . % and . % bound to monosomes. thus, pkr protein depletion during mav- infection does not appear to stem from a decrease in pkr mrna translation. we also assayed whether pkr mrna might have a signal that would reduce its translation during mav- infection. we constructed a plasmid that positioned sequence corresponding to the = untranslated region (utr) of pkr mrna upstream of a reporter nanoluciferase gene ( ) , transfected it into c bl/ mefs, and then infected with mav- . compared to cells transfected with a control plasmid with the human ␤-globin = utr upstream of the reporter nanoluciferase, there was no significant difference in luciferase activity between mock-infected and infected samples (fig. s ). these data suggest that mav- was not affecting pkr translation through interaction with the = utr of pkr. the data are consistent with the ribosome pellet and polysome data indicating that mav- infection does not reduce pkr mrna translation. pkr is depleted by proteasomal degradation during mav- infection. there are two main proteolysis pathways in cells: proteasomal degradation and lysosomal degradation ( ) . to determine whether mav- depletes pkr by either protein degradation pathway, we first assayed whether pkr is lysosomally degraded as follows. cmt cells were mock infected or infected with mav- and treated at the time of infection with a lysosome inhibitor (ammonium chloride or chloroquine) or water (as a control). at hpi, we collected lysates and analyzed them by immunoblotting with antibodies to pkr. in the presence of the lysosomal degradation inhibitors, pkr was depleted by hpi (fig. a ), indicating that lysosomal degradation was not the cause of pkr depletion during mav- infection. we confirmed that the inhibitor treatment did block lysosomal degradation by incubating cells with dye-quenched bovine serum albumin (dq bsa) in addition to the lysosomal inhibitors. dq bsa is self-quenched until it is digested in the lysosome ( , ) , and imaging confirmed that the cells treated with lysosome inhibitors did not fluoresce but that the cells treated with the vehicle control (h o) did, as expected (fig. b ). next, we examined whether proteasomal degradation is responsible for the degradation of pkr by using proteasome inhibitors mg and bortezomib. these inhibit mav- degrades pkr during infection ® proteasome activity by binding to the active sites in the s subunit and blocking the proteolytic activity ( - ). we mock infected or infected c bl/ mefs with mav- and treated with mg or bortezomib and dimethyl sulfoxide (dmso) at the time of infection. at hpi, we collected lysates and analyzed them by immunoblotting for pkr protein levels. while pkr was depleted in the control dmso-treated mav- -infected cells as expected, pkr protein was present in the mg -and bortezomib-treated cells at levels comparable to those in the mock-infected cells (fig. ). to rule out the possibility that pkr was present (not depleted) because the virus infection itself was inhibited by mg or bortezomib, we assayed viral replication of mav- with mg and bortezomib treatment by qpcr of viral dna. viral replication levels were equivalent in all three treatment groups (fig. s ) , indicating that the treatments did not affect the ability of the virus to productively infect the cells. taken together, these data indicate that mav- infection results in pkr depletion by causing pkr to be degraded by the proteasome during infection. a signal for proteasomal degradation is the conjugation of ubiquitin to a protein ( , ) . we examined by immunoblotting whether pkr is ubiquitinated. we detected ubiquitination of a positive control, mouse p , which is degraded in the presence of mav- proteins ( ). however, even with the use of epitope-tagged ubiquitin ( ) and mg treatment, we were unable to detect pkr ubiquitination during infection (fig. s ). this is consistent with an inability to detect pkr ubiquitination when it is degraded during rvfv infection ( ) . although rvfv nss is known to recruit an e ligase to pkr, the authors of that study reported that ubiquitinated pkr is undetectable. therefore, the cellular degradation signal for pkr remains unclear. pkr is actively depleted early in infection. we investigated the time point at which proteasomal degradation of pkr occurs. early viral proteins are expressed prior to viral dna replication, which is then followed by late viral protein expression. first, we examined the kinetics of pkr degradation to determine whether an early or late viral protein was likely responsible. we mock infected and infected cmt cells with mav- at an moi of , collected lysates every h for h, and analyzed them by immunoblotting with antibodies to pkr or mav- early region a (e a) protein, the first viral protein made during infection ( ) . pkr degradation in the infected cells was first detected at hpi (fig. a) , and quantitation of the results of five independent experiments showed that ϳ % of the starting levels of pkr protein remained at hpi (fig. b) . in parallel, to determine the half-life of pkr in uninfected cmt cells, we treated cmt cells with cycloheximide to halt protein translation and thus production of new pkr. we collected lysates every h for h and analyzed by immunoblotting with antibodies to pkr. after h of cycloheximide treatment, approximately % of the starting levels of pkr protein remained (fig. a, bottom, and b ). comparing the results from mav- infection (fig. a, top, and b ) and cycloheximide treatment of uninfected cells (fig. a, bottom, and b) , we conclude that mav- was actively depleting pkr protein early in infection. e a was detected by immunoblotting at hpi (fig. c) , whereas viral dna replication was first detected at hpi in cmt cells (fig. s ) . thus, the -hpi time point is considered an early time point during mav- infection of cmt cells, prior to dna replication, suggesting the involvement of an early viral protein in pkr depletion. an early viral function is required for pkr depletion by mav- . to determine whether viral gene expression or dna replication is required for pkr degradation during infection, we infected c bl/ mefs and cmt cells with uv-inactivated mav- (which does not replicate viral dna; fig. s ). we infected cells at an moi of with wt mav- or uv-inactivated mav- and analyzed lysates from and hpi by immunoblotting for pkr protein levels. in both cell types, while pkr was degraded by hpi in the cells infected with wt mav- , pkr protein levels were unaffected at both time points in cells infected with uv-inactivated mav- (fig. a ). this suggested that either gene expression or dna replication was required for pkr degradation during mav- infection. we addressed whether viral dna replication is needed for pkr degradation. we mock infected or infected cmt cells with mav- at an moi of and treated them with cytosine arabinoside (arac) at the time of infection to inhibit dna synthesis ( , ) . this would allow the virus to infect the cell and produce early viral proteins, but would inhibit viral dna replication and prevent late protein synthesis. we collected lysates at and hpi and analyzed them by immunoblotting. we confirmed that arac treatment resulted in no late protein synthesis by immunoblotting for late virion proteins (fig. s ) . in samples treated with arac, pkr degradation was seen at and hpi (fig. b) , indicating that dna replication was not required for pkr degradation. taken together, the results shown in fig. are consistent with early viral gene expression prior to dna replication being involved in induction of pkr degradation by mav- . we have demonstrated here that pkr is antiviral in mav- infections of cultured cells. surprisingly, mav- infection of primary and established cultured cells depleted pkr. the depletion was not due to reduced steady-state levels or reduced translation of pkr mrna. instead, we showed that pkr depletion is inhibited by proteasome inhibitors, implicating proteasomal degradation of pkr. several lines of evidence suggest that the degradation is due to a viral early function. pkr is an ifn-inducible gene product that is an important component of the innate immune response ( , ) . however, not all viruses, including emcv and vaccinia virus, have increased virulence in pkr Ϫ/Ϫ mefs ( , ) . while hads produce va rnas that inhibit pkr antiviral activity during infection ( , ), mav- does not produce such va rnas, and how mav- infection is affected by pkr is first described in this report. when we infected pkr Ϫ/Ϫ mefs with mav- , viral dna yields were to times higher than the viral dna yields from wild-type mefs (fig. ) , indicating that pkr plays an antiviral role during mav- infection. the viral dna yield from the n-pkr Ϫ/Ϫ mefs was nearly times higher than the yield from the c-pkr Ϫ/Ϫ mefs at hpi, but by hpi the viral dna yields from the two types of pkr Ϫ/Ϫ mefs were similar to each other and were significantly increased compared to the yield seen with wild-type mefs (fig. ) . this difference in viral replication kinetics between the two types of pkr Ϫ/Ϫ mefs may be due to differences in the expression and activity levels of the pkr fragments reportedly produced by them; we have not assayed pkr fragment production in our cells. we examined pkr protein levels during mav- infection and found that pkr was depleted from the cells as early as hpi (fig. ) . depletion was seen in a wide variety of cell types, including immortalized c bl/ mefs, primary c bl/ peritoneal macrophages, and cmt mouse colon carcinoma cells. once depleted, the pkr protein levels never returned to the levels measured for the mock-infected cells during infection. activation (transautophosphorylation) of pkr ( - ) was not required for this depletion, because kinase-dead mouse pkr was also depleted from k r sv mefs during infection (fig. c) . both pkr and phospho-pkr were depleted in all cell types examined. we examined several possible explanations for the depletion of pkr protein, including pkr mrna levels and alterations in translation. pkr mrna levels remained unchanged during mav- infection in c bl/ mefs at hpi and were increased in primary c bl/ peritoneal macrophages during mav- infection (fig. ) , corresponding to the times when the pkr protein levels were depleted ( fig. a) . while the pkr mrnas in c bl/ mefs were depleted % at hpi and % at hpi compared to mock-infected lysates, this was not sufficient to explain the % and % reductions, respectively, in pkr protein levels at those time points (fig. b ). more likely, the reduction in pkr steady-state mrna levels at the late infection time points can be attributed to other effects from viral infection, including the degradation or inhibition of proteins that induce pkr expression. for example, p is capable of binding to the pkr promoter and inducing its expression ( ), but mav- proteins cause p proteolysis ( ) . together, our results from c bl/ mefs and macrophages suggest that the virus does not cause pkr protein depletion by reducing pkr steady-state mrna levels. the differences in total pkr mrna levels between c bl/ mefs and primary peritoneal macrophages during infection (fig. ) were possibly due to the fact that macrophages are immune cells whereas mefs are not. pkr protein took almost times as long to be completely degraded during infection in macrophages than in mefs ( h versus h) ( fig. a) . total pkr mrna levels were to times higher in mav- -infected macrophages than in mock-infected macrophages, unlike the mefs, where total pkr mrna levels were unchanged or were reduced % to % during mav- -infection compared to the mock-infected mefs. since pkr is an ifn-stimulated gene ( , ) , the higher levels of total pkr mrna seen during infection in the macrophages suggest ifn induction. this suggests that the immune response mounted by the macrophages was greater than the immune response in the mefs and could help explain why pkr took longer to degrade in macrophages than in mefs. we considered whether the reduced pkr levels were due to reduced pkr protein translation. there was no change in the total amount of pkr mrna bound to ribosomes during infection compared to uninfected cells, and there was also no significant change in the amount of actively translating pkr mrna during infection (fig. ) . we also found that the = utr of mouse pkr placed upstream of a reporter gene produced the same amounts of reporter with and without mav- infection. these data indicate that there are not translational effects of mav- infection on pkr protein levels that could explain the depleted pkr levels that we observed. inhibiting lysosomal degradation resulted in no change in pkr depletion in infected cells (fig. a ), but adding proteasome inhibitors preserved pkr protein within cells ( fig. a and b) . this indicates that pkr is degraded not by lysosomal degradation during viral infection but by proteasomal degradation. though pkr degradation was due to proteasome activity during mav- infection, we were unable to demonstrate pkr ubiquitination, although we did detect ubiquitination of mouse p (see fig. s in the supplemental material). this inability to demonstrate pkr ubiquitination could be explained if at any given moment there were only low levels of ubiquitinated pkr present in the cell. perhaps increasing the time spent under conditions of mg treatment could increase the amounts of ubiquitinated proteins enough that pkr ubiquitination could be seen. however, our inability to detect ubiquitinated pkr is consistent with a similar inability to identify pkr ubiquitination by rvfv nss, even though nss is known to recruit an e ligase to pkr ( ) . alternatively, it is possible that in mav- infection, pkr is degraded in a ubiquitin-independent manner, possibly because of the presence of intrinsic disordered regions of pkr or binding of regulating proteins to pkr that target proteins to the proteasome ( , ) . our experiments indicated that mav- actively depletes pkr early in infection. ongoing experiments are focused on determining the mav- early protein(s) responsible for pkr degradation. two possibilities are represented by e proteins, the homologs of had e orf and e orf , which we originally termed e orfa/b and e orfa/c, respectively ( ) . in human adenovirus, e orf interacts with another early had protein, e b k, to participate in an e ligase complex that ubiquitinates and degrades p via proteasomal degradation ( , ) . when mav- e orf , e b k, and mouse p are introduced by transfection into human cells, all three proteins interact and mouse p is degraded ( ) . if mav- e orf and e b k form a similar complex in mouse cells, it may also degrade pkr. we have preliminary evidence indicating that mouse p is ubiquitinated in c bl/ mefs during mav- infection, which suggests that the mouse p degradation seen in human cells could be paralleled by degradation of endogenous mouse p and mouse pkr in mouse cells, mediated by mav- e orf and e b k during infection. another had e protein, e orf , causes proteasomal degradation of transcriptional intermediary factor ␥ ( ) and general transcription factor ii-i ( ) in a manner independent of had e orf and e b k. e orf has sumo e ligase and e elongase activity and induces sumoylation of general transcription factor ii-i, leading to its proteasome-dependent degradation ( ) . mav- e orf may similarly have sumoylation activity that results ultimately in proteasome-dependent pkr degradation. another possibility of a viral protein involved in pkr degradation is the protease encoded by mav- . the had protease is encapsidated in virions and proteolytically processes viral proteins iiia, vi, vii, viii, mu, and tp ( ) ( ) ( ) ( ) . however, we think it is unlikely that the mav- protease degrades pkr, because we showed that uv-inactivated virus was unable to degrade pkr. we assume that uv treatment would not destroy the mav- protease activity, just as hsv- vp activity is not altered by uv inactivation of hsv- ( ) , but we have not tested this directly. in summary, we demonstrated that pkr has an antiviral role during mav- infection in vitro, because when pkr is mutated, viral replication in mefs is significantly higher than that seen in wild-type mefs. analysis of global pkr steady-state protein levels during infection showed complete pkr depletion by hpi in multiple cell types, including immortalized and primary cells, with even faster kinetics in some. pkr transcription and translation were not decreased by mav- infection, whereas proteasomal inhibition prevented pkr degradation. taken together, these data suggest that mav- causes pkr to be proteasomally degraded at a posttranslational level. this work provides new insight into possible mechanisms of adenovirus inhibition of pkr by dna viruses. pkr degradation may be induced by other adenoviruses that do not produce va rna, which includes all animal adenoviruses except primate adenoviruses and one type of fowl adenovirus ( ) . cells, virus, and infections. cmt cells (ccl- ) and c bl/ mefs (scrc- ) were obtained from the american type culture collection and passaged in dulbecco's modified eagle medium (dmem) containing % and % heat-inactivated fetal bovine serum (fbs), respectively, before use. primary peritoneal macrophages were obtained from -to- -week-old c bl/ mice purchased from jackson laboratory (catalog no. ) as described previously ( ) . briefly, -to- -week-old c bl/ mice were injected intraperitoneally with . ml % thioglycolate and euthanized to days later. the abdominal skin was carefully removed, exposing the peritoneum, which was then injected with ml of sterile phosphate-buffered saline (pbs). the abdomen was massaged gently, and then the pbs containing the peritoneal macrophages was carefully withdrawn. the macrophages were centrifuged at ϫ g for min, and red blood cells were lysed in lysis buffer ( . m ammonium chloride, mm potassium bicarbonate, . mm edta disodium salt) for min at room temperature, centrifuged at ϫ g for min, washed twice in pbs, resuspended in dmem- % heat-inactivated fbs, and plated in -well plates. wt and pkr Ϫ/Ϫ mefs (termed pkr wt mefs and n-pkr Ϫ/Ϫ mefs, respectively, throughout this paper) were obtained from robert silverman, cleveland clinic ( ) , and were passaged in dmem containing % heat-inactivated fbs before use. pkr Ϫ/Ϫ mefs stably transfected with empty vector (termed c-pkr Ϫ/Ϫ mefs throughout this paper) were obtained from gokhan hotamisligil, harvard university ( ) , and were passaged in dmem containing % heat-inactivated fbs before use. wt (sv mefs) and k r pkr mutant (k r sv mefs) mefs were obtained from anthony sadler, hudson institute of medical research ( ) , and were passaged in dmem containing % heat-inactivated fbs before use. wild-type mouse adenovirus type (mav- ) stock was prepared, and titers were determined on mouse nih t fibroblasts as described previously ( ) . wt mav- was subjected to uv inactivation by uv treatment of l of virus for min at mj/cm . uv inactivation was confirmed by qpcr and plaque assay. for infection assays, medium was removed from cells and adsorption procedures were performed with . ml of inocula in -well plates with -mm-diameter wells (unless otherwise noted) for h at °c at the indicated mois (pfu/cell). after min, ml of dmem- % fbs was added without removal of inocula; that time point was designated hpi. for arac experiments, g/ml arac (sigma c ) was added at hpi and replenished every to h. immunoblotting. at room temperature, cells were washed once with pbs, and pierce radioimmunoprecipitation assay (ripa) lysis buffer (thermo scientific catalog no. ) with ϫ protease inhibitors (protease inhibitor cocktail kit; thermo scientific catalog no. ) was added to the plate. the cells were allowed to lyse at room temperature for min before being harvested and centrifuged at °c at , ϫ g for min to remove debris. equivalent amounts of protein, determined by a bicinchoninic acid (bca) assay (pierce bca protein assay kit; thermo scientific catalog no. ), were subjected to acetone precipitatation by incubation with a ϫ volume of ice-cold acetone overnight at Ϫ °c. precipitated proteins were pelleted at °c at , ϫ g for min, and the pellets were dried for min at room temperature. pellets were resuspended in a mixture of l pierce ripa lysis buffer (thermo scientific catalog no. (li-cor biosciences) or enhanced chemiluminescent substrates (pierce ecl western blotting substrate; catalog no. ) and x-ray film (dot scientific catalog no. bdb ). densitometric quantification was performed using .tif files and imagej software from nih ( ) . to attempt to demonstrate pkr ubiquitination status during mav- infection, c bl/ mefs were transfected with green fluorescent protein (gfp) (addgene catalog no. )-tagged or hemagglutinin (ha) epitope (addgene catalog no. )-tagged ubiquitin plasmids h before infection. we used polyplus jetprime transfection reagent (polyplus catalog no. - ) with g plasmid dna and l jetprime reagent per -cm-diameter plate. at hpi ( h posttransfection), we treated mock-infected and infected c bl/ mefs with m mg (sigma m ) for h before collecting lysates at hpi in hcn buffer ( mm hepes, mm nacl, mm cacl , % triton x- [sigma t ]), ϫ protease inhibitors [protease inhibitor cocktail kit; thermo scientific catalog no. ], mm n-ethylmaleimide). the lysates were split into two aliquots, and g pkr (santa cruz d- sc- ; discontinued) or g isotype rabbit polyclonal antibody (jackson immuno catalog no. - - ) was added to lysates. after rocking samples overnight at °c, l of a protein a agarose suspension (calbiochem/millipore catalog no. ip - . ml) was added to each sample and the samples were rocked at °c for h. after incubation, the agarose was washed times with ml hcn buffer, resuspended in l ϫ laemmli buffer (bio-rad catalog no. - )- % -mercaptoethanol (sigma m ), and boiled for min. the lysate supernatants remaining after the initial pkr immunoprecipitation were then immunoprecipitated again using the same procedure but with g anti-p mouse monoclonal antibody (do- ; santa cruz sc- ) or g isotype mouse monoclonal antibody (thermofisher scientific catalog no. - ). immunoprecipitated proteins were subjected to immunoblotting for gfp or ha epitope-tagged ubiquitin with antibodies for gfp (roche catalog no. ) ( : , ) or ha (abcam catalog no. ab ) ( : , ). no ubiquitin signal was detected from the pkr immunoprecipitations, though the positive control, p , showed ubiquitin signal with both epitope-tagged ubiquitins (fig. s in the supplemental material). blots were also probed for pkr (pkr b- sc- ) ( : ), p (anti-p sc- ) ( : ), and irdye cw anti-mouse (li-cor - ) ( : , ) to confirm that the immunoprecipitations had been successful, and signals for both proteins were detected (fig. s ) . viral dna yield analysis by qpcr. cells were washed twice with room temperature pbs and harvested by scraping into pbs, centrifuging at ϫ g for min at °c, and resuspending in pbs. total cellular dna was purified using an invitrogen purelink dna purification kit (thermo scientific catalog no. k - ) and quantitated using a nanodrop spectrophotometer. a -ng volume of total cellular dna was analyzed by qpcr using custom primers specific to mav- e a (me agenomic fwd [ = gca ctc cat ggc agg att ct =] and me agenomic rev [ = ggt cga agc aga cgg ttc ttc =]), and the results were normalized to gapdh (glyceraldehyde- -phosphate dehydrogenase), which was analyzed using a gapdh-specific primer/probe set (thermo fisher scientific mm _g ; catalog no. ). mrna analysis by qpcr. cells were harvested by scraping into media, centrifuging at ϫ g for min at °c, and washing the cell pellet three times with ice-cold pbs. rna was purified using a qiagen rneasy minikit (qiagen catalog no. ) and stored at Ϫ ˚c. a -ng volume of rna per sample was used to make cdna using a high-capacity cdna reverse transcription (rt) kit (applied biosystems catalog no. ), and l of the cdna was analyzed by qpcr using a primer/probe set specific to mouse pkr sequence (thermo fisher mm _m ; catalog no. ). the results were normalized to gapdh, which was analyzed using a gapdh-specific primer/probe set (thermo fisher scientific mm _g ; catalog no. ). arbitrary units were calculated as follows: mean threshold cycle (c t ) pkr Ϫ mean c t gapdh ϭ Δc t for sample (arbitrary unit ϭ ϪΔct ). proteasome inhibition. c bl/ mefs were infected at an moi of , and dmso, m mg (sigma m ), or m bortezomib (selleckchem catalog no. s ) was added to the media after a h adsorption. at hpi, cells were washed once with room temperature pbs, and pierce ripa lysis buffer (thermo scientific catalog no. ) with ϫ protease inhibitors (protease inhibitor cocktail kit; thermo scientific catalog no. ) was added to the plate. the cells were allowed to lyse at room temperature for min before being harvested and centrifuged at °c at , ϫ g for min to remove debris. lysosome inhibition and dq bsa assay. cmt cells were infected at an moi of . after h of adsorption, l water, mm (final concentration) nh cl (baker chemical company catalog no. - ), or m (final concentration) chloroquine (sigma c ) was added to the media. at hpi, at room temperature, cells were washed once with pbs, and pierce ripa lysis buffer (thermo scientific catalog no. ) with ϫ protease inhibitors (protease inhibitor cocktail kit; thermo scientific catalog no. ) was added to the plate. the cells were allowed to lyse at room temperature for min before being harvested and centrifuged at °c at , ϫ g for min to remove debris. the dq bsa assay was performed as described previously ( ) . briefly, c bl/ mefs and cmt cells were plated at . ϫ cells/plate and ϫ cells/plate, respectively, in mattek glass-bottom microwell dishes (part no. p g- . - c) with ml of dmem- % and dmem- % fbs, respectively. the next day, the cell medium was treated with l water, mm (final concentration) nh cl, or m (final concentration) chloroquine (sigma c ). four hours after addition of inhibitors, dq red bsa (invitrogen catalog no. d ) was added to the media to reach a final concentration of g/ml in ml dmem plus % and % fbs, respectively. at h posttreatment, the cells were imaged on a nikon te inverted microscope equipped with a mercury arc lamp; a plan-apochromat ϫ, . -numerical-aperture (na) objective; a cooled digital charge-coupled-device (ccd) camera (quantix photometrics, tucson, az); and a temperature-controlled stage (set at °c). to image the dq-bsa, we used an excitation filter centered at nm and an emission filter centered at nm. the exposure times were the same for all images. ribosome pelleting. ribosomes were pelleted as described previously ( ) . briefly, c bl/ mefs were plated on -cm-diameter plates at ϫ cells per plate. the next day, the cells (ϳ % confluent) were infected with mav- at an moi of . c bl/ mef lysates were collected at hpi by scraping the cells in ice-cold pbs containing g/ml cycloheximide (sigma c ), pelleting, and resuspending in lysis buffer, which contained mm hepes (ph . ), mm kcl, mm mgcl , mm dtt, . % np- , g/ml cycloheximide, u/ml rnasin (promega catalog no. n ), % sucrose, and ϫ protease inhibitors (protease inhibitor cocktail kit; thermo scientific catalog no. ). cells were lysed by passage through a chilled -gauge needle five times and were cleared by centrifugation for min at , ϫ g at °c. a -l volume of cleared lysate (optical density at nm [od ] of ) was layered onto % sucrose and centrifuged at , rpm in an sw rotor (average relative centrifugal force [rcf], , ) for h at °c. after pelleting, the supernatant was removed by the use of a micropipette, and l of buffer rlt plus (from qiagen rneasy minikit) ( °c) was added to the pellet for collection of the rna. the rna was purified immediately using an qiagen rneasy minikit (qiagen catalog no. ) and stored at Ϫ ˚c until analysis. polyribosome gradients. c bl/ mefs were plated on -cm-diameter plates ( ϫ cells per plate). the next day, the cells were infected with mav- at an moi of . following a standard protocol ( ), min prior to collection, cycloheximide was added at a final concentration of g/ml and the reaction mixture was incubated at °c. cells were collected at hpi by scraping in ice-cold pbs containing g/ml cycloheximide, pelleting, and resuspending in l lysis buffer ( mm ). cells were lysed by passage through a chilled -gauge needle times, vortex mixing for s, and then incubating on ice for min. lysates were cleared by centrifugation for min at , ϫ g at °c. a -l volume of cleared lysate (od of ) was layered onto a %-to- % sucrose gradient and centrifuged at , rpm in an sw rotor ( , rcf) for h at °c. after centrifugation, gradients were pumped out of the top with a brandel br- density gradient fractionation system with a continuous reading of the od . from to fractions ( to l) were collected. rna was purified from selected fractions immediately using a qiagen rneasy minikit (qiagen catalog no. ) and stored at Ϫ ˚c until analysis by rt-qpcr. statistical analyses. data were analyzed with graphpad prism . software. for qpcr and densitometry analyses, the data were analyzed by individual mann-whitney tests. a p value of Ͻ . was considered significant. supplemental material for this article may be found at https://doi.org/ . /mbio . - . molecular cloning and characterization of the human double-stranded rna-activated protein kinase induced by interferon mechanism of interferon action: identification of a rna binding domain within the n-terminal region of the human rna-dependent p /eif- alpha protein kinase identification of doublestranded rna-binding domains in the interferon-induced doublestranded rna-activated p kinase isolation of the interferon-inducible rnadependent protein kinase pkr promoter and identification of a novel dna element within the '-flanking region of human and mouse pkr genes identification of the double-stranded rnabinding domain of the human interferon-inducible protein kinase dsrna-dependent protein kinase pkr and its role in stress, signaling and hcv infection endogenous inhibitors of the dsrnadependent eif- alpha protein kinase pkr in normal and rastransformed cells the double-stranded rna-dependent protein kinase is also activated by heparin autophosphorylation of the protein kinase dependent on double-stranded rna adenoviridae: the viruses and their replication role of eif alpha kinases in translational control and adaptation to cellular stress phosphorylation of initiation factor elf- and the control of reticulocyte protein synthesis effect of hemin on site-specific phosphorylation of eukaryotic initiation factor viral proteins targeting host protein kinase r to evade an innate immune response: a mini review identification of a conserved motif that is necessary for binding of the vaccinia virus e l gene products to doublestranded rna roles of vaccinia virus genes e l and k l and host genes pkr and rnase l during intratracheal infection of c bl/ mice recombinant vaccinia virus k l gene product prevents activation of double-stranded rnadependent, initiation factor alpha-specific protein kinase 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 biochemical and genetic evidence for complex formation between the influenza a virus ns protein and the interferoninduced pkr protein kinase ebola virus vp protein binds double-stranded rna and inhibits alpha/beta interferon production induced by rig-i signaling inhibition of pkr activation by the proline-rich rna binding domain of the herpes simplex virus type us protein the herpes simplex virus us protein effectively compensates for the gamma ( . ) gene if present before activation of protein kinase r by precluding its phosphorylation and that of the alpha subunit of eukaryotic translation initiation factor hiv-i tat inhibits pkr activity by both rna-dependent and rna-independent mechanisms hiv- tat directly interacts with the interferon-induced, double-stranded rna-dependent kinase tar rna-binding protein is an inhibitor of the interferon-induced protein kinase pkr adenovirus vai rna is required for efficient translation of viral mrnas at late times after infection virus-associated rnas of naturally occurring strains and variants of group c adenoviruses rna of low molecular weight in kb cells infected with adenovirus type toscana virus nss protein promotes degradation of double-stranded rna-dependent protein kinase nss protein of rift valley fever virus induces the specific degradation of the double-stranded rnadependent protein kinase rift valley fever virus nss protein promotes post-transcriptional downregulation of protein kinase pkr and inhibits eif alpha phosphorylation protein kinase r degradation is essential for rift valley fever virus infection and is regulated by skp -cul -f-box (scf)fbxw -nss e ligase degradation of the interferoninduced , -m(r) protein kinase by poliovirus requires rna the cellular , -mr protein kinase is highly autophosphorylated and activated yet significantly degraded during poliovirus infection: implications for translational regulation esterase d enhances type i interferon signal transduction to suppress foot-and-mouth disease virus replication foot-and-mouth disease virus induces lysosomal degradation of host protein kinase pkr by c proteinase to facilitate virus replication middle east respiratory coronavirus accessory protein a inhibits pkr-mediated antiviral stress responses rapid decrease in the levels of the double-stranded rnadependent protein kinase during virus infections dsrna binding domain of pkr is proteolytically released by enterovirus a to facilitate viral replication nss virulence factor of rift valley fever virus engages the f-box proteins fbxw and beta-trcp to degrade the antiviral protein kinase pkr susceptibility and signs associated with mouse adenovirus type infection of adult outbred swiss mice mouse adenoviruses adenovirus myocarditis in mice. an electron microscopic study proinflammatory effects of interferon gamma in mouse adenovirus myocarditis mouse adenovirus type causes a fatal hemorrhagic encephalomyelitis in adult c bl/ but not balb/c mice adenovirus virus-associated rna and translation control completion of the dna sequence of mouse adenovirus type : sequence of e b, l , and l ( - map units) essential role for the dsrna-dependent protein kinase pkr in innate immunity to viral infection the murine double-stranded rnadependent protein kinase pkr is required for resistance to vesicular stomatitis virus blockade of interferon induction and action by the e l double-stranded rna binding proteins of vaccinia virus functional characterization of pkr gene products expressed in cells from mice with a targeted deletion of the n terminus or c terminus domain of pkr deficient signaling in mice devoid of double-stranded rna-dependent protein kinase characterization of transgenic mice with targeted disruption of the catalytic domain of the double-stranded rna-dependent protein kinase, pkr the kinase activity of pkr represses inflammasome activity inhibition of protein synthesis in reticulocytes by antibiotics. ii. the site of action of cycloheximide, streptovitacin a and pactamycin cgg repeat-associated non-aug translation utilizes a cap-dependent scanning mechanism of initiation to produce toxic proteins protein degradation, the cell: a molecular approach dq-red bsa trafficking assay in cultured cells to assess cargo delivery to lysosomes the utilization of extracellular proteins as nutrients is suppressed by mtorc mg , a proteasome inhibitor, induces apoptosis in tumor cells bortezomib as the first proteasome inhibitor anticancer drug: current status and future perspectives activity probe for in vivo profiling of the specificity of proteasome inhibitor bortezomib atpdependent conjugation of reticulocyte proteins with the polypeptide required for protein degradation proposed role of atp in protein breakdown: conjugation of protein with multiple chains of the polypeptide of atp-dependent proteolysis using the e orf -based e ubiquitin ligase as a tool to analyze the evolution of adenoviruses a deubiquitinase negatively regulates retro-translocation of nonubiquitinated substrates interaction of mouse adenovirus type early region a protein with cellular proteins prb and p inhibitors of dna synthesis (aphidicolin and arac/hu) prevent the recovery of rna synthesis after uv-irradiation a study of the mechanisms of cytotoxicity of ara-c on three human leukemic cell lines interferon action in triply deficient mice reveals the existence of alternative antiviral pathways structure, function, and evolution of adenovirusassociated rna: a phylogenetic approach pkr, a p target gene, plays a crucial role in the tumor-suppressor function of p proteasomes can degrade a significant proportion of cellular proteins independent of ubiquitination proteasomes and protein conjugation across domains of life transcription mapping of mouse adenovirus type early region degradation of p by adenovirus e orf and e b k proteins occurs via a novel mechanism involving a cullin-containing complex analysis of the adenovirus e b- k-anchored proteome reveals its link to ubiquitination machinery adenovirus e orf targets transcriptional intermediary factor gamma for proteasome-dependent degradation during infection the adenovirus e -orf protein functions as a sumo e ligase for tif- gamma sumoylation and poly-sumo chain elongation genetic analysis of adenovirus type iii. temperature sensitivity of processing viral proteins processing of adenovirus -induced proteins identification of the gene coding for the precursor of adenovirus core protein x the proteinase polypeptide of adenovirus serotype virions herpes simplex virus type immediate-early gene expression is required for the induction of apoptosis in human epithelial hep- cells virus taxonomy. classification and of nomenclature viruses. ninth report of the international committee on taxonomy of viruses mouse adenovirus type infection of macrophages alphavirus minus-strand synthesis and persistence in mouse embryo fibroblasts derived from mice lacking rnase l and protein kinase r a critical role for pkr complexes with trbp in immunometabolic regulation and eif alpha phosphorylation in obesity construction of mouse adenovirus type mutants distribution of mouse adenovirus type in intraperitoneally and intranasally infected adult outbred mice novel expression of mouse adenovirus type early region gp k at late times after infection the imagej ecosystem: an open platform for biomedical image analysis polyribosome analysis for investigating mrna translation in xenopus oocytes, eggs and embryos polysome profiling analysis we thank robert silverman, gokhan hotamisligil, and anthony sadler for pkr mutant and wild-type cells. we thank katelyn green, becky billmire, and peter todd for the use of and assistance with the brandel br- density gradient fractionation system and for providing the nanoluciferase and firefly luciferase plasmids. we thank zachary mendel and joel swanson for assistance with imaging the dq bsa assay. we thank jason weinberg, michael imperiale, christiane wobus, billy tsai, andrew mehle, mitch ledwith, mike mathews, chuck samuel, and britt glaunsinger for helpful discussions. we thank michael imperiale and christiane wobus for comments on the manuscript. key: cord- -x yswoua authors: lipsitch, marc; inglesby, thomas v. title: reply to “studies on influenza virus transmission between ferrets: the public health risks revisited” date: - - journal: mbio doi: . /mbio. - sha: doc_id: cord_uid: x yswoua nan between and the value obtained by dividing by the number of observed lab-years ( ) . moreover, while u.s. labs working with select agents show no reports of accidental viral infections in to , accidental lais have occurred in or from bsl , bsl agricultural (bsl ag), or bsl laboratories in china, singapore, the united kingdom, russia, and taiwan ( ) . finally, underreporting of lai is internationally the rule rather than the exception, in part because serosurveillance is not routinely performed in many highcontainment labs, and in part because reporting systems, if they exist, are inadequate. the netherlands has been singled out as notable for inadequate surveillance and reporting of lai ( ) . also, as a general principle, it is self-evident that the number of potentially serious laboratory exposures is greater than the number of actual confirmed laboratory infections. for example, the cdc has reported a number of potentially serious laboratory exposures this year, but none of them would have been factored into lai calculations. at the time of this writing, it is unclear whether the exposure of a cdc technician to ebola virus due to an error of switching live and inactivated samples has resulted in infection; whatever the outcome, this incident reinforces the idea that accidental exposures are possible in the best virologic laboratories. fouchier proposes another measure of accident rate, the number of accidents per worker-year, where a worker is defined as a person with access approval to a bsl , - , or - lab that handles select agents. from these data he calculates a rather low risk of Ͻ per , worker-years, using the henkel et al. ( ) denominator, including all agents at all biosafety levels, and the numerator of known viral infections. this suffers from the same conceptual and statistical problems noted above and from the additional problem that the individuals "approved" to have access to a select agent facility will be highly heterogeneous in the amount of time they actually spend there. a more relevant metric, we suggest, is that estimated for the intramural labs in niaid, which experienced known lais in , person-hours of actually work in a bsl lab, or about a % risk for every , h of work in a bsl lab ( ) . fouchier lists a number of enhancements to standard bsl practices that are in place in the erasmus medical center facility and proposes that these provide an increase in safety of at least a factor of above that of standard bsl labs. this factor of is arbitrary. using a factor of to represent an unknown value in the absence of data to support this strikes us as inconsistent with the rules of caution that apply to dealing with unknown hazards of high-consequence events. we agree that it is difficult to quantify the impact of these practices, and we agree that some account should be taken of these enhancements, for laboratories that use them. we leave it to impartial risk assessors to decide how these enhancements should be accounted for. any risk assessment, however, must explicitly account for the possibility of human error or malicious removal of the agents from the source lab, circumventing these enhanced safeguards ( ) . as observed by kimman et al., "in the majority of cases of lais a direct cause could not be assigned. . ., suggesting that a failure was not noticed in many cases or that containment may have been insufficient" ( ) . in relation to the gain-of-function risk assessment, gavin huntley-fenner, a speaker at the national academy of sciences symposium on gain-of-function, wrote "we need to plan as though human error is inevitable. research suggests that even the most experienced and knowledgeable workers sometimes cut corners and that everyone is susceptible to distraction, fatigue and faulty reasoning" ( ) . in this regard, it is notable that all three recently publicized cdc laboratory incidents ( , ) involved removal of infectious material from the bsl containment facilities in which they were handled, in the false belief that the material was not infectious (the anthrax agent and ebola virus) or in the false belief that the material did not contain a highly pathogenic avian influenza (hpai) contaminant (influenza virus). under such circumstances, even the highestfunctioning mechanical systems and best-trained personnel in the source lab cannot prevent accidents in the destination lab. fouchier states that vaccination of laboratory workers in his laboratory reduces the risk of infection for such workers and that heightened surveillance of such workers and provision of antiviral drugs in case of an exposure will reduce their risk further. the benefits of vaccination and antivirals in preventing infection are overstated for several reasons. (i) vaccination of workers and heightened surveillance and treatment can be effective if the accidental exposure involves a member of that laboratory, but not necessarily if it involves personnel from other laboratories, as in the three cdc incidents ( , ) . probability calculations must separate exposures in the source laboratory from exposures in other laboratories. (ii) the availability and effectiveness of vaccines are not certain. in general, vaccine efficacy even against well-matched seasonal influenza virus varies widely ( ) . some ferret gain-oftransmission experiments involve subtypes for which there are (at the time of the experiment) no licensed vaccines ( ) ( ) ( ) . moreover, the effectiveness of vaccines and antivirals against laboratory-engineered strains in these experiments is uncertain, even when they are effective against the starting strain; this is why the initial report of ferret-transmissible strains described assays of vaccine neutralization (experiment ) and antiviral susceptibility (experiment ) ( ) . after performing the experiment, one can retrospectively infer that these protections would have been effective, but at the time of proposing an experiment, neither is certain. indeed, even after obtaining in vitro results, fouchier stated that the effectiveness of drugs and vaccines in vivo against the strains produced in his experiments was in doubt and needed testing ( ) . (iii) an additional crucial point is that the rate of laboratoryacquired infections in bsl labs, which we cite as at least . % per laboratory-year, already reflects the routine use of vaccination and prompt treatment of suspected exposures for many of the pathogens considered. hence, the . %/lab-year figure is, to a large degree, the rate at which breakthrough, detectable infections occur in laboratory workers who have immunologic ( ) and pharmacological ( ) protection, and therefore, for many pathogens it is not a rate pertaining to unprotected workers. to adjust downwards from this rate double-counts the protective benefits of those vaccines and drugs. fouchier further argues that the risk of onward transmission from an lai would be reduced relative to those we posited, a range of to %. he suggests that prophylaxis and vaccination should not only reduce the probability of infection (discussed above) but also reduce the probability of onward transmission by a factor of . the effect of prophylaxis and vaccination should be accounted for, as we noted in our article ( ), but again only if the infection occurs in the source laboratory where workers are prepared; moreover, the factor of reduction is much too optimistic, for the following reasons. (i) it assumes the infection is detected, which may or may not occur before spread. (ii) it assumes that vaccines and antivirals are given and effective against the labengineered virus, which is not guaranteed or in some cases even likely for the reasons noted above. (iii) if infection is detected before spread, and if vaccines and antivirals are given to the exposed person rapidly, and if the strain is susceptible to the antiviral, the reduction in infectiousness of a vaccinated, antiviraltreated case is probably closer to a factor of to than a factor of . this estimate is based on an assumption of multiplicative effects of antivirals and vaccination, using clinical data to estimate that oseltamivir reduces infectiousness approximately ϫ (by %) ( ) and a meta-analysis of published studies showing no reduction in infectiousness ( ) yet suggesting a "best guess" of a . -fold ( %) reduction in infectiousness from a well-matched inactivated vaccine ( ) . if infection is not detected before spread, or if vaccines and antivirals are not given to the infected person(s) rapidly enough to prevent spread, or if the strain is not susceptible to the antiviral or vaccine, then there is no reduction in risk. fouchier suggests that quarantine of laboratory workers would reduce transmission by another factor of . using a factor of to represent an unknown value in the absence of data to support this again strikes us as inconsistent with the rules of caution that apply to dealing with unknown hazards of high-consequence events. once again, this assumes that any exposure or infection is detected before transmission, something that has not occurred in a number of past lais. it also assumes that the exposures occur inside his laboratory, which is not guaranteed due to the possibility of erroneous or malicious removal of the strain from the lab. notably, the one published study designed to estimate risk of uncontrolled spread given an lai incorporated the assumption that detected lais would be subjected to nonpharmaceutical interventions, which would be somewhat effective against the first few cases of a flu-like agent. the risk of uncontrolled spread in that study came from scenarios in which such measures were not taken, for example, because the infection was not detected ( ) (around a to % chance depending on parameters), as well as from scenarios in which it was detected but not successfully con-author reply trolled, particularly for viruses with basic reproductive numbers exceeding . . of course, no effects of quarantine should be included in scenarios involving accidents outside the source lab. moreover, fouchier suggests that the viruses made transmissible in ferrets would be less transmissible in humans than ordinary pandemic or seasonal viruses, because they transmit less well in ferrets than do human seasonal or pandemic viruses and because their adaptation is to ferrets, not humans. we have two points to make in response. (i) these claims run counter to the original rationale for ferretgain-of-transmission studies. that rationale was to predict pandemic potential of natural isolates, which fouchier earlier argued was associated with "airborne transmission" best studied in mammalian nonhuman hosts ( ) . likewise, yoshihiro kawaoka described the purpose of his ferret gain-of-transmission studies as "[t]o determine whether h n viruses could be transmitted between humans" ( ) , and the original reports of ferret transmission experiments say that pandemic potential is associated with the changes observed. for example: "whether this virus may acquire the ability to be transmitted via aerosols or respiratory droplets among mammals, including humans, to trigger a future pandemic is a key question for pandemic preparedness . . . identification of the minimal requirements for virus transmission between mammals may have prognostic and diagnostic value for improving pandemic preparedness" ( ) . similarly, in another ferret transmission study on synthetic -like viruses from the kawaoka lab, mutations conferring ferret transmissibility are specifically called "human-adaptive mutations" ( ) . the cdc considers ferret transmissibility and human alpha- , receptor binding to be of the top predictors of the threat of emergence of influenza viruses ( ) . a recent publication by cdc influenza virologists suggests that they interpret specific mutations found in the ferret gain-of-transmission studies as signaling human adaptation, not specifically ferret adaptation ( ) . (ii) the claim of lack of transmission in humans is a sharp departure from earlier claims. fouchier initiated public discussion of these studies by claiming he had created "probably one of the most dangerous viruses you can make" ( ) . current denials that this is possible seem to be designed to reduce perceived risk from the experiments rather than to describe new scientific data or understanding. paul keim, the chair of the national science advisory board on biosecurity who reviewed the original submission of the h n paper, stated, "i can't think of another pathogenic organism that is as scary as this one" ( ) . the reaction of another member of the nsabb, michael imperiale, was as follows: "[imperiale] also says it was news to him that the mutated virus did not spread between ferrets via the aerosol route as readily as seasonal strains, as fouchier showed at the asm meeting. 'that really didn't come across to me in the paper,' he says. 'i didn't see that kind of comparison.'" ( ) . without understanding why this change in interpretation has occurred, it is difficult to incorporate into a risk analysis a speculation that ferret adaptation reduces human adaptation, as fouchier argues in his paper. moreover, whether or not this occurred in the particular experiment involving h n in the fouchier lab, it cannot be assumed to be a reliable outcome of future studies. fouchier argues that the consequences of onward transmission would be less than assumed in the upper-bound estimate we use for the case fatality ratio ( %), though he does not indicate what estimate he thinks would be more appropriate. assertions that wild-type h n is much less than % lethal are not well founded. the estimate that several percent of persons in large areas of asia were asymptomatically infected with h n , used to support a lower estimate of wild-type h n lethality, comes from work by wang et al. ( ) which has been directly refuted by influenza serology experts and epidemiologists ( ) and further refuted by a separate analysis that was similarly critical of the data used by wang et al. ( ) . we do not regard the case fatality risk (cfr) of h n in naturally exposed humans as a settled issue, and well-conducted serosurveys may support the idea that asymptomatic or subclinical infections are more common than previously estimated, at least in some populations ( ) . yet for the moment there is little evidence that the observed~ % cfr in humans for h n is the result of missing large numbers of milder infections, in contrast to the situation, for example of h n , where detected cases are thought to be a small fraction of the total ( ). fouchier further cites evidence of human attenuation when other viruses have been passaged in nonhuman hosts and implies that the viruses passaged in ferrets in his laboratory are attenuated, stating, "[i]n addition, it is important to note that fatalities in ferrets infected with a/h n virus via respiratory droplets or aerosols have not occurred, contrary to when ferrets received large dosages of a/h n virus directly in the (lower) airways" ( ). our response to this point is threefold. (i) there is no direct evidence that the ferret-passaged variants of h n from fouchier's laboratory are less virulent for humans, or indeed for ferrets, than wild-type h n . such a comparison would require lower mortality from the ferret-transmissible strain following inoculations of the same doses by the same route. in table of reference , it is shown that / ferrets died from wild-type or ferret-transmissible virus when exposed by the intratracheal route: in this assay, their virulence was indistinguishable. table gives no data on wild-type h n administered by the intranasal route, suggesting that ferret-passaged (but nontransmissible) wild-type h n can be used as a stand-in. even this suboptimal comparison, using four different isolates, is not statistically significant ( / versus / ; p ϭ . ). when the engineered viruses were transmitted by aerosol to ferrets, / died, consistent with a % confidence interval for the probability of lethality in ferrets of to %. we do not know the inoculum in these transmission experiments and how it compares to inocula in humans if they were infected by aerosol, or how this translates into fatality risk in humans. from a risk assessment perspective, the conservative assumption that human lethality of evolved strains is similar to that of the starting strains is well justified. (ii) as with transmissibility, reduced ferret lethality of ferrettransmissible strains is a falsifiable hypothesis only when the experiment is undertaken, not a known result. it might occur or it might not occur, and one cannot tell without doing the experiments. it is certainly not a law of nature that transmissibility brings reduced lethality; such reduction did not occur, for example, when h n viruses were made transmissible in ferrets ( ) . (iii) as with reduced transmissibility, the assertions of reduced lethality are inconsistent with early statements about the experiments. nsabb member michael imperiale was quoted in science as saying in , "what ron [fouchier] is saying now is not what was in the paper. we were led to believe by the paper that aerosol transmission is also lethal." ( ) . this view was shared by at least one reporter who attended the malta presentation of the results ( ) . fouchier asserts that his claims of the likely low human transmissibility and lethality of the ferret-adapted strains should not be interpreted as reducing the likely benefits of the work for public health. we disagree. given the uncertainties about whether the strains created in any given laboratory are indeed transmissible and virulent for humans, there should indeed be some probability assigned to the scenarios in which they are not and some probability assigned to those in which they are. this does reduce the overall risk by some factor, though for reasons stated above, we believe the reduction would be modest, rather than the orders-ofmagnitude reduction suggested by fouchier. while the impact on risk assessment might be to assign less than % weight to the scenario of a virulent, pandemic-like strain being released, we believe the same uncertainty negates or even reverses the principal public health benefits claimed for this work. these purported benefits depend on the assumption that mutations found in ferret passage experiments reliably predict pandemic risk. cdc experts state that they have deployed teams to cambodia based on the presence in h n isolates there of mutations identified in ferret passage experiments ( ) and relied on these markers for pandemic threat assessment of h n : "early detection of these molecular markers in h n viruses isolated from humans gave public health authorities evidence that these viruses posed an immediate pandemic threat" ( ) . yet there is no evidence that this reliance has improved decisions by cdc or other public health officials, because we do not know if the strains they identified as high risk actually are higher risk than average. this condition of ignorance stems from the fact that there is no validated predictive algorithm for pandemic risk ( ) . to take a simplified example, suppose it were the case that % of the time, strains produced in ferret passage experiments were highly lethal and transmissible in humans, and % of the time they were attenuated. we would not know which instances are which, but suppose we knew these are the overall frequencies. in this case, it would be appropriate to multiply our pandemic risk calculations by about %, because out of ferret passage experiments would produce strains not very harmful to humans. twenty-five percent of the risk we estimated ( ) is still exceptionally high. yet now consider the use of this information by public health authorities. at best, three out of every four times they identified a veterinary or zoonotic isolate as high risk, they would actually be targeting a strain with features that make it attenuated in humans. they would be deploying resources to contain a strain that is, unbeknownst to them, human attenuated. one in four times, they might identify a strain with somewhat increased risk for humans, albeit not necessarily the strain most deserving of attention. in fact, because the prediction of mutational effects becomes more uncertain with changes in the genetic background, the predictive power of such targeting activities is even lower. in summary, while the possibility that ferret gain-of-transmission strains are attenuated in humans modestly reduces the risk estimate associated with producing and using them, it may nullify and even reverse the utility of such studies for public health. considering a particular sequence change may help to further illuminate this issue. the cdc team's description of the public health benefits of gof experiments refers to the lysine mutation at pb position as an important factor in raising the level of concern for animal or zoonotic human virus isolates ( ) . the h n strain of created a pandemic that caused over , to , respiratory deaths globally ( , ) despite lacking this mutation. had there been surveillance in place for the viruses giving rise to that pandemic, the lack of this mutation might have misled experts into thinking the virus carried a lower risk and focusing attention on other viruses-a false negative. indeed, fouchier's lab was the first to demonstrate that in that genetic background, there was no detectable effect of the mutation ( ) . this is just one anecdote-though arguably the most pertinent, as it is the only modern pandemic-supporting the general fact that interpreting surveillance through the lens of particular mutations remains an unproven and error-prone technique ( ) . fouchier repeatedly describes his adjustments to the probability estimates we proposed as "conservative," implying that the actual risk is even less than his figures show. his analysis is not conservative. his estimate of one lai per~ , worker-years is dramatically lower than that currently estimated for any category of laboratory, and current estimates themselves are too low due to underreporting ( ) . moreover, describing the estimates as conservative is at odds with the use of large factors to stand for unknown effects of safety enhancements, inconsistent use of numerators and denominators to favor lower probabilities, and the assumption that safety enhancements used in the erasmus mc laboratory will be effective in the face of evidence that many laboratory infections have no traceable cause and that many mishaps involving infectious exposures may occur outside the "home laboratory." the assumptions of antiviral and vaccine effectiveness and reduced human transmissibility and virulence of selected strains range from uncertain (in the case of much of the published work) to unknowable (in the case of experiments not yet done) and false (in the case of reduced virulence and vaccine availability in examples such as h n ) ( ) . such assumptions are "anticonservative," giving too-optimistic predictions. further problems include unsupported claims that the implementation of the select agent program necessarily strengthens biosafety. for example, in the cdc report on the lab accident involving h n , the description of the event indicates that scientists were making their decisions in reference to the select agent rule, as opposed to whether there was a biosafety breach ( ) . the quality of "targeted risk assessments" undertaken before each study is performed is unclear; such assessments have not been quantitative to date ( , ) . some of the disagreements discussed here could be clarified by a clearer understanding of the data. it would be extremely helpful for cdc to tabulate incidents with select agents, including lais, by the biosafety level of the laboratory involved, so that proper denominators can be used for calculations rather than having to rely on bounding arguments ( ) . critical evaluation of claims about the safety of particular laboratories-not only the erasmus mc laboratory discussed by fouchier but others where potential pandemic pathogen experiments are proposed or conducted-is impossible without transparent reporting of potential loss, release, and theft events at these particular facilities and in laboratories more generally ( ) . if the cdc incidents of have any lesson, it is that state-of-the-art biosafety and biosecurity in highly respected facilities are no guarantee against human error, so there author reply is a limit to the reassurance one should take from lists of preventive measures in place at any particular facility. finally, there are previously published general recommendations regarding risk analysis and catastrophic events. ord et al. have noted that when one performs a risk analysis and estimates an exceptionally low probability (p) of a catastrophic outcome, it is crucial to consider the probability q (which may exceed p) that the model used to derive that probability is itself wrong, in a way that understates the true probability of the outcome ( ) . in such a circumstance a correction is needed, adjusting the estimate upward to account for this uncertainty. the combination of an implausibly low estimate of lai risk with assumptions that are difficult to defend, in a field where underreporting of accidents is thought to be routine ( ) , would seem to make the assessment suggested by fouchier's letter ( ) a prime candidate for such adjustment. studies on influenza virus transmission between ferrets; the public health risks revisited the unacceptable risks of a man-made pandemic the consequences of a lab escape of a potential pandemic pathogen. front pub health : ethical alternatives to experiments with novel potential pandemic pathogens moratorium on research intended to create novel potential pandemic pathogens monitoring select agent theft, loss and release reports in the united states probability of adverse events that have not yet occurred: a statistical reminder national bio and agro-defense facility final environmental impact statement, appendix b. accessed evidence-based biosafety: a review of the principles and effectiveness of microbiological containment measures ebola lapses show lab safety protocols should factor in human error report on the potential exposure to anthrax ebola sample is mishandled at c.d.c. lab in latest error, p a efficacy and effectiveness of influenza vaccines: a systematic review and meta-analysis limited airborne transmission of h n influenza a virus between ferrets airborne transmission of highly pathogenic h n influenza virus in ferrets minimal molecular constraints for respiratory droplet transmission of an avianhuman h n influenza a virus airborne transmission of influenza a/h n virus between ferrets flu researcher ron fouchier: "it's a pity that it has to come to this risk of occupationally acquired illnesses from biological threat agents in unvaccinated laboratory workers management guidelines for laboratory exposures to agents of bioterrorism antiviral effects on influenza viral transmission and pathogenicity: observations from household-based trials estimating influenza vaccine efficacy from challenge and community-based study data containing the accidental laboratory escape of potential pandemic influenza viruses predicting "airborne" influenza viruses: (trans-) mission impossible? h n : flu transmission work is urgent circulating avian influenza viruses closely related to the virus have pandemic potential influenza risk assessment tool (irat) use of highly pathogenic avian influenza a(h n ) gain-of-function studies for molecularbased surveillance and pandemic preparedness controversial studies give a deadly flu virus wings nsabb members react to request for second look at h n flu studies seroevidence for h n influenza infections in humans: meta-analysis comment on "seroevidence for h n influenza infections in humans: meta-analysis assessment of serosurveys for h n avian influenza a(h n ) and a(h n ) seroprevalence and risk factors for infection among egyptians: a prospective, controlled seroepidemiological study human infection with avian influenza a h n virus: an assessment of clinical severity five easy mutations to make bird flu a lethal pandemic improving pandemic influenza risk assessment global mortality estimates for the influenza pandemic from the glamor project: a modeling study estimated global mortality associated with the first months of pandemic influenza a h n virus circulation: a modelling study introduction of virulence markers in pb of pandemic swine-origin influenza virus does not result in enhanced virulence or transmission report on the inadvertent cross-contamination and shipment of a laboratory specimen with influenza virus h n statement near : . nas/iom gain-of-function symposium report of the ibc subcommittee on dual use research of concern probing the improbable: methodological challenges for risks with low probabilities and high stakes we thank several colleagues for critical reading of sections of this reply. m.l.'s work was supported by national institute of general medical sciences of the national institutes of health under award no. u gm .the content is solely the responsibility of the authors and does not necessarily represent the official views of the national institutes of health. key: cord- - o tb h authors: furmanski, martin title: the h n influenza virus reemergence demonstrated gain-of-function hazards date: - - journal: mbio doi: . /mbio. - sha: doc_id: cord_uid: o tb h nan r ozo and gronvall, in "the reemergent h n strain and the gain-of-function debate" ( ), confirmed the laboratory origin of the influenza pandemic and judged it was unintentional, but they concluded that its "relevance to gof research is greatly diminished if the epidemic was the result of a vaccine trial or vaccine development gone awry; these are both more plausible explanations than a single laboratory accident." separating the risks of vaccine development from those of basic gain-of-function (gof) research is inappropriate, because gof research seeks to discover antigenic and genomic changes that facilitate human-to-human transmission and/or augment virulence, with the aim of preemptively producing vaccines. clearly, if and when such changes are identified, candidate viruses possessing novel potential pandemic traits would be sent to vaccine laboratories for further development, prior to their appearance in nature. because it is unlikely that such traits would be unique, and because the natural appearance of any particular trait is unpredictable, a stable of multiple potentially pandemic virus lineages, and artificial hybrids thereof, could enter the vaccine development laboratories, multiplying the opportunities for novel gof viruses to escape. this emphasizes rather than diminishes the implications of the h n escape, since the - h n virus progenitor of the virus was likely in vaccine development preemptively because of the unrealized threat of a swine flu pandemic. i disagree with the authors' statement: "it remains likely that to this date, there has been no real-world example of a laboratory accident that has led to a global epidemic." the h n virus caused a global epidemic, and as rozo and gronvall themselves concluded, it originated in a microbiology laboratory and its release was unintentional. which laboratory is responsible matters little in the gof debate. rozo and gronvall also stated that, "in , influenza research was performed without modern biosafety regulations and protective equipment, making the lab accident hypothesis much less relevant to the modern gof debate." however, the current record of containment of high-consequence pathogens is hardly reassuring. my review of relevant events ( ) found that escapes of highconsequence pathogens causing community infections typically occur from state-of-the-art laboratories, including six outbreaks of severe acute respiratory syndrome and one of foot-and-mouth disease since . since , four extramural escapes of high-consequence pathogens have originated from prestigious u.s. laboratories. vir-ulent anthrax and avian influenza virus sent from the cdc ( ) and anthrax sent from dugway proving ground ( ) by public carriers entered nonsecure areas of other laboratories. activities at the select agent laboratory at the tulane national primate research center remain suspended after burkholderia pseudomallei, the agent of melioidosis, escaped containment and caused multiple primate infections in an outdoor primate facility ( , ) . the incidence of laboratory accidents that offer an exit path for high-consequence pathogens into the community through worker infection is unsettlingly high. in , the most recent year for which data have been reported ( ), the cdc received reports of releases (intramural breaches of containment) of select agents from state-of-the-art select agent-approved laboratories. in these elite laboratories, a breach of containment happens about twice weekly. the reemergent h n strain and the gain-of-function debate threatened pandemics and laboratory escapes: selffulfilling prophecies c.d.c. closes anthrax and flu labs after accidents review committee report: inadvertent shipment of live bacillus anthracis spores by dod escape of dangerous bacterium leads to halt of risky studies at news release. conclusion of select agent inquiry into burkholderia pseudomallei release at tulane national primate research center monitoring select agent theft, loss and release reports in the united states- - key: cord- - hvq qaj authors: nguyen-contant, phuong; embong, a. karim; kanagaiah, preshetha; chaves, francisco a.; yang, hongmei; branche, angela r.; topham, david j.; sangster, mark y. title: s protein-reactive igg and memory b cell production after human sars-cov- infection includes broad reactivity to the s subunit date: - - journal: mbio doi: . /mbio. - sha: doc_id: cord_uid: hvq qaj the high susceptibility of humans to severe acute respiratory syndrome coronavirus (sars-cov- ) infection, the cause of coronavirus disease (covid- ), reflects the novelty of the virus and limited preexisting b cell immunity. igg against the sars-cov- spike (s) protein, which carries the novel receptor binding domain (rbd), is absent or at low levels in unexposed individuals. to better understand the b cell response to sars-cov- infection, we asked whether virus-reactive memory b cells (mbcs) were present in unexposed subjects and whether mbc generation accompanied virus-specific igg production in infected subjects. we analyzed sera and peripheral blood mononuclear cells (pbmcs) from non-sars-cov- -exposed healthy donors and covid- convalescent subjects. serum igg levels specific for sars-cov- proteins (s, including the rbd and s subunit, and nucleocapsid [n]) and non-sars-cov- proteins were related to measurements of circulating igg mbc levels. anti-rbd igg was absent in unexposed subjects. most unexposed subjects had anti-s igg, and a minority had anti-n igg, but igg mbcs with these specificities were not detected, perhaps reflecting low frequencies. convalescent subjects had high levels of igg against the rbd, s , and n, together with large populations of rbd- and s -reactive igg mbcs. notably, igg titers against the s protein of the human coronavirus oc were higher in convalescent subjects than in unexposed subjects and correlated strongly with anti-s titers. our findings indicate cross-reactive b cell responses against the s subunit that might enhance broad coronavirus protection. importantly, our demonstration of mbc induction by sars-cov- infection suggests that a durable form of b cell immunity is maintained even if circulating antibody levels wane. t he betacoronavirus severe acute respiratory syndrome coronavirus (sars-cov- ), the causative agent of a respiratory disease termed coronavirus disease , emerged in china in late and rapidly spread worldwide ( ) . a pandemic was declared in march , and global deaths from covid- now exceed , . the rapid increase in cases in many countries has challenged health care systems, and shutdowns and quarantine measures introduced to slow virus spread have caused major disruptions to society and economies ( ) . sars-cov- infection produces a wide spectrum of outcomes. a proportion of infections, likely more than %, remain asymptomatic. most clinical cases develop mild to moderate respiratory symptoms, but up to % progress to a more severe disease with extensive pneumonia ( , ) . when sars-cov- emerged and began to spread, the severity of the threat was primarily attributed to the novelty of the virus to the human immune system and, consequently, a lack of preexisting immune memory to quickly clear virus and limit disease progression. four types of common cold coronavirus are endemic in humans, including the alphacoronaviruses e and nl and the betacoronaviruses oc and hku . however, limited relatedness between key structural proteins of these human coronaviruses (hcovs) and those of sars-cov- suggested that significant crossreactive immunity was unlikely ( , ) . initial studies of non-sars-cov- -exposed individuals found negligible levels of igg against the sars-cov- spike (s) protein, the viral attachment protein that binds receptor angiotensin converting enzyme (ace ) on host cells to initiate infection ( ) . more recently, however, studies have provided evidence of sars-cov- -reactive b and t cell memory in unexposed subjects that could confer some protection against sars-cov- or modulate disease pathogenesis ( ) ( ) ( ) . sera from non-sars-cov- -exposed individuals have been screened for igg binding to the s and s subunits of the sars-cov- s protein. the membrane-distal s subunit contains the receptor binding domain (rbd) for receptor recognition, and the membrane-proximal s subunit, which has higher homology among coronaviruses than does s ( , ) , mediates membrane fusion to release viral rna into the host cell. in two large cohorts of unexposed subjects, approximately % had igg that bound s but not s or the rbd. approximately % of the subjects had igg against the sars-cov- nucleocapsid (n) protein, which is highly conserved among coronaviruses ( , ) . although n is an internal viral protein and not a target of neutralizing antibodies (abs), coronavirus infections typically elicit strong anti-n ab production ( ) . the idea that circulating hcovs elicit igg that cross-reacts with sars-cov- is supported by the finding that sars-cov- infection increases igg titers against the s proteins of multiple hcovs ( ) . in t cell studies, cd ϩ t cells in up to % of non-sars-cov- -exposed donors responded to epitopes in s and non-s proteins of sars-cov- ( , ) . notably, s-reactive cd ϩ t cells in unexposed subjects were mostly reactive to the conserved s subunit, consistent with cross-reactivity to circulating hcovs ( ) . sars-cov- -reactive cd ϩ t cells were also detected in unexposed donors, but the response was less marked than for cd ϩ t cells ( ) . sars-cov- -reactive memory b cells (mbcs) generated in b cell responses to hcovs are also likely to be present in non-sars-cov- -exposed individuals. indeed, mbcs might be more important than preexisting cross-reactive abs as a source of protection against sars-cov- . igg mbcs are more broadly reactive than bulk serum abs generated against the same antigen, they persist after circulating ab levels wane, and they are readily activated to generate strong ab responses or seed germinal centers for additional rounds of affinity maturation ( ) . concurrent early production of virusspecific igm and igg in the response to sars-cov- infection suggests a response mediated by igg mbcs as well as by naive b cells ( , ( ) ( ) ( ) . this picture is supported by identification of b cell subsets with high and low immunoglobulin v gene mutation frequencies during the response to sars-cov- infection ( ) . however, little direct analysis of sars-cov- -reactive mbcs in unexposed subjects has been performed. characterization of populations of mbcs generated and/or expanded by sars-cov- infection can also provide insights into cross-reactivity between coronaviruses and participation of preexisting mbcs in the response. wec et al. ( ) used cells from a survivor of the sars-cov outbreak as a source of mbcs that bound the s protein of sars-cov- ; a comprehensive panel of abs expressed by the mbcs were cloned and characterized. notably, most of the highly mutated mabs bound the s subunit of multiple hcov s proteins, often with higher affinity than to the s of sars-cov- . a screening of healthy donors identified low frequencies of mbcs reactive to the s proteins of the sars-cov and sars-cov- ( ) . findings suggest that s -reactive mbcs generated by hcovs were activated and expanded by the sars-cov. rbd-binding mbcs sampled in the convalescent phase of sars-cov- infection expressed abs with relatively low numbers of v gene mutations, suggesting that this component of the response largely reflected naive b cell activation by novel epitopes ( ) . to extend our understanding of the b cell response to sars-cov- infection, the current study compared ab and mbc immunities to sars-cov- in unexposed individuals and individuals in the convalescent phase of infection. in particular, we were interested in the presence of sars-cov- -reactive mbcs in unexposed subjects that could confer some protection against sars-cov- and in formation of mbcs by sars-cov- infection to provide durable protection against reinfection. most importantly, we demonstrate that sars-cov- infection generates both igg and igg mbcs reactive to the novel rbd and the conserved s subunit of the s protein. long-lived mbcs are thus likely to be available to mediate rapid protective ab responses if circulating ab levels wane and reinfection occurs. our results also draw attention to preexisting sars-cov- -cross-reactive b cell memory corresponding to the s subunit in sars-cov- -naive subjects. we speculate that the strong response to s after sars-cov- infection reflects preexisting s -reactive mbc activation and strengthens broad coronavirus protection. igg against sars-cov- proteins in unexposed subjects primarily targets the s subunit of the s protein. to investigate preexisting b cell immunity to sars-cov- in unexposed individuals and sars-cov- -reactive b cell immunity generated by infection, we analyzed sera and peripheral blood mononuclear cells (pbmcs) from (i) healthy donors sampled prior to the emergence of sars-cov- and (ii) nonhospitalized covid- convalescent subjects sampled to weeks after symptom onset. reactivity was measured against the s protein (including the rbd and s subunit) and n protein of sars-cov- and the s proteins of the human alphacoronavirus e and betacoronavirus oc . h influenza virus hemagglutinin and tetanus toxoid (ttd) were included as control antigens that humans are commonly exposed to through infection and vaccination. serum igg levels were measured by enzyme-linked immunosorbent assay (elisa). approximately one-third of non-sars-cov- -exposed subjects in the healthy donor cohort had low levels of serum igg against the s and n proteins of sars-cov- , likely reflecting cross-reactivity with seasonal hcovs (fig. a) . notably, % of unexposed subjects had igg against the highly conserved s subunit of the s protein. it is possible that inherent features of the bulky s reagent used in our analysis reduced binding by anti-s abs. igg that bound the highly novel rbd was not detected in unexposed subjects. all non-sars-cov- -exposed subjects had igg against s proteins of hcovs e and oc , indicating previous infection, and against control proteins h and ttd ( fig. c to f). s-and n-specific igg production following sars-cov- infection includes a strong response to the s subunit. levels of igg against s, rbd, s , and n were markedly higher in convalescent subjects than in unexposed subjects, indicating strong induction of these abs by sars-cov- infection (fig. a) . in a lower number of convalescent subjects, high anti-s igg titers were associated with low levels of anti-n igg. indeed, more than % of convalescent subjects had anti-n igg levels within the range seen in unexposed subjects, questioning the reliability of using anti-n igg measurement to identify previous sars-cov- infection in recovered patients ( ) . notably, serum igg titers against s were consistently higher than against the rbd in convalescent subjects, perhaps reflecting the novelty of the rbd and a response dependent on naive b cell activation (fig. b) . interestingly, titers of igg were higher against the s protein of the hcov oc in convalescent subjects than in unexposed subjects, but this was not the case for the s protein of hcov e (or for the control proteins h and ttd) (fig. c to f) . the anti-oc s igg titers correlated with those against the sars-cov- s (r s ϭ . , p ϭ . ), rbd (r s ϭ . , p ϭ . ), and s (r s ϭ . , p Ͻ . ), indicating a relationship with sars-cov- infection (fig. g) . the particularly strong correlation between igg titers against oc s and the sars-cov- s suggests a cross-reactive response to the s subunit. since the healthy donor samples in our analysis were collected to years before the emergence of sars-cov- , we considered the possibility that a recently circulating hcov was responsible for the higher anti-oc s igg titers in the convalescent subjects. to exclude this possibility, we measured anti-oc s igg titers in sera collected from health care workers in . the health care workers had cared for hospitalized sars-cov- patients, but all were negative for igg against sars-cov- s and rbd, consistent with the effectiveness of personal protective equipment and appropriate work practices. oc s-reactive igg levels in health care worker sera were similar to those in non-sars-cov- -exposed healthy donor sera and significantly lower than those in sera from convalescent subjects (fig. c) . taken together, our results indicate that sars-cov- infection generates a strong igg response that cross-reacts with the s of human betacoronaviruses. strong s-reactive mbc formation following sars-cov- infection includes reactivity to the rbd and s subunit. pbmcs from non-sars-cov- -exposed subjects and convalescent subjects were analyzed for the presence of mbcs reactive to sars-cov- proteins. circulating antigen-specific igg mbc populations were measured by in vitro stimulation of mbcs to induce differentiation into ab-secreting cells (ascs). poststimulation antigen-specific measurement of levels of mbc-derived ascs (mascs) by enzyme-linked immunosorbent spot (elispot) assay or of mbc-derived polyclonal abs (mpabs) by elisa provided a measure of the levels of precursor mbcs ( ) . analysis of mascs by elispot assay was performed against the sars-cov- s, rbd, and n proteins and against influenza virus h and ttd. mpab levels were measured against those of antigens used in the elispot assay, as well as sars-cov- s and the s proteins of hcovs oc and e. antigen-specific igg mpab concentrations correlated strongly with the frequency of igg mascs derived from stimulated mbcs (determined for sars-cov- s, sars-cov- rbd, influenza virus h , and ttd, r s ϭ . , . , . , and . , respectively, p Յ . ), validating the use of the mpab concentration as a measure of the size of specific mbc populations. the presence of a low level of igg against the sars-cov- s, rbd, and n proteins in a proportion of unexposed subjects suggested that igg mbcs with the same specificity had also been formed. however, these mbcs were not detected (fig. c) , possibly because of very low frequencies in the circulation. in contrast, igg mbcs reactive to the s proteins of the hcovs oc and e and the control proteins h and ttd were detected in nearly % or more of non-sars-cov- -exposed subjects, consistent with the higher levels of serum igg against these antigens (fig. e to h) . as expected, sars-cov- rbd-reactive mbcs were not detected in unexposed subjects. in marked contrast to non-sars-cov- -exposed subjects, the vast majority of convalescent subjects had circulating igg mbcs reactive to sars-cov- s, rbd, and s , indicating strong induction by sars-cov- infection of mbcs reactive to novel and conserved regions of the s protein ( fig. a and c) . notably, numbers of igg mbcs reactive to the s protein of the hcov oc were higher in convalescent subjects than in unexposed subjects (fig. e) , but there was no difference between the two subject groups in the levels of igg mbcs reactive to the hcov e s protein or influenza virus h or ttd (fig. b and f to h) . s -reactive igg mbc numbers correlated well with levels of igg mbcs reactive to sars-cov- s (r s ϭ . , p Ͻ . ) and rbd (r s ϭ . , p ϭ . ) and to s of hcov oc (r s ϭ . , p ϭ . ) but not with those reactive to s of hcov e (r s ϭ Ϫ . , p ϭ . ), influenza virus h (r s ϭ . , p ϭ . ), or ttd (r s ϭ . , p ϭ . ). the findings of our mbc analysis are consistent with serum igg measurement and indicate that sars-cov- infection generates igg mbcs reactive to the sars-cov- s that cross-react with the s of human betacoronaviruses. interestingly, only a small proportion of the convalescent subjects generated detectable n-reactive igg mbcs, even though most subjects produced high levels of anti-n igg in serum ( fig. c and d) . it is unclear whether this reflects a real difference between s-reactive mbc formation and n-reactive mbc formation or an effect of the sampling time. overall, we demonstrate that sars-cov- infection induces strong s-reactive mbc formation that would be expected to provide lasting protection against reinfection and, potentially, broad protection against betacoronaviruses. our goals in this study were to investigate sars-cov- -reactive b cell memory in unexposed subjects that could provide some protection against sars-cov- infection and the generation of b cell memory by sars-cov- infection that could provide lasting protection against reinfection. in particular, we were interested in igg mbcs, which respond to cognate antigens with rapid, vigorous, and high-affinity ab production. importantly, mbcs are long-lived cells that continue to provide strong protection when circulating ab levels wane. our approach was to analyze circulating igg as well as igg mbcs from the sars-cov- -naive and sars-cov- -convalescent subject groups. our key findings are as follows: (i) the presence of igg reactive to the s subunit of sars-cov- in most unexposed subjects, likely reflecting cross-reactivity to hcovs; (ii) markedly increased levels of igg against the sars-cov- s and n proteins, including reactivity to the rbd and s subunit of s, in convalescent subjects; (iii) increased igg binding to the s protein of the oc hcov, but not the e hcov, in convalescent subjects, reflecting greater cross-reactivity between s subunits of betacoronaviruses; (iv) strong formation of igg mbcs reactive with the rbd and s subunit of the sars-cov- s protein in convalescent subjects; and (v) formation of igg mbcs reactive with the s protein of oc , but not with that of e, in convalescent subjects, consistent with s subunit cross-reactivity between betacoronaviruses. approximately one-third of our cohort of non-sars-cov- -exposed subjects had low levels of igg against the sars-cov- s and n proteins. the low anti-n igg level likely reflects infection with hcovs, which have low-level ( % to %) homology with the sars-cov- n protein ( ) . however, a protective function for anti-n abs has not been established ( ) . notably, % of unexposed subjects had igg against the s subunit, reflecting homology with hcovs, but none had igg against the highly novel sars-cov- rbd ( , , ) . abs that target the s subunit have been shown to have virus-neutralizing activity, raising the possibility that the presence of preexisting anti-s igg confers some protection against sars-cov- ( ) . the processes that generate anti-s igg are also likely to generate s -reactive igg mbcs, and these might provide more significant protection than low levels of anti-s abs. however, s -reactive mbcs (or s-reactive and n-reactive mbcs) were not detected in non-sars-cov- -exposed subjects. taking those findings together with the identification of s-reactive mbcs in unexposed healthy donors ( ) , it is likely that the levels of s -reactive mbcs were below the limit of detection in our assays. on the basis of an estimate of to igg mascs generated per igg mbc after in vitro stimulation ( ) , our analysis suggests that the frequency of s -reactive mbcs, if present in unexposed healthy donors, would be Ͻ / pbmcs. most mbcs are resident in lymphoid tissues and, except for mbcs against frequently seen immunogenic antigens (for example, the influenza virus h or ttd in this study), are at very low frequencies in the circulation in the steady state ( , ) . anti-rbd, anti-s, and anti-n igg levels were markedly higher in the convalescent subjects than in non-sars-cov- -exposed subjects, indicating strong induction by sars-cov- infection. perhaps notably, the majority of convalescent subjects had higher igg titers against the s than against the rbd. this is particularly surprising because of the accessibility of the rbd to b cells and the expected immunodominance over the s subunit ( , ) . our demonstration of strong anti-s igg production is consistent with the activation of a preexisting population of igg mbcs against the conserved s subunit in the absence of mbcs reactive to the novel rbd. however, we cannot exclude the possibility of inherent differences in the stability or antigenicity of rbd and s reagents as an explanation. igg levels against the s protein of hcov oc (but not e) were significantly higher in convalescent subjects than in non-sars-cov- -exposed subjects and correlated strongly with anti-s igg levels. these findings support the idea of stronger b cell cross-reactivity between the s subunits of sars-cov- and human betacoronaviruses than alphacoronaviruses ( ) . importantly, we demonstrated that sars-cov- infection generates rbd-reactive and s -reactive igg mbcs. recently, long et al. ( ) found that levels of sars-cov- reactive abs, including neutralizing abs, start to decrease within to weeks of infection, especially when the infection is asymptomatic. since mbc populations are maintained for many years, perhaps decades, our findings indicate that mbcs generated by sars-cov- infection would be available to rapidly generate protective abs if waning ab levels were to allow reinfection to occur ( ) . notably, three convalescent subjects in our analysis had undetectable rbd-reactive igg levels but nevertheless had rbd-reactive igg mbcs. this might reflect mbc production by germinal centers that remained active after recovery from infection ( ) . the proportion of subjects with mbcs reactive to the hcovs oc and e was greater for the convalescent group than for the unexposed group, likely reflecting the increase in levels of s -reactive mbcs in the convalescent group and cross-reactivity with hcovs. s -reactive mbc expansion mediated by sars-cov- infection could enhance protection against a broad range of coronaviruses ( ) . the level of n-reactive mbc formation in convalescent subjects was lower than expected given the large number of subjects with high titers of n-reactive igg, but additional sampling times are required to confirm this observation. the antigen-specific b cell response to infection and vaccination in humans is characterized by entry into the circulation of recently proliferated class-switched b cells, termed activated b cells (abcs), which are phenotypically and transcriptionally distinct from ascs ( ) . circulating abc frequencies peak at to weeks after antigen exposure and have substantially decreased by months. frequencies of antigen-specific resting mbcs (negative for markers of recent proliferation) increase together with those of abcs and decrease much more slowly ( , ) . abcs, like resting mbcs, were activated by the in vitro stimulation conditions used in our study to divide and differentiate into ascs ( ) . we therefore cannot exclude the possibility that abc activation contributes, to some degree, to measurement of what we designate mbcs. on the basis of the kinetics of abc and resting mbc formation and maintenance of immunoglobulin gene clonal lineages in the two populations, ellebedy et al. ( ) suggested that at least a subset of abcs form resting mbcs. however, the differentiation pathways of abcs are not well established ( ) and the proportion that becomes part of long-maintained mbc populations remains uncertain. in conclusion, our analysis investigated ab and mbc immunity to sars-cov- in unexposed subjects and individuals soon after recovery from sars-cov- infection. the findings emphasized the novelty of the sars-cov- s protein rbd in unexposed subjects. however, igg reactive to the s was widespread in unexposed subjects and likely resulted from exposure to hcovs. although our approach was unable to directly identify s -reactive mbcs in the unexposed subjects, we suggest that these cells were present and strongly contributed s -reactive igg early in the response to sars-cov- infection. the igg response in convalescent sars-cov- subjects was also strong against the rbd and, less consistently, against the n protein. importantly, the convalescent sars-cov- subjects had generated rbd-reactive and s -reactive igg mbcs. the rbd-reactive mbcs are likely to provide strong long-term protection if rbd-reactive neutralizing ab levels wane and reinfection occurs. additional studies are required to establish the importance of s -reactive igg in providing broad anticoronavirus activity and the influence of expanded s -reactive mbc populations on a de novo b cell response to the rbd. study participants and clinical samples. all study participants were recruited at the university of rochester medical center, rochester, ny, and provided written informed consent prior to inclusion in the studies. the studies were approved by the university of rochester human research subjects review board (protocols - , - , and - ) and conducted in accordance with the principles of good clinical practice. a prepandemic cohort of healthy donors (median age, years; interquartile range [iqr], to years) were enrolled from to (non-sars-cov- -exposed subjects). a cohort of health care workers (median age, years; iqr, to years) at strong memorial hospital, rochester, ny, were enrolled in may . the health care workers had not been diagnosed with covid- prior to enrollment. a cohort of nonhospitalized covid- convalescent subjects ( males and females) (median age, years; iqr, to years) were enrolled in may and consisted of pcr-confirmed patients and non-pcr-confirmed subjects who were contacts of confirmed cases or displayed covid- -like symptoms. the convalescent subjects were sampled to weeks after symptom onset. symptoms reported (percentages of subjects) were fever ( %), cough ( %), sore throat ( %), stuffy/runny nose ( %), difficulty breathing ( %), fatigue ( %), headache ( %), body aches ( %), nausea/vomiting ( %), and diarrhea/loose stool ( %). recombinant proteins. rbd and stabilized ectodomain s protein from sars-cov- (isolate wuhan-hu- ) were expressed in-house in hek cells using pcaggs plasmid constructs kindly provided by florian krammer (icahn school of medicine at mount sinai) ( ) . baculovirus-expressed s subdomain and hek cell-expressed n protein were obtained from sino biological (chesterbrook, pa) and raybiotech (peachtree corners, ga), respectively. baculovirus-expressed s proteins from seasonal hcovs oc and e were obtained from sino biological. in-house hek cell-expressed hemagglutinin from eggderived h n a/california/ / and ttd (milliporesigma, burlington, ma) were used as noncoronavirus control proteins. mbc analysis. measurement of levels of antigen-specific mbcs was essentially performed as described previously ( ) . briefly, cryopreserved pbmcs were thawed and rested overnight at °c in complete medium. rested pbmcs were stimulated for days at ϫ pbmcs/well in -well plates to induce mbc expansion and differentiation into ascs. the stimulation cocktail consisted of complete medium supplemented with g/ml r (sigma, st. louis, mo), ng/ml interleukin- (il- ) (gibco, gaithersburg, md), and ng/ml il- (stemcell technologies, vancouver, canada). after stimulation, cells were harvested and pelleted by centrifugation. the undiluted supernatant containing abs secreted by ascs generated from stimulated mbc precursors (mpabs) was collected and stored for analysis by elisa. supernatants from unstimulated cultures of rested pbmcs were collected to control for abs produced by preexisting ascs. antigen-specific ascs in the cell pellet (mascs) were enumerated by elispot assay. for each antigen, , stimulated pbmcs were analyzed by elispot assay and the limit of masc detection was set at spots (mascs)/ pbmcs. on the basis of elispot assay results, antigen-specific mbcs in peripheral blood were quantified as antigen-specific igg mascs as a proportion of stimulated pbmcs. antigen-specific igg concentrations in mpab samples (after subtraction of ab concentrations in supernatants from the levels seen in unstimulated pbmc control cultures) were also used as a measure of the relative sizes of reactive mbc populations. enzyme-linked immunosorbent assay (elisa). concentrations of ag-specific serum abs and mpabs were measured by elisa as previously described ( ) . briefly, nunc maxisorp -well plates (thermo fisher, waltham, ma) were coated overnight with optimized concentrations of antigens. serially diluted samples were added to blocked plates and incubated for h at room temperature. alkaline phosphatase-conjugated anti-human igg (clone mt ; mabtech, stockholm, sweden) and p-nitrophenyl phosphate substrate (thermo fisher) were subsequently added to detect bound antigen-specific abs. absorbance was read at nm after color development. a weight-based concentration method was used to quantify antigen-specific ab levels in test samples as described previously ( , ) . sera from healthy donors and convalescent subjects with high titers for test antigens were used to establish human serum standards. the cutoff for assay positivity was set at approximately ϫ the mean optical density (od) value for negative wells. statistical analyses. the medians (with q and q ) were summarized by subject group and compared by the wilcoxon rank sum test. spearman correlation analysis was used together with corresponding robust regression models to assess monotonic associations among ab responses. multiple-test adjustment was not applied for this explorative study; thus, a p value of Ͻ . was considered significant for all analyses. statistical analyses were performed using sas . software (sas institute inc, cary, nc). we thank the staff of the university of rochester infectious disease research clinic for subject enrollment and sample collection and bei resources for providing some of the reagents used in this study. this project was funded in part with federal funds from the national institute of a pneumonia outbreak associated with a new coronavirus of probable bat origin sars-cov- vaccines: status report clinical features of patients infected with novel coronavirus in wuhan clinical and immunological 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activation dynamics and immunoglobulin evolution of pre-existing and newly generated human memory b cell responses to influenza hemagglutinin assignment of weight-based antibody units to a human antipneumococcal standard reference serum, lot -s allergy and infectious diseases, national institutes of health, department of health and human services, under ceirs contract no. hhsn c.we declare no conflicts of interest. key: cord- -n b r e authors: zhao, min; liu, kefang; luo, jiejian; tan, shuguang; quan, chuansong; zhang, shuijun; chai, yan; qi, jianxun; li, yan; bi, yuhai; xiao, haixia; wong, gary; zhou, jianfang; jiang, taijiao; liu, wenjun; yu, hongjie; yan, jinghua; liu, yingxia; shu, yuelong; wu, guizhen; wu, aiping; gao, george f.; liu, william j. title: heterosubtypic protections against human-infecting avian influenza viruses correlate to biased cross-t-cell responses date: - - journal: mbio doi: . /mbio. - sha: doc_id: cord_uid: n b r e against a backdrop of seasonal influenza virus epidemics, emerging avian influenza viruses (aivs) occasionally jump from birds to humans, posing a public health risk, especially with the recent sharp increase in h n infections. evaluations of cross-reactive t-cell immunity to seasonal influenza viruses and human-infecting aivs have been reported previously. however, the roles of influenza a virus-derived epitopes in the cross-reactive t-cell responses and heterosubtypic protections are not well understood; understanding those roles is important for preventing and controlling new emerging aivs. here, among the members of a healthy population presumed to have previously been infected by pandemic h n (ph n ), we found that ph n -specific t cells showed cross- but biased reactivity to human-infecting aivs, i.e., h n , h n , h n , and h n , which correlates with distinct protections. through a t-cell epitope-based phylogenetic analysis, the cellular immunogenic clustering expanded the relevant conclusions to a broader range of virus strains. we defined the potential key conserved epitopes required for cross-protection and revealed the molecular basis for the immunogenic variations. our study elucidated an overall profile of cross-reactivity to aivs and provided useful recommendations for broad-spectrum vaccine development. using the crystal structures of the major humoral antigen ha, we investigated the humoral antigenic variations of different aivs compared to a(h n )/california/ / (fig. c) . humoral immunogenic sites on the ha protein of influenza a virus are mainly distributed over five conformational sites (fig. d) , including four humoral antigenic sites (sa, sb, ca, and cb) on the ha "head" and a subdominant b-cell epitope on the ha "stem." structure-based conservancy analysis of these epitopes revealed that the four antigenic sites on the ha head of the four aivs a(h n )/vietnam/ / , a(h n )/ taiwan/ / , a(h n )/anhui/ / , and a(h n )/hong kong/ / were distinct from those of a(h n )/california/ . the stem-derived epitope in the has of each aiv was comparatively conserved but still contained specific substitutions. to expand our discovery to other aiv strains, we performed phylogenetic analyses based on ha sequences from different human-infecting h n , h n , h n , and h n strains (fig. e ). different clusters of aiv has were generated. though h and h are adjacent to each other in phylogeny, there was no cross-reactive serological reactivity to h n among the putative ph n -infected population. thus, the results are consistent with respect to comparisons between the divergent b cell epitopes on the humoral ha antigens of different aivs and the phenomenon that no cross-reactive antibody titers were observed between the tested aivs and ph n . biased t-cell cross-reactivities. to investigate potential preexisting t-cell responses to different aivs among the ph n -infected subjects, we stimulated freshly isolated pbmcs from the ph n ab ϩ cohort via an enzyme-linked immunosorbent spot (elispot) assay with live influenza viruses as stimulators. we found that certain levels of cross-reactive t-cell responses against h n , h n , h n , and h n viruses were detected. cross-reactive t-cell responses against h n ( . spot-forming cells [sfcs]/ pbmcs) were found at levels similar to those determined for ph n -specific t-cell responses ( . sfcs/ pbmcs), but the levels for both were higher than those seen with other aivs, i.e., h n ( . sfcs/ pbmcs), h n ( sfcs/ pbmcs), and h n ( . sfcs/ pbmcs) ( fig. a) . lee et al. synthesized the overlapping peptides covering all proteins and found that m and np possess the major immunogenic sites ( ) . we evaluated cross-reactive t-cell responses to m proteins from different aivs among the ph n m -cultured pbmcs for days. though cross-t-cell reactivity was observed for all four tested aivs, the cross-reactivity to m of h n ( sfcs/ pbmcs) shown by the ph n -specific t cells was higher than that to other aivs (for h n , sfcs/ pbmcs; for h n , sfcs/ pbmcs) (fig. b ). in intracellular cytokine staining (ics) assays, both the cross-cd ϩ t cells ( . % gamma interferon positive [ifn-␥ ϩ ] cd ϩ cd ϩ ) and cd ϩ t cells ( . % ifn-␥ ϩ cd ϩ cd ϩ ) displayed a bias with respect to h n ; in contrast, h n presented the lowest cross-t-cell reactivity level, with . % ifn-␥-secreting cells in cd ϩ t cells and . % ifn-␥secreting cells in cd ϩ t cells (fig. c to f), while the t-cell responses of ph n antibody negative individuals did not show such differences in statistics (see fig. s a in the supplemental material). m is the dominant contributor to biased t-cell reactivities. the cross-reactivity to h n was significantly lower than that to other aivs among the ph n antibodypositive (ab ϩ ) subjects. thus, to investigate the contribution of different cellular antigens of influenza viruses to biased cross-t-cell reactivities, we compared t-cell responses to h n and ph n among the members of the healthy population using overlapping peptides covering the m and np proteins. we found that t-cell reactivity to h n np was of the same level as the t-cell reactivity to ph n np as detected in the pbmcs ex vivo by elispot assay (fig. i ). subsequently, we established h n -and ph n -specific t-cell lines in vitro by stimulating the pbmcs with an np peptide pool for days. the responses to nps of either h n ( sfcs/ pbmcs) or ph n ( sfcs/ pbmcs) of the t-cell lines remained at the same level (fig. j) . in ics assays, the results seen with ifn-␥-secreting cells in both cd ϩ and cd ϩ t cells were similar under conditions of stimulation of h n np ( . % ifn-␥ ϩ /cd ϩ ) and ph n np ( . % ifn-␥ ϩ /cd ϩ ) ( fig. k and l) . in contrast, the cross-reactivity to h n m ( sfcs/ pbmcs) was significantly lower than the reactivity to ph n m ( sfcs/ pbmcs) itself ( fig. g and h) . subsequently, six immunogenic individual peptides from the ph n m pool were identified as responsible for the m -specific t-cell responses among the subjects via a matrix assay. a biased responsive magnitude against the corresponding peptides from different aivs was observed, consistent with the diverse substitutions in these peptides (see fig. s ). further analysis indicated that these peptides contained previously identified hla class i-or class ii-restricted epitopes (see fig. s d and h) and that the immunogenicity can be influenced by the substitutions in different aivs. phenotypes of the cross-reactive t-cell. to further investigate the functional subset of the cross-reactive t cells, we detected the memory phenotypes of ph n specific ifn-␥ ϩ t cells. memory phenotypes of the ifn-␥-secreting cells with respect to all of the different antigens, including live ph n virus and the m pool and np pool of ph n , were determined by analysis of the cd ra and cd l data ( fig. a and b) . the results showed that for all the antigens, the effector memory (cd ra Ϫ cd l Ϫ ) t-cell subset dominated the ifn-␥-secreting cells both among cd ϩ and cd ϩ t cells. for instance, . % (median) of the ifn-␥ ϩ cd ϩ t cells and . % (median) of the ifn-␥ ϩ cd ϩ t cells specific for m presented an effector memory phenotype (fig. c) . . t-cell responses were investigated using freshly isolated pbmcs from individuals (n ϭ ) through ifn-␥ elispot assays with live viruses. (b) ph n -specific t cells in pbmcs from individuals (n ϭ ) were expanded by stimulation with the ph n m peptide pool for days, and the t-cell responses were determined through ifn-␥ elispot assays using pools of overlapping peptides derived from influenza virus m protein. (c and d) the ratios of influenza virus-specific ifn-␥-secreting cells in cd ϩ (c) and cd ϩ (d) t-cell levels were determined using ics and flow cytometry. the frequencies of virus-specific t-cell responses to aivs were compared with the frequencies of responses to the ph n m pool (n ϭ ). (e and f) representatives of virus-specific t cells in cd ϩ (e) and cd ϩ (f) t cells are shown. (g to j) t-cell responses to the m antigen and np antigen of ph n and h n before and after culturing with m pools or np pools of ph n and h n were compared through ifn-␥ elispot and ics assays. (i) the t-cell responses of freshly isolated pbmcs from the healthy donors to the ph n np pool and h n np pool were compared using ifn-␥ elispot assays. (j) pbmcs were cultured with the ph n np pool and tested on the ninth day using ifn-␥ elispot assays. the t-cell responses to m pools of these two viruses were tested at the same time as the control (g, fresh pbmcs; h, pbmcs cultured in vitro with m ). (k and l) np-specific t-cell responses to ph n and h n tested in ics assays using pbmcs cultured in vitro with the ph n np pool for days. data in panels a, b, and g to j are shown as means ϩ sem (standard errors of the means), and data in panels c and d are shown as medians. the differences among multiple groups were compared using anova (a to d), and differences between two groups were compared using student's t test (e to h). *, p Ͻ . ; **, p Ͻ . ; ***, p Ͻ . . immunity plays an important role in protection from heterosubtypic influenza infections when antibodies do not work well. to better investigate the correlation of the internal proteins and t-cell cross-reactivities, we performed phylogenetic analyses of the predominant influenza virus t-cell immunogens, i.e., m , np, and pb , based on the full-length protein sequences ( fig. a and c and e). we found that the ha relationship of the aivs did not reflect the biased cross-t-cell reactivities. the m protein from different h n strains and that from ph n were not very clearly closely related to each other, and neither were np and pb . cross-reactive t cells targeted different cd ϩ or cd ϩ t-cell epitopes of aivs covering special sequences of the antigens. thus, we hypothesized that the phylogenetic relationship in terms of the t-cell epitopes from different aivs may correspond to the biased cross-t-cell reactivities. we retrieved the previously defined hla class i-restricted peptides located within the internal m , np, and pb proteins of ph n and the corresponding peptides within different aivs and plotted their phylogenetic relationship ( fig. b and d and f) . the t-cell epitope evolution of ph n is close to that of h n but distant from those of h n , h n , and h n , which is consistent with the biased cross-t-cell reactivity to h n but not to the other strains. to trace the full view of cd ϩ immunogenic features of ph n and aivs, we performed bioinformatic analyses of potential cellular antigenicity for different viruses based on previously determined t-cell epitopes available from the immune epitope data base (iedb). we analyzed representative human-infecting aiv strains as well as the recent seasonal h n stains. a total of ctl epitopes were retrieved from iedb and then mapped to each strain (up to december ). we filtered out internal protein epitopes to perform further analysis (see table s ). clustering analysis indicated that the h n stains are located in one cluster (see fig. s a ). for different humaninfecting aiv strains, h n strains remained close to ph n compared to the distance of other aivs (i.e., h n , h n , h n ) to ph n (fig. g) . the h n and h n strains were intermixed with each other, which may have been related to the shared internal genes of these aiv subtypes in china ( ) . seasonal h n influenza viruses before , such as a(h n )/brisbane/ / , were located in a cluster that was adjacent to but distinct from that of ph n , indicating a cellular antigenic transition of ph n compared to previous h n stains. table s and s . eleven epitopes that are conserved in h n and ph n strains but that different from those in other aivs are framed with a black rectangle. (h) the sequences of each strain were compared to the sequence of a(h n )/california/ / virus, and mutant sites are highlighted in red (potential tcr docking sites) or yellow (anchor residue sites). peptide m ( - ; underlined) indicates peptide h -p . the sequence information from the peptides is presented in table s . ph n , pandemic influenza virus cluster; sh n , seasonal h n strains before (but the data also include the h n strain). comparing the different variations of the t-cell epitopes between ph n and various aivs, a cluster that included t-cell epitopes was found to be conserved in h n and ph n strains but presented different variations in other aivs, either in major histocompatibility complex class i (mhc-i)-anchoring or t-cell receptor (tcr)-docking positions ( ) (fig. g and h; see table s ). this cluster of epitopes may have a key role in determining the bias of cross-t-cell reactivities to different aivs in the healthy population. we also performed bioinformatic analyses using influenza virus-derived t-cell epitopes with restrictions of different mouse mhc alleles (h- d b , h- k b ). both the clustering and phylogenetic analyses (see fig. s b and c) showed that the mouse epitope-based h n lineage was still adjacent to h n but far from other aivs, including h n . molecular basis of the biased t-cell cross-reactivities. as mentioned above, there was a region covering nonconserved short peptides which was highlighted in the heat map (fig. h ). the epitopes had a common feature: the amino acids which showed substitutions at the anchoring positions and/or at the exposed positions were substituted frequently (see table s ). this indicates that the variable antigenicity of the nonconserved peptides may be contributed by substitution-dependent intervention of mhc binding and/or tcr recognition. in this study, more than half ( . %) of the subjects had the hla-a* allele, and m protein-derived peptide h -p (m - ; lykklkreitf in ph n ) with hla-a* restriction is of the peptides. p is conserved between h n and h n (named peptide h -p ) but has a dominant mutation with substitutions at position from ile to met (i m) (named peptide h -p ) in h n and h n and ile to val (i v) in h n . in hla-a* ϩ individuals, we confirmed that peptide h -p could induce lower cross-reactivity than peptide h -p only in ph n -specific t cells (fig. a) . to characterize the binding of peptide h -p derived from ph n (or h n ) and h /h variant h -p to hla-a* , in vitro renaturing (fig. b ) and circular dichroism ( ) assays of the hla-a* /peptide complexes were performed (fig. c ). the refolding efficiencies of both the hla-a* /h -p and hla-a* /h -p complexes were high (fig. b) . further, the hla-a* /h -p complex was more thermally stable (with a melting temperature [t m ] of . °c) than the hla-a* /h -p complex (with t m ϭ . °c) (fig. c ). despite the fact that the i m mutation is not located in the traditional primary anchoring positions of hla i binding peptides, peptide h -p displayed a minor decreased binding affinity for hla-a* compared to h /h -derived peptide h -p . to further interpret the immunogenicity transition at the molecular level, we determined the crystal structures of hla-a* in complex with peptide h -p or h -p (data set ) (see table s ). the overall conformations of main chains were similar in the two structures ( fig. d and e) . however, in the hla-a* /h -p structure, residue ile inserts inside the e pocket of the hla-a* groove (fig. f ). in contrast, in the hla-a* /h - complex, mutated residue met is too large to be accommodated in the e pocket of hla-a* and, thus, its side chain protrudes from the peptide binding groove and might be contacted by the tcr (fig. g) . another structural variation is contributed by lys . although this residue is conserved between the p peptides of ph n and h n , the salt bridge formed between lys and glu in hla-a* /h -p is not present in the structure of hla-a* /h -p . without the constraint of the salt bridge, lys of peptide h -p in hla-a* /h -p is exposed to solvent instead of being partially buried in the c pocket as in the hla-a* /h -p structure. due to the low resolution ( . Å) of the first data set from hla-a* /h -p , we collected another data set which had higher resolution ( . Å) (see table s ). though no clear electron density for residues in the middle portion of h - was observable on the basis of data set of hla-a* /h -p , the conformations of residues lys and met from h -p in data set were still different from those from h -p but were similar to those from h -p in data set (see fig. s b ). taking these data together, though the h -p peptide has only one dominant i m mutation, this mutation influences the antigenicity of the peptide, most likely by altering both hla binding and tcr recognition. this may illustrate a common mode of antigenic variation of the nonconserved peptides between the h n /h n cluster and other aivs (h n , h n , and h n ) that induce a biased scale of cross-reactive t-cell responses. biased t-cell cross-reactivities provided distinct cross-protection efficacies against aivs. considering the unequal cross-reactivities to different aivs exhibited by ph n -specific t cells, especially with respect to the difference between h n and h n , the next issue that we addressed was whether these t-cell immunogenicity variations can lead to different heterosubtypic protections against these aivs. we used ph n virus at sublethal doses to prime mice, and days later, the mice were intranasally infected with lethal doses of ph n , h n , or h n . for homologous challenge of ph n , the protection ratio was % (fig. a) . the rate of heterosubtypic protection against h n challenge was also % (fig. d ), but the rate of protection against h n challenge was % (fig. g) . the mice in the homologous ph n challenge group (fig. b ) and the ph n primed-h n challenge group (fig. e ) n and o) ph n -specific cd ϩ t cells of ph n -primed mice were stained with tetramers h -np Ϫ (complex between h- d b and peptide from h n ), h /h -np Ϫ (complex between h- db and peptide from h n /h n ), h -pa Ϫ (complex between h- d b and peptide from h n ), and h -pa Ϫ (complex between h- d b and peptide from h n /h n ). cd ϩ t cells were subsequently selected for the analysis of tetramers (n ϭ ). differences between two groups were compared using student's t test (j to l and n), and differences among multiple groups were compared using anova (m). *, p Ͻ . . displayed no or minor body weight losses, while the mice in the ph n primed-h n challenge group (fig. h ) had a body weight loss of Ͼ % in the first days after h n challenge. concerning virus shedding in the lungs after infection, no virus was detected by days postinfection (d.p.i.) or afterward for the homologous challenge of ph n (fig. c) . as for the ph n -primed h n group (fig. f) , the h n virus load was much lower than that seen with the unprimed group by dpi and disappeared by dpi. in the h n groups (fig. i ), the primed group had a detectable virus load by and dpi. to distinguish the contributions of neutralizing antibodies and t-cell responses in the heterosubtypic protection against aivs, we performed mn and t-cell response detection assays in mice infected with different influenza viruses at sublethal dosages. in the ph n -infected group, ph n -specific neutralizing antibodies were detectable on day , and no cross-neutralization antibody titers to h n or h n could be detected (fig. j) and similarly, there was no cross-neutralization antibody titers to ph n detected in serum of the h n -or h n -infected group (fig. k and l) . the ph n -specific t-cell response in the mice was found to be sfcs/ splenocytes at dpi with ph n (fig. m ). meanwhile, cross-reactive t-cell responses to either h n or h n were found in the ph n -infected mice. however, the cross-reactive t-cell responses to h n ( sfcs/ splenocytes) were lower than those against h n ( sfcs/ splenocytes). these results indicated that bias of cross-reactive t-cell responses induced by ph n may lead to different protection efficacies against h n and h n . we also prepared the tetramers of two immunodominant h- d brestricted t-cell epitopes, np - and pa Ϫ , and their h n /h n mutants. the tetramer staining of the splenocytes from the mice infected by h n for days showed that the mice possessed robust h -np Ϫ and h -pa Ϫ peptidespecific cd ϩ t cells ( . % and . %), whereas certain t cells cross-recognizing the h n /h n mutants could also be detected ( . % and . %) (fig. n and o) . however, the ratios of mutant peptides h /h -np Ϫ and h /h -pa Ϫ were lower, which may still have contributed to the cross-protection. here, we found that ph n -specific t cells had biased cross-reactivities to different human-infecting aivs, while no preexisting neutralizing antibodies were detected. the cross-cellular immune response to h n in previously ph n -infected subjects was higher than those to h n , h n , and h n , correlating with heterosubtypic protection in an animal model. epitope-based phylogenetic analysis demonstrated that the h n subtype possesses a cluster of conserved epitopes with ph n that may lead to the observed cross-antigenicity. peptide-mhc (pmhc) structure determination indicated a molecular basis for the immune cross-reactivity between ph n and h n , as well as the lack of cross-reactivities toward other aivs, especially h n . antibody-mediated neutralization is the direct inhibition of viral infection ( ) . the elicitation of a neutralizing-antibody response is a correlate of protection for vaccines and contributes to protection against many viral infections ( ). in particular, ha imprinting in childhood could provide lifelong protection against severe infection and death from emergent viruses ( ) . however, a previous study showed that no h n specific antibody titers were detected in the , serum samples collected before the emergence of h n from poultry workers, most of whom were born after , when h n emerged ( ) . this indicated that cross-reactive serological immunity against h n virus did not preexist among healthy young populations. in our study, the levels of neutralizing antibodies that cross-recognized h n and h n were also undetectable, which may indicate a critical role of t cells in heterosubtypic protection. recent studies indicated that ha-specific cd ϩ t cells do not possess a dominant role compared to the internal proteins such as m and np ( , ) . therefore, ha-specific immunity may not be sufficient to explain the cross-protection against aiv by the immunity to previous seasonal influenza viruses. according to our bioinformatics analyses, the heat map of all available t-cell epitopes among human-infecting viruses indicated that the accumulation of varied epitopes may hinder cross-t-cell reactivities. in particular, a cluster of conserved t-cell epitopes shared by h n and h n may be related to cross-protection against h n . these epitopes could be considered for use in the development of vaccines preventing h n and h n infections. in contrast, t-cell recognition of the mutant viral epitopes in h n was significantly decreased due to the poor t-cell activation threshold and disrupted peptide-hla interactions. although low cross-reactivity of the variable peptides may also exist due to tcr conformational plasticity, their protective effect remains to be determined ( ) . we also detected a certain level of cross-reactive t-cell responses against aivs existing among different individuals, which may be contributed by conserved t-cell epitopes. greenbaum et al. showed that a large fraction of conserved t-cell epitopes in seasonal influenza virus could induce significant t-cell responses; as such preexisting t-cell immunity may decrease the severity of a variant strain infection ( ) . our previous work also confirmed a dominant role for conserved t-cell epitopes in anti-influenza virus responses ( ) . although there have been hundreds of influenza virus-specific epitopes identified across proteins using a range of epitope identification techniques, the majority of the conserved epitopes are derived from the internal proteins m , np, and pb ( ) ( ) ( ) . lee and others reported that m and np possess the major immunogenicities among the internal proteins, followed by pb and pa ( ) . they also found that the recognition frequency of m protein was higher than those of the other internal proteins. however, the identity of the internal proteins which possess a dominant influence on t-cell cross-reactivity was still unknown. on the basis of previous researches, we compared the t-cell responses against h n and h n among healthy donors with overlapping peptide pools of np and m , respectively, in the present study. np-specific t-cell cross-reactivity against h n showed a high level among the members of the ph n ab ϩ population. interestingly, we found biased t-cell cross-reactivities in responses to m proteins derived from different aivs. the similarity of the m sequences in the two strains was lower ( %) than those of np ( %) and other internal proteins (pb [ %], pb [ %], and pa [ %]). besides, the influences of the substitutions on immunogenicity would be different among different proteins, which may also contribute to varied cross-reactivity of the internal proteins between h n and h n . mutations in the m protein might have a larger influence on its immunogenicity. considering the immunogenicity and dissimilarity conservation of these two proteins, we proposed that m might have a more dominant influence in eliciting t cell immunity among influenza viruses and chose m as the stimulus in the experiments that followed that assessed t cell cross-reactivity and heterogeneous protection. thus, like the humoral response in ha, minor variations among the immunodominant epitopes may impact cross-t-cell reactivities, which should be carefully considered during the development of universal vaccines based on the m proteins of influenza viruses. the activation of t cells could be affected mainly by two factors: the stability of the peptide-mhc (pmhc) complex and the interaction between tcr and pmhc. minor substitutions of the residues on the middle bulged region of the peptides can abrogate t-cell recognition ( ) . also, the thermal stability of pmhc could influence t-cell activation. peptide gag - derived from hiv could be recognized by hla-b* : , hla-b* : , hla-b* : , and hla-b* : . however, gag - showed poor thermal stability with hla-b* : and elicited weaker t-cell responses ( ) . also, the substitutions of the primary or secondary anchor residues may completely change the antigenicity of the peptides ( ) . as previously determined in structural studies, mutational escape in t-cell epitopes contributed to the antigenic disaccord ( , ) . gras et al. investigated the cross-reactive t-cell responses against hla-b supertyperestricted variable epitope np Ϫ in humans ( ) . they found that cross-reactive cd ϩ t-cell immunity did not exist between the ph n virus and recent seasonal influenza viruses due to structural variation of the solvent-exposed residues in t-cell epitopes that can be recognized by tcrs. mutations of anchor residues (or partially solvent-exposed secondary anchors) can also dramatically decrease cd ϩ t-cell responses and result in delayed viral clearance ( ) . moreover, the substitutions may also lead to an induced fit on the helices of mhc-i that form the peptide-binding groove ( ) and thus impact tcr recognition ( ) . in the structures of hla-a* with h /h derived peptide h -p and h /h -specific epitope h -p , the i m substitution influenced the solvent-exposed surface and decreased the force of anchoring of the peptide to hla-a* . this indicated that variable antigenicities of nonconserved peptides could be largely influenced by both exposed positions for tcr docking and anchoring positions for mhc binding, which should be investigated in the hla-a* cohort. influenza virus-specific effector memory t cells were shown to be able to efficiently reduce the pulmonary viral titer early during the secondary infection as they accumulated in the lungs with rapid kinetics ( ) . a previous study reported the correlation of preexisting t cells with memory phenotype to the conserved ph n epitopes in core proteins m and np with clinical outcomes after incident ph n infection ( ) . we also found that most of the functional cross-reactive t cells were dominated by the effector memory phenotype. in recent years, while hundreds of aiv-infected cases had been reported, a large number of latent aiv infections also existed among lpm workers, as revealed by serological surveillance ( ) . our study showed that prior ph n infection led to heterosubtypic protection, and others indicated that the cross-reactive memory t cells were critical in heterosubtypic protection against h n with different hierarchies in mice ( , ) . determining whether the memory t cells induced by seasonal influenza viruses provide cross-protection against aiv infection in humans requires further investigation. overall, our study revealed preexisting but biased t-cell reactivity of ph n influenza virus to human-infecting aivs which provided distinct protection toward each subtype. this cross-reactive t-cell recognition had a regular pattern depending on the t-cell epitope matrix derived from aivs and seasonal influenza viruses. thus, efforts to develop heterosubtypic protection-oriented universal vaccines against influenza viruses should consider the pattern of cross-t-cell immunity. ethics statement. the ethics review committee, national institute for viral disease control and prevention, chinese center for disease control and prevention (china cdc), approved this study [project approval number ivdc ( )]. all of the subjects provided written informed consent for the studies performed on their samples and publication of their cases. the donors were identified anonymously as donor to donor . animals and eggs used in this study ( -to- -week-old female c bl/ mice and -to- -day-old embryonated chicken eggs) were bought from beijing vital river laboratory animal technology co., ltd. animal experiments were approved by animal experimental ethics board, china cdc (approval number ). the study was conducted in accordance with the principles of the declaration of helsinki and the standards of good clinical practice (as defined by the international conference on harmonization). subjects and samples. a total of healthy volunteers were recruited from november to april . serum samples were collected from coagulation-promoting tubes (bd vacutainer) from all donors, and pbmcs were isolated from anticoagulant blood by ficoll-paque density centrifugation from of the samples (see table s in the supplemental material). volunteers were given questionnaires to confirm whether they had received influenza vaccination or had caught cold. none of the subjects had previously received influenza vaccines. and no symptoms of influenza virus infection were reported during the sampling period. hla class i genotyping of the donors was performed using labtype sso (one lambda). ( ) . the peptides with substitutions within these viruses were divided into corresponding pools (see table s ). overlapping peptides covering nps of a(h n )/california/ / and a(h n )/anhui/ / were synthesized according to a similar strategy. heterosubtypic protections to aivs and t-cell immunity ® pbmcs from donors were incubated with influenza virus peptide pools in rpmi (gibco) containing % fetal bovine serum (hyclone) at °c with % co at a density of . ϫ cells/ml in a -well plate. on day , u/ml recombinant human il- (rhil- ) was added to the medium ( ) . half of the medium was changed on day with supplementation by rhil- . cells were harvested and tested for the presence of influenza virus-specific t cells on day . elispot assay. antigen-specific t lymphocyte responses were detected via an ifn-␥-secreting elispot assay (bd). briefly, -well elispot plate membranes were coated with anti-human ifn-␥ antibody at °c overnight before use. pbmcs from donors were incubated in wells ( . ϫ /well ex vivo for freshly isolated pbmcs and ϫ /well for in vitro-cultured t-cell lines) along with viruses (multiplicity of infection [moi] of ) or peptide pools ( m for individual peptides) for h at °c with % co , as well as with phytohemagglutinin (pha) as a positive control for nonspecific stimulation. cells incubated without stimulation were employed as a negative control. after incubation, cells were removed and, in turn, plates were processed with biotinylated ifn-␥ detection antibodies, streptavidinhorseradish peroxidase (hrp) conjugate, and substrate. the development of the spots was stopped by thoroughly rinsing with water. the numbers of the spots were captured and quantified with an automatic elispot reader and image analysis software (cellular technology limited). the threshold of the positive responses set for ex vivo elispot was Ն sfcs/ pbmcs. intracellular cytokine staining and flow cytometry. after ph n -specific t cells were expanded in vitro for days, t-cell lines were washed and rested for h. these cells were then stimulated with a specific peptide pool for h and incubated with golgistop/monensin (bd bioscience) for an additional h at °c in % co . then, cells were harvested and stained with a panel of surface mabs in fluorescence-activated cell sorter (facs) buffer ( . % bovine serum albumin) for min on ice, including fluorescein isothiocyanate (fitc)-anti-cd (bd pharmingen ), peridinin chlorophyll protein (percp)-anti-cd (biolegend , clone okt ), phycoerythrin (pe)-anti-cd l (bd pharmingen ), and v -anti-cd ra (bd biosciences ). subsequently, cells were fixed with bd fix/perm buffer on ice for min and then stained with the intracellular marker allophycocyanin (apc)-anti-ifn-␥ (bd pharmingen ). after two washes, cells were resuspended in % paraformaldehyde (pfa) facs wash buffer for flow cytometry (bd influx). pbmcs stimulated with ph n virus were prepared as described above. samples were analyzed with flowjo software. tetramer preparation and staining. h- d b -restricted tetramers of peptides np Ϫ and pa Ϫ and hla-a* -restricted tetramers of peptides h -p and h -p were prepared as previously described ( ) . preparation and staining of h- d b -restricted tetramers were performed as follows. briefly, to produce biotinylated peptide-mhc protein, h- d b was modified by the addition of a substrate sequence for biotinylating enzyme bira at the c terminus of the ␣ domain. in vitro-renatured h- d b / peptide complexes were then purified and biotinylated by incubation with d-biotin, atp, and the biotin protein ligase bira (avidity) at °c for h. the biotinylated h- db was further purified using a superdex / gl gel-filtration column (ge healthcare) to remove excess biotin and then mixed with pe-streptavidin (sigma). cells from the subjects were stained with pe-tetramer, fitc-conjugated anti-cd antibody, and percp-cy . anti-cd antibody. all samples were analyzed with a facscalibur flow cytometer (becton, dickinson) after staining. six-to- -week-old female c bl/ mice were used for virus infections. for the primary infection, mice were lightly anesthetized by inhalation of drikold and intranasally (i.n.) infected with . % tissue culture-infective doses (tcid ) of ph n in l of phosphate-buffered saline (pbs). the same operation was performed on the control group of mice with l of pbs. for the secondary challenge, mice were challenged with a high dose of h n ( . tcid ) and a lethal dose of h n ( . tcid ) or h n ( . tcid ) or with pbs weeks later and grouped as h n -pbs, h n -h n , h n -h n , h n -h n , pbs-pbs, pbs-h n , pbs-h n , and pbs-h n . inoculated animals were assessed daily. the mice with severe manifestations after the virus challenge (Ͼ % weight loss plus severe clinical impairment) were humanely euthanized according to our approved protocol. tissue sampling and cell preparation. three mice from each group were euthanized at , , , and dpi after secondary challenge. lungs were collected, weighed, and homogenized in ml of cold dmem using an ika t homogenizer under sterile conditions. then, solid debris was pelleted by centrifugation at , ϫ g for min, and the homogenates were used for virus titrations in mdck cells. splenocytes were filtered through cell strainers and were lysed with . % ammonium chloride lysis solution to remove erythrocytes ( ) . immunoinformatics. human host and mouse b host mhc-i t-cell epitopes of internal proteins of influenza a viruses were downloaded from iedb ( ) . epitopes with peptide lengths of Ͻ residues were selected, and their positions in proteins were renumbered using isolate a/california/ / as a reference. after merging of duplicates was performed, unique human-host mhc-i t-cell epitopes and unique mice b -host mhc-i t-cell epitopes were obtained (see table s and s ). protein sequences of representative strains were downloaded from the gisaid epiflu database, and strains were analyzed (see table s ). subsequently, multiple-sequence alignment was performed for each protein with the alignment tool muscle v . . ( ) . peptides were extracted as predicted t-cell epitopes of the representative sequences according to the unique epitopes mentioned above. the numbers of different amino acids of each predicted t-cell epitope compared with those from strain a/california/ / were counted. the maximum-likelihood phylogenetic trees for full-length proteins and epitope joint sequences were constructed using molecular evolutionary genetics analysis mega software ( ) with the jtt model and , bootstrap replicates. protein expression, refolding, and purification. the ectodomain of the hla-a* heavy chain and human ␤ microglobulin (␤ m) were expressed in escherichia coli as inclusion bodies and subsequently refolded in vitro in the presence of different peptides. briefly, the dissolved hla heavy chain, ␤ m inclusion body, and peptides were diluted at a molar ratio of : : , respectively, into a refolding buffer ( mm tris-hcl, mm l-arginine, mm edta-na, mm glutathione [gsh], . mm glutathione disulfide [gssg] ) and slowly stirred for h at °c. the refolded complexes were then concentrated and purified by superdex / gl (ge healthcare) chromatography, further purified on an ionexchange resource q column (ge healthcare), and manipulated for crystal screening. thermal stability assay. the stability of each hla/peptide complex was tested using circular dichroism (cd) spectroscopy. all complexes were refolded and purified as described above and measured at g/ml in a solution consisting of mm tris-hcl (ph . ) and mm nacl. cd spectra at nm were measured on a chirascan spectrometer (applied photophysics) using a thermostatically controlled cuvette at temperature intervals of . °c at a rate of °c/min between and °c. the denaturation curves were generated by nonlinear fitting with origin . software. crystallization, data collection, and structure determination. crystals of hla-a* /h -p and hla-a* /h -p were grown by the sitting-drop, vapor diffusion method at °c with a protein/ reservoir drop ratio of : and at a concentration of mg/ml in a mixture containing mm tris-hcl (ph . ) and mm nacl. the hla-a* /h -p complex crystal grew in a mixture of . m imidazole (ph . ) and % (wt/vol) polyethylene glycol . the hla-a* /h -p complex crystal grew in a reaction mixture containing . m mes (morpholineethanesulfonic acid) monohydrate (ph . ) and % (wt/vol) polyethylene glycol . reservoir solutions containing % glycerol were used for cryoprotection. the x-ray diffraction data were collected at the shanghai synchrotron radiation facility (ssrf) u beamline. data were indexed and scaled using denzo and the hkl software package. the structures were determined using molecular replacement with the program cns with the structure of protein data bank (pdb) code i l as the model. extensive model building was performed by hand using coot, and restrained refinement was performed using refmac . additional rounds of refinement were performed using the phenix.refine program implemented in the phenix software package with anisotropic displacement parameter (adp) refinement and bulk solvent modeling. the stereochemical quality of the final model was assessed with the program procheck. the structure-based antigenic analyses of ha proteins ph n , h n , h n , h n , and h n were performed using the structures with pdb codes jtv for h n ( ), znk for h n ( ), yy for h n ( ) , kol for h n ( ) , and jsd for h n ( ) . structure-related figures were generated using pymol (http://www.pymol.org/). statistical analysis. one-way analysis of variance (anova) was used in multiple comparisons. the two-tailed student's t test was used to compare data that were normally distributed and the mann-whitney test for nonparametric analyses. asterisks in each figure indicate statistical significance (*, p Ͻ . ; **, p Ͻ . ; ***, p Ͻ . ; ****, p Ͻ . ). analyses were performed with graphpad prism software (graphpad software, inc., la jolla, ca). accession number(s). the coordinates and structure factors of peptides complexed to hla-a* have been deposited in the protein data bank under accession codes wwu (hla-a* /h -p ), wxd (hla-a* /h -p , data set ), and wxc (hla-a* /h -p , data set ). supplemental material for this article may be found at https://doi.org/ . /mbio . - . two years after pandemic influenza a/ /h n : what have we learned? influenza vaccine effectiveness against pandemic influenza a(h n ) virus differed by vaccine type during - in the united states estimating age-specific cumulative incidence for the influenza pandemic: a meta-analysis of a(h n )pdm serological studies from countries human infection with influenza h n phylogenetics of varied subtypes of avian influenza viruses in china: potential threat to humans influenza and the live poultry trade genesis, evolution and prevalence of h n avian influenza viruses in china highly pathogenic h n influenza virus infection in migratory birds preexisting influenza-specific cd ϩ t cells correlate with disease protection against influenza challenge in humans preexisting cd ϩ t-cell immunity to the h n influenza a virus varies across ethnicities universal immunity to influenza must outwit immune evasion cellular immune correlates of protection against symptomatic pandemic influenza human t-cell immunity against the emerging and re-emerging viruses multipronged cd ( ϩ ) t-cell effector and memory responses cooperate to provide potent immunity against respiratory virus cytotoxic t-cell immunity to influenza induction of partial specific heterotypic immunity in mice by a single infection with influenza a virus a potent broad-spectrum protective human monoclonal antibody crosslinking two haemagglutinin monomers of influenza a virus serologic study for influenza a (h n ) among high-risk groups in china bat-derived influenza-like viruses h n and h n memory t cells established by seasonal human influenza a infection cross-react with avian influenza a (h n ) in healthy individuals cross-recognition of avian h n influenza virus by human cytotoxic t-lymphocyte populations directed to human influenza a virus human cytotoxic t lymphocytes directed to seasonal influenza a viruses cross-react with the newly emerging h n virus il- adjuvanted multivalent vaccinia-based universal influenza vaccine requires cd ϩ t cells for heterosubtypic protection protection against influenza a virus by memory cd t cells requires reactivation by bone marrow-derived dendritic cells examination of influenza specific t cell responses after influenza virus challenge in individuals vaccinated with mva-np ϩ m vaccine lack of prominent peptide-major histocompatibility complex features limits repertoire diversity in virus-specific cd ϩ t cell populations cross-immunity against avian influenza a(h n ) virus in the healthy population is affected by antigenicity-dependent substitutions protective t cell responses featured by concordant recognition of middle east respiratory syndrome coronavirus-derived cd ϩ t cell epitopes and host mhc 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responses acute emergence and reversion of influenza a virus quasispecies within cd ϩ t cell antigenic peptides a molecular switch in immunodominant hiv- -specific cd t-cell epitopes shapes differential hla-restricted escape the l v variation in hepatitis b virus core protein elicits new epitope-specific cytotoxic t lymphocytes and enhances viral replication cross-reactive cd ϩ t-cell immunity between the pandemic h n - and h n - influenza a viruses protective efficacy of cross-reactive cd ϩ t cells recognising mutant viral epitopes depends on peptide-mhc-i structural interactions and t cell activation threshold modification of mhc anchor residues generates heteroclitic peptides that alter tcr binding and t cell recognition loss of t cell antigen recognition arising from changes in peptide and major histocompatibility complex protein flexibility: implications for vaccine design t cell receptor cross-reactivity directed by antigen-dependent tuning of peptide-mhc molecular flexibility t cell responses to influenza virus infection: effector and memory cells antibodies against h and h avian influenza among poultry workers in china diverse heterologous primary infections radically alter immunodominance hierarchies and clinical outcomes following h n influenza challenge in mice memory t cells generated by prior exposure to influenza cross react with the novel h n influenza virus and confer protective heterosubtypic immunity comparison of overlapping peptide sets for detection of antiviral cd and cd t cell responses screening and identification of severe acute respiratory syndrome-associated coronavirus-specific ctl epitopes generation of murine ctl by a hepatitis b virus-specific peptide and evaluation of the adjuvant effect of heat shock protein glycoprotein and its terminal fragments structure and receptor binding of the hemagglutinin from a human h n influenza virus muscle: multiple sequence alignment with high accuracy and high throughput mega : molecular evolutionary genetics analysis version . molecular basis of the receptor binding specificity switch of the hemagglutinins from both the and pandemic influenza a viruses by a d g substitution recognition of sulphated and fucosylated receptor sialosides by a/vietnam/ / (h n ) influenza virus structures and receptor binding of hemagglutinins from human-infecting h n influenza viruses h avian and h swine influenza virus haemagglutinin structures: possible origin of influenza subtypes gao contributed to the overall concept and the experimental design and hypothesis and wrote the manuscript.this study was supported by grants from national natural science foundation of china ( , the funding sources had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. we declare no competing financial interests. we thank tong zhao at institute of microbiology, chinese academy of sciences, for support in flow cytometry assays. key: cord- -l eisi authors: park, su-jin; yu, kwang-min; kim, young-il; kim, se-mi; kim, eun-ha; kim, seong-gyu; kim, eun ji; casel, mark anthony b.; rollon, rare; jang, seung-gyu; lee, min-hyeok; chang, jae-hyung; song, min-suk; jeong, hye won; choi, younho; chen, weiqiang; shin, woo-jin; jung, jae u.; choi, young ki title: antiviral efficacies of fda-approved drugs against sars-cov- infection in ferrets date: - - journal: mbio doi: . /mbio. - sha: doc_id: cord_uid: l eisi due to the urgent need of a therapeutic treatment for coronavirus (cov) disease (covid- ) patients, a number of fda-approved/repurposed drugs have been suggested as antiviral candidates at clinics, without sufficient information. furthermore, there have been extensive debates over antiviral candidates for their effectiveness and safety against severe acute respiratory syndrome cov (sars-cov- ), suggesting that rapid preclinical animal studies are required to identify potential antiviral candidates for human trials. to this end, the antiviral efficacies of lopinavir-ritonavir, hydroxychloroquine sulfate, and emtricitabine-tenofovir for sars-cov- infection were assessed in the ferret infection model. while the lopinavir-ritonavir-, hydroxychloroquine sulfate-, or emtricitabine-tenofovir-treated group exhibited lower overall clinical scores than the phosphate-buffered saline (pbs)-treated control group, the virus titers in nasal washes, stool specimens, and respiratory tissues were similar between all three antiviral-candidate-treated groups and the pbs-treated control group. only the emtricitabine-tenofovir-treated group showed lower virus titers in nasal washes at days postinfection (dpi) than the pbs-treated control group. to further explore the effect of immune suppression on viral infection and clinical outcome, ferrets were treated with azathioprine, an immunosuppressive drug. compared to the pbs-treated control group, azathioprine-immunosuppressed ferrets exhibited a longer period of clinical illness, higher virus titers in nasal turbinate, delayed virus clearance, and significantly lower serum neutralization (sn) antibody titers. taken together, all antiviral drugs tested marginally reduced the overall clinical scores of infected ferrets but did not significantly affect in vivo virus titers. despite the potential discrepancy of drug efficacies between animals and humans, these preclinical ferret data should be highly informative to future therapeutic treatment of covid- patients. low sn titers, resulting in a prolonged infection. as several fda-approved or repurposed drugs are being tested as antiviral candidates at clinics without sufficient information, rapid preclinical animal studies should proceed to identify therapeutic drug candidates with strong antiviral potential and high safety prior to a human efficacy trial. keywords covid- , severe acute respiratory syndrome coronavirus (sars-cov- ), antiviral therapeutics, immunosuppression, serum neutralization, ferrets i n december of , a novel coronavirus (cov) disease , identified in wuhan, hubei province, china, in patients with pneumonia, was found to be caused by a previously unknown betacoronavirus. the outbreak rapidly spread to other provinces in mainland china, and despite great efforts, the epidemic continued to spread from china to europe, north america, and other asian countries. the world health organization (who) announced that the severe acute respiratory syndrome cov (sars-cov- ) epidemic was a public health emergency of international concern on january , and on march , the who director general characterized covid- as a pandemic (http://www.euro.who.int/en/health-topics/health -emergencies/coronavirus-covid- /news/news/ / / -ncov-outbreak-is-an -emergency-of-international-concern; https://www.who.int/dg/speeches/detail/who -director-general-s-opening-remarks-at-the-media-briefing-on-covid- --- -march - ). currently, the number of people diagnosed with sars-cov- infection is increasing by approximately , cases a day. as of april , approximately , , cases had been diagnosed as covid- and , deaths had occurred ( ) . further, recent studies reported the detection of sars-cov- rna in various clinical specimens, suggesting other transmission routes for this virus besides respiratory secretions ( ) ( ) ( ) . unfortunately, to date, no vaccines or antiviral drugs have been approved for the treatment of sars-cov- infection by regulatory agencies. researchers are fervently working to develop vaccines specifically for this virus, as well as potential treatments for covid- . although many clinical trials are ongoing, there are no specific therapeutic agents approved for covid- . developing sars-cov- -specific antiviral drugs from scratch could take years; drugs that have already been approved by the u.s. food and drug administration (fda) have the potential to reach patients more quickly. during the sars outbreak in , screening of approved drugs identified lopinavir-ritonavir, a human immunodeficiency virus type (hiv- ) aspartate protease inhibitor, as effective against sars-cov replication ( ) . the antiviral activity of lopinavir-ritonavir against middle east respiratory syndrome coronavirus (mers-cov) both in vitro ( ) and in an animal model ( ) has been reported, and case reports suggest that the combination of lopinavir-ritonavir with ribavirin and interferon alpha results in virologic clearance and survival ( , ) . chloroquine (cq), a widely used antimalarial with immunomodulatory effects ( ), was found in a recent study to inhibit the growth of sars-cov- in vitro ( ) . however, this finding has not been strongly supported by clinical studies of approximately sars-cov- -infected patients ( , ) . a derivative of chloroquine, hydroxychloroquine (hcq) sulfate, was first synthesized in by adding a hydroxyl group to cq, resulting in a compound found to be much less toxic than cq in an animal study ( ) . in autoimmune diseases, hcq sulfate works by reducing inflammation ( ) . however, recent reports have also shown heart risk concerns with the use of cq and hcq sulfate for covid- treatment. emtricitabine-tenofovir (truvada) is a prescription medicine for hiv approved by the u.s. fda for preexposure prophylaxis to reduce the risk of hiv infection in adults and adolescents. as a nucleotide analogue, it is reported that the active triphosphate form of this tenofovir diphosphate inhibits activity for rna-dependent rna polymerase (rdrp) of hiv and hepatitis b virus (hbv) ( , ) . still, even these existing drugs will need rigorous testing for efficacy and safety and ultimately ramped-up production before they can be deployed widely against covid- . generally, immunocompromised patients are more susceptible to bacterial, fungal, viral, and parasitic infections than healthy persons due to their inability to mount successful immune responses. this can be caused by impairment or weakening of the immune system by a number of conditions, including diseases (e.g., diabetes or hiv infection), malnutrition, and the use of certain medications. it has become apparent that sars-cov- infection also affects immunocompromised individuals more severely. a majority of covid- patients who were clinically diagnosed are older than ϳ years and have underlying complications, including heart disease, diabetes, hypertension, or cancer, indicating that age and reduced immune activity are the critical risk factors or determinants for covid- morbidity and mortality. we have recently established a ferret model for sars-cov- infection and transmission that highly recapitulates aspects of the human infection ( ) . elevated body temperatures and virus replication were readily detected in sars-cov- -infected ferrets. sars-cov- -infected ferrets shed the virus through nasal washes and in saliva, urine, and fecal specimens. sars-cov- was transmitted readily to naive direct-contact ferrets but less efficiently to naive indirect-contact ferrets ( ) . further, acute bronchiolitis was observed in infected lungs. in this report, we evaluated the efficacy of oral administration of lopinavir-ritonavir, hcq sulfate, and emtricitabine-tenofovir for sars-cov- infection in ferret infection models. we also treated ferrets with azathioprine, an immunosuppressive drug, and evaluated the replication kinetics of sars-cov- . while most drug treatments reduced clinical symptoms (cs), none of them led to a significant reduction of in vivo virus titers in ferrets. thus, a drug candidate study in a robust preclinical animal model should greatly facilitate testing the efficacies and safety of therapeutic treatments for covid- patients. clinical features of sars-cov- -inoculated ferrets treated with antivirals. in order to determine the antiviral efficacies of lopinavir-ritonavir, hydroxychloroquine (hcq) sulfate, or emtricitabine-tenofovir for treatment of sars-cov- infection, sars-cov- antibody-free ferrets ( /group) were inoculated with . % tissue culture infective doses (tcid )/ml of an nmc-ncov strain through the intranasal (i.n.) route ( fig. ). at day postinfection (dpi) with sars-cov- , ferrets were administered lopinavir ( mg/kg of body weight)-ritonavir ( mg/kg), hydroxychloroquine sulfate ( . mg/kg), or emtricitabine ( mg/kg)-tenofovir ( mg/kg) daily via oral gavage for days (fig. ). in addition, to test the effect of immunosuppression on viral infection and clinical outcome, a group (n ϭ ) of ferrets was also treated with phosphatebuffered saline (pbs) (as a control) or azathioprine, an immunosuppressive drug ( mg/kg), for days prior to sars-cov- infection (fig. ) . while all groups of sars-cov- -infected ferrets showed elevated temperatures at to dpi, the lopinavirritonavir-or emtricitabine-tenofovir-treated group exhibited mild fever compared with the pbs-treated group ( fig. a) . as with the pbs-treated group, the hydroxychloroquine sulfate-or azathioprine-treated group showed ϳ % body weight loss at dpi, while the lopinavir-ritonavir-or emtricitabine-tenofovir-treated group showed a Ͻ % change in body weight on average (fig. b ). to compare clinical features of sars-cov- infection following treatment with each drug, we developed an arbitrary scoring method to generate clinical symptom values based on -min observations of cough, rhinorrhea, and reduced activity and compared these cs values among the ferret groups as described in table . the average cs value of pbs-treated control ferrets was . at dpi, remained relatively high (Ͼ ) for to dpi, and returned to normal (less than ) by dpi (table ). the lopinavir-ritonavir-treated ferrets showed the reduced overall cs values (less than ) that peaked at to dpi and resolved by dpi (table ) . the hcq sulfate-treated ferrets also showed reduced cs values at to dpi compared with those of the pbs-treated group, but overall clinical symptoms were similar to those of the pbs-treated control group (table ) . although the emtricitabine-tenofovir-treated ferrets initially demonstrated clinical symptoms similar to those of the pbstreated control group, their overall cs values were relatively low at to dpi, and clinical symptoms were ultimately resolved by dpi. finally, the azathioprine-treated immunosuppressed ferrets also showed cs values similar to those of the pbs-treated control group, but this group's clinical symptoms lasted slightly longer than those of the pbs-treated control group (table ) . these results collectively showed that the lopinavir-ritonavir-, hcq sulfate-, or emtricitabine-tenofovir-treated group exhibited lower overall clinical scores than the pbs-treated control group. the overall clinical symptoms of immunosuppressed ferrets were similar but persisted slightly longer than those of pbs-treated control ferrets. comparisons of virus titers and shedding periods in antiviral-drug-treated ferrets. to evaluate the antiviral activity of each drug against sars-cov- , we measured infectious virus titers in nasal washes from drug-treated ferrets (fig. c ). sars-cov- was isolated from all infected ferrets regardless of drug treatment from dpi to dpi, with similar virus titers ( . to . log tcid /ml). at dpi, the emtricitabinetenofovir-treated ferrets exhibited reduced virus titers compared with those of the pbs-treated control group. although infectious virus was not detected in ferrets of the pbs-or antiviral-drug-treated groups at dpi, three of four azathioprine-treated ferrets were positive for virus even at dpi (fig. c) , suggesting delayed virus clearance in the upper respiratory tracts of immunocompromised ferrets. because gastrointestinal involvement has been documented in coronavirus infections of animals and humans ( ) , we also collected fecal specimens and performed quantitative real-time pcr (qrt-pcr) to determine whether any of the drug treatments affected sars-cov- shedding in the gastrointestinal system (fig. d) . the results showed that the viral rna was present in fecal specimens of all groups from to dpi, with peak viral rna copy numbers observed at to dpi. however, there was no statistical difference in viral rna copy numbers among the groups during the experimental period. by dpi, viral rna copy numbers declined in all drug-treated ferrets. to further evaluate virus titers in tissues, three ferrets from each group were euthanized at and dpi, and virus titers were measured in nasal turbinate and lungs. at dpi, all groups of ferrets showed high virus titers of more than . log tcid /g in nasal turbinate tissues, and the virus was also isolated from their lung tissues (fig. ) . at dpi, while all nasal turbinate tissues were positive for virus, the azathioprinetreated group showed a much higher virus titer (ϳ . log tcid /g) than those of the other groups (fig. a) . at dpi, the azathioprine-treated group still had detectable virus titers in their lung tissues, whereas the rest of groups were negative for the virus (fig. b) . to compare the serum neutralization (sn) antibody titers among drug-treated groups, blood was collected from each group of ferrets at , , and dpi. at dpi, the pbs-treated control and drug-treated groups demonstrated sn titers greater than (fig. ) . the antiviral-treated groups showed sn titers similar to those of the pbs-treated control group until dpi, but they exhibited lower sn titers at dpi than the pbs-treated control group (fig. ) . it is noteworthy that the azathioprine-treated immunosuppression group showed geometric mean sn titers of . and . at and dpi, respectively, suggesting the continuously reduced sn antibody response of the immunosuppressed group. in this study, we evaluated the antiviral efficacies of three fda-approved drug candidates against sars-cov- infection using a ferret infection model which has previously proven to be highly susceptible to sars-cov- infection ( , ) . although several clinical trials continue to evaluate these drug candidates, most of the enrolled patient populations are considered heterogeneous with regard to the duration and severity of illness at enrollment. further, given the rapid spread of covid- around the efficacies of fda-approved drugs against sars-cov- ® world, there are relatively higher mortality rates in some regions and in certain age groups. therefore, the use of highly susceptible animal models and controlled experimental settings should be an effective approach prior to human clinical trials to evaluate the in vivo antiviral effects of potential therapeutics. ferrets treated with antivirals showed relatively reduced overall clinical scores compared with those of the pbs-treated control group, which displayed high overall clinical scores. of the three drugs, the emtricitabine-tenofovir-treated group showed a noticeable reduction in overall clinical scores (Յ ) and a shorter duration ( dpi) of clinical symptoms. although attenuation of the overall clinical scores was observed in lopinavir-ritonavir-and hcq-treated groups at some time points, their clinical durations were comparable with those of the pbs-treated control group. these results suggest that treatment with emtricitabine-tenofovir, a nucleotide analogue that inhibits rnadependent rna polymerase activity, may be the most likely candidate to reduce clinical symptoms, including the cough and morbidity of sars-cov- -infected hosts. while the lopinavir-ritonavir protease inhibitor and a cq/hcq sulfate autophagy inhibitor have been shown to be effective against sars-cov, mers-cov, or sars-cov- in in vitro culture ( ), several reports have already described no benefit for clinical improvement of covid- patients. furthermore, hqc has been reported to be associated with a number of side effects, including a heart rhythm problem, severely low blood pressure, and muscle or nerve damage. thus, these existing fda-approved drugs still need rigorous testing for efficacy and safety in an animal model prior to clinical trials of covid- patients. since the emergence of sars-cov- in china in december , the number of cases has rapidly increased as the disease has spread globally. the increase in the number of cases is alarming and specially compounded by the possibility of viral transmission from asymptomatic individuals. several studies indicate that asymptomatic patients can transmit the virus to persons in close contact ( , ) . therefore, although clinical symptoms were attenuated in ferret groups treated with antiviral candidates, we also evaluated virus titers in respiratory and gastrointestinal tracts using nasal washes and stool samples, respectively, from sars-cov- -infected ferrets. regardless of antiviral candidate treatment, sars-cov- was detected at more than log tcid /ml until dpi, and there was no statistical difference between the pbs-treated control group and the antiviral-drug-treated groups. however, the emtricitabine-tenofovir-treated group showed relatively low virus tiers and shortened periods of virus shedding in nasal wash specimens compared with those of the other groups. currently, the role of sn antibody in the pathogenesis and disease clearance of sars-cov- is unclear. wu et al. ( ) recently reported that the sn antibody levels in covid- patients were variable, depending on the immune status of the patient, and that about % of patients failed to develop high sn titers after sars-cov- infection, although the disease durations of these patients were comparable to those of others. this suggests that the sn antibody titer may be closely associated with immune activity rather than with the virus titer and disease duration in patients. interestingly, we found that the antiviral-treated groups showed lower sn antibody titers than the pbs-treated control group. it is possible that as the antiviral treatment reduced the overall clinical symptoms of sars-cov- -infected ferrets, it might evoke weak immune responses and thereby lead to reduced neutralizing antibody responses. nevertheless, further immunological studies are needed to understand the detailed mechanisms of the low sn antibody titers in antiviral-treated ferrets. efficacies of fda-approved drugs against sars-cov- ® while covid- is typically characterized by respiratory symptoms, gastrointestinal symptoms have been reported in some cases. moreover, there is evidence of viral rna in the stools of sars-cov- patients. emtricitabine-tenofovir has reportedly shown antiviral efficacy in the gastrointestinal tract as well as in the respiratory tract ( ) . however, qrt-pcr analysis of stools revealed no statistical difference in virus titers in stools among the pbs-treated control and the antiviral-treated groups, suggesting that none of the tested antiviral candidates significantly diminished gastrointestinal sars-cov- replication in infected ferrets. in conclusion, although there may be some discrepancies in drug efficacy between animals and humans, these results of a preclinical ferret infection study should aid in the selection of antiviral treatments of covid- patients. this also suggests that a robust preclinical animal model for sars-cov- infection is valuable in order to identify antiviral drugs for future human efficacy trials. a sars-cov- strain, nmc-ncov , was propagated in vero cells in dulbecco's modified eagle medium (dmem; gibco, grand island, ny) supplemented with % penicillin-streptomycin (gibco) and tpck (tosylsulfonyl phenylalanyl chloromethyl ketone)-treated trypsin ( . g/ml; worthington biochemical, lakewood, nj) in a °c incubator supplemented with % co for h. propagated virus was stored at Ϫ °c as the working virus stock for animal studies. the % tissue culture infective dose (tcid ) was determined through fixation and crystal violet staining. immunosuppression and antiviral-drug candidate treatments. ten ferrets were treated orally with azathioprine ( mg/kg) (celltrion) daily for days prior to sars-cov- infection, and treatment continued until days postinfection (dpi) to reduce the immune response. as a control, pbs in the same volume was administered to ferrets. to confirm the immunosuppressed status of ferrets, blood samples were collected from azathioprine-treated ferrets, and the percentage of lymphocytes was assessed at and days preinfection and at , , and dpi. the reduction in lymphocyte numbers in azathioprine-treated ferrets compared with lymphocyte numbers in the pbs control group was confirmed (see fig. s in the supplemental material). for treatment with candidate antiviral drugs, groups of ferrets ( /group) were administered lopinavir ( mg/kg)-ritonavir ( mg/kg) (abbott), hydroxychloroquine sulfate ( mg/kg) (elyson), or emtricitabine ( mg/kg)-tenofovir ( . mg/kg) (gilead) daily via oral gavage starting at dpi of sars-cov- infection and continuing until dpi. experimental infection of ferrets. groups of -to -month-old female ferrets ( /group), seronegative for sars-cov- and sars-cov- , were intranasally inoculated with . tcid /ml of nmc-ncov under anesthesia. the body weights and temperatures of infected ferrets were monitored every other day until dpi. nasal washes and stool specimens were collected every other day from the inoculated ferrets. blood was collected at , , and dpi to measure the serum neutralization titer. three ferrets per group were euthanized at and dpi, and nasal turbinate and lungs were collected to measure tissue virus titers and examine lung histopathology. virus titers in nasal washes and tissues were determined by % tcid assessment in vero cells, while the virus titers in stool specimens were measured with quantitative real-time pcr (qrt-pcr). briefly, total rna was extracted using trizol reagent (thermo fisher scientific) or an rneasy kit (qiagen), and cdnas were generated with a sars-cov- specific primer by reverse transcription using quantitect reverse transcription (qiagen). qrt-pcrs were performed using a sybr green supermix (bio-rad) and a cfx touch real-time pcr detection system (bio-rad) with a spike gene-based, sars-cov- -specific primer set as previously described ( ) , and virus rna copy numbers were calculated as a ratio with respect to the standard control. clinical scoring methods of sars-cov- -infected ferrets. the ferrets were monitored daily over a -day period for temperature change, weight loss, clinical symptom, and movement and activity change. briefly, the frequency of cough and rhinorrhea was assessed in each group of ferrets and scored on the basis of the following criteria: no evidence of cough (score, ), occasional cough (score, ), and frequent cough (score, ) and no nasal rattling or sneezing (score, ), moderate nasal discharge on external nares (score, ), and severe nasal discharge on external nares (score, ). a change in a ferret's activity was assessed and scored on the basis of the following criteria: normal movement and activity (score, ), mildly reduced movement and activity (score, ), and considerably reduced movement and activity (score, ) for at least min. neutralizing assay. sera were collected from each group of ferrets to detect the serum neutralization titer. heat-inactivated -fold-diluted serum samples were serially diluted by -fold. an equal volume of sars-cov- at tcid was added to all diluted samples. the mixture of serum and virus was incubated at °c for h and then added to vero cells in a -well tissue culture plate for min. the mixture of serum and virus was then removed, followed by two washes with cold pbs. fresh medium was added to infected cells, and cells were incubated at °c in % co for days. supernatants were removed, fixed with a % formalin solution, and stained with crystal violet to determine the titer. statistical analysis. to assess significant differences in values for weight loss, temperature, viral titers, and serum neutralization titers, statistical analyses were done. asterisks indicate the statistical significance between pbs-administered and treated ferrets determined by two-way analysis of variance (anova) and a subsequent dunnett test (*, p Ͻ . ; **, p Ͻ . ; and ***, p Ͻ . ). all statistical analyses were performed using graphpad prism version . for windows 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transmission of efficacies of fda-approved drugs against sars-cov- ® -ncov infection from an asymptomatic contact in germany a familial cluster of pneumonia associated with the novel coronavirus indicating person-to-person transmission: a study of a family cluster neutralizing antibody responses to sars-cov- in a covid- recovered patient cohort and their implications drug-susceptible hiv- infection despite intermittent fixed-dose combination tenofovir/emtricitabine as prophylaxis is associated with low-level viremia, delayed seroconversion, and an attenuated clinical course all animal experiments were approved by the medical research institute, a member of the laboratory animal research center of chungbuk national university (larc) (approval number cbnur- - ), and were conducted in strict accordance with and adherence to relevant policies regarding animal handling as mandated under the guidelines for animal use and care of the korea centers for disease control (kcdc). the handling of virus was performed in an enhanced biosafety level (bsl ) containment laboratory as approved by the korea centers for disease control and prevention (protocol kcdc- - - ). supplemental material is available online only. key: cord- -m fwefuy authors: rivera-hernandez, tania; rhyme, mira syahira; cork, amanda j.; jones, scott; segui-perez, celia; brunner, livia; richter, johanna; petrovsky, nikolai; lawrenz, maria; goldblatt, david; collin, nicolas; walker, mark j. title: vaccine-induced th -type response protects against invasive group a streptococcus infection in the absence of opsonizing antibodies date: - - journal: mbio doi: . /mbio. - sha: doc_id: cord_uid: m fwefuy recent global advocacy efforts have highlighted the importance of development of a vaccine against group a streptococcus (gas). combo is a non-m protein-based vaccine that provides protection against gas skin infection in mice and reduces the severity of pharyngitis in nonhuman primates. however, combo with the addition of aluminum hydroxide (alum) as an adjuvant failed to protect against invasive gas infection of mice. here, we show that formulation of combo with adjuvants containing saponin qs significantly improves protective efficacy, even though all adjuvants tested generated high antigen-specific igg antibody titers, including alum. detailed characterization of combo formulated with smq adjuvant, a squalene-in-water emulsion containing a tlr agonist and qs , showed significant differences from the results obtained with alum in igg subclasses generated following immunization, with an absence of gas opsonizing antibodies. smq, but not alum, generated strong interleukin- (il- ), gamma interferon (ifn-γ), and tumor necrosis alpha (tnf-α) responses. this work highlights the importance of adjuvant selection for non-m protein-based gas vaccines to optimize immune responses and protective efficacy. streptococcal sequelae can manifest following repeated mild infections in the form of acute rheumatic fever (arf) and rheumatic heart disease (rhd). the global burden of streptococcal diseases has been long neglected despite being responsible for an estimated , deaths annually ( ) . recent advocacy efforts have highlighted the urgent need for the development of a vaccine that prevents gas-related infections ( ) ( ) ( ) . m protein-based vaccines have been considered strong candidates, despite concerns over insufficient serotype coverage (n-terminal-based vaccines) and the association of m protein with the generation of cross-reactive antibodies linked to arf and rhd ( ) . on the other hand, non-m protein-based vaccines have emerged as alternative candidates that overcome such concerns. regardless of the choice of gas antigen used as a vaccine candidate, the choice of adjuvant, a significant element of the final vaccine formulation, has generally been overlooked. aluminum salts (alum) have represented the most common adjuvant used to test gas vaccine candidates ( ) ( ) ( ) ( ) . for m-protein based vaccines, alum has proven effective for the generation of opsonizing antibodies, which are associated with protection against infection in animal models ( , ) . however, less is known about potential correlates of protection for non-m proteinbased vaccines and the role that different adjuvants may play in the protection afforded by these vaccines. our research has focused on the development of combo vaccine, a combination of gas protein antigens (arginine deiminase, [adi], c a peptidase [scpa], streptolysin o [slo], interleukin- protease [spycep] , and trigger factor [tf]). we previously tested combo formulated with alum in murine and nonhuman primate (nhp) models of infection with various results. while immunization with combo /alum decreased the severity of clinical signs but did not reduce colonization in a nhp pharyngitis model of infection ( ) and provided protection in a murine superficial skin infection model, the same formulation failed to protect in a murine invasive model of disease ( ) . in this study, taking advantage of the opportunity presented by these findings, we examined the protective capacity of combo formulated with a panel of different adjuvants (table ) using the invasive gas disease model. the level of protection from lethal challenge ranged from % to % survival among the different formulations. we further characterized the protective immune response elicited by combo formulated with smq adjuvant and compared this to the nonprotective response elicited by combo /alum and to the protective response elicited by the "gold standard" homologous m protein formulated with alum. antigen-specific antibody responses. groups of humanized plasminogen mice were immunized with combo formulated with alum, advax- , advax- , swe, lq, lmq, or smq adjuvant (table ) , while negative-control groups were immunized with phosphate-buffered saline (pbs) plus the corresponding adjuvant. one primary and two booster immunizations were delivered via intramuscular injection into the thigh muscle. serum samples from day were used to measure antigen-specific antibodies against all proteins contained in combo . antigen-specific total igg endpoint titers against all antigens (adi, scpa, slo, spycep, and tf) with all adjuvanted vaccines (fig. a , blue circles) were significantly higher than the titers seen with their corre- sponding negative controls (fig. a , black circles). immunization with m /alum was used as positive control in all experiments, and fig. b shows anti-m igg titers from one representative experiment. survival against invasive challenge. on day , mice were infected subcutaneously with gas strain (dose range, . ϫ cfu to . ϫ cfu). mice were monitored for days postinfection, and animals showing signs of disease, pain, or distress were humanely euthanized and classified as having succumbed to infection. as shown in a previous study ( ) , immunization with combo /alum did not provide significantly greater protection than treatment with pbs/alum (fig. ). mice immunized with combo /swe or combo /advax- showed no protection compared to mice immunized with adjuvant alone, mirroring the situation for combo /alum (fig. ). mice immunized with combo formulated with advax- showed % survival against lethal challenge, although the results were not significant compared to the % survival rate seen with mice immunized with advax- alone (fig. ). combo formulated with lq, lmq, and smq adjuvants showed significantly higher levels of survival, with %, %, and % protection, respectively, than that observed for controls treated with adjuvant alone (fig. ). mice immunized with m /alum were consistently protected against infection, with % to % survival across the experiments (fig. ) . characterization of protective immune response using smq adjuvant. we further characterized the immune response induced by the formulation that provided the highest protection against invasive lethal challenge and compared it to the immune response induced by combo /alum and m /alum. four mice per group were immunized with combo /smq, combo /alum, and m /alum, and naive mice were used as negative controls. at day following one primary immunization and two booster immunizations, animals were humanely euthanized to obtain blood and spleens for further analyses. levels of antigen-specific igg subclasses (igg , igg b, igg c, and igg ) were measured in serum samples. igg serum titers against adi were significantly higher in combo /alum-immunized mice than in those immunized with combo /smq (fig. a) . on the other hand, anti-adi igg c titers were significantly higher in combo /smq-immunized mice than in combo /alum-immunized mice. titers for anti-adi igg b and igg were comparable in the two groups. anti-scpa and anti-tf igg b and igg c titers were significantly higher in mice that received the combo /smq formulation than in those that received combo /alum, while titers against the same antigens were comparable for igg and igg (fig. a) . igg b, igg c, and igg titers against slo were significantly higher in combo /smq-immunized mice than in those treated with combo /alum, whereas the igg titers were equivalent for the two formulations (fig. a) . anti-spycep igg , igg b, and igg antibody titers were not significantly different between combo formulations, but a significant difference was observed in igg c titers, which were between and in the combo /smq group, while the igg c titers were undetectable in the combo /alum group (fig. a) . the anti-m igg subclass response following immunization with m /alum was characterized by high igg titers followed by igg b titers and low titers for igg c and igg antibodies (fig. b ). igg /igg c ratios were calculated for each antigen in both combo and m protein formulations (fig. c) . ratios closer to were observed for combo /smq, indicating a more balanced th /th response (fig. c ). on the other hand, higher ratios were obtained for combo and m antigens formulated with alum, suggesting a th -biased antibody response (fig. c) . binding of immune serum to live gas m t was assessed using flow cytometry. sera from combo /smq-immunized, combo /alum-immunized, and m /alumimmunized mice showed significantly higher fluorescence than sera from naive mice as expressed by calculated t(x) values (fig. a ). functionality of antibodies in sera was evaluated using an in vitro opsonophagocytic killing assay. only m immune sera was able to promote opsonophagocytic killing of gas ( fig. b ) with an opsonic index significantly higher than that measured for naive mice. finally, we investigated cytokine secretion by splenocytes from immunized mice following antigen stimulation. combo /smq-immunized and combo /alumimmunized mice splenocytes were stimulated with combo antigens, m /alumimmunized mice splenocytes were stimulated with m protein, and naive splenocytes were stimulated with both combo and m protein. igg subclasses generated by smq and alum. naïve mice or mice immunized with combo /smq, combo /alum or m /alum (n ϭ per group) were euthanized on day . serum samples were used to measure levels of igg , igg b, igg c, and igg (x axes) specific for adi, scpa, slo, spycep, and tf (a) and for m protein (b) as indicated. (c) igg /igg c ratios for each antigen. antigen-specific titers generated by different adjuvants were compared using a two-tailed mann-whitney u test (*, p Ͻ . ). bars represent geometric mean titers Ϯ geometric sd. vaccine-induced th -type response against invasive gas ® (fig. ). splenocytes from combo /alum-immunized mice secreted il- , il- , il- , il- , and il- and low levels of tnf-␣ (fig. ) . splenocytes from the combo /smq group secreted il- , il- , il- , and il- and high levels of il- , ifn-␥, and tnf-␣ (fig. ) . a comparison between the cytokine concentrations secreted by splenocytes from com-bo /alum and combo /smq immune mice showed that the smq-adjuvanted formulation induced significantly higher secretion of il- , il- , ifn-␥, and tnf-␣ than the alum-adjuvanted formulation. the licensure of a gas vaccine would bring global public health benefits. prevention of a number of clinical manifestations caused by gas would not only lower disease burden but also reduce the prescription of antibiotics and therefore positively contribute to the fight against antimicrobial resistance ( , ) . the non-m protein combo vaccine contains conserved gas proteins (adi, scpa, slo, spycep, and tf) that have low sequence variation and high coverage among gas isolates from different geographical regions ( ) . using alum as an adjuvant, we have tested combo in different animal models with various results. immunization with combo /alum provided protection in a superficial skin infection model in mice ( ) and decreased the severity of pharyngitis and tonsillitis clinical signs in a pharyngitis model in nonhuman primates ( ) . on the other hand, the same formulation failed to provide protection using the humanized plasminogen mouse model of invasive lethal infection ( ) . given that the humanized plasminogen mouse model is a well-established model to evaluate systemic gas dissemination ( , ) and that vaccine-induced protection was evident in this model when purified m protein was used as an antigen, we decided to investigate whether alternative adjuvants could improve the efficacy of combo . in this study, we tested six different adjuvants (table ) , each containing molecules with immunomodulatory properties. advax- contains delta inulin, a plant-based polysaccharide that has been shown to enhance antibody titers and cellular immune responses ( ) , and cpg, a tlr agonist that provides additional immunostimulatory properties ( ) . in addition to delta inulin and cpg, advax- contains murabutide, which stimulates the nod pattern recogni- values between sample replicates, and a t(x) value of Ͼ was considered significant (p Ͻ . ). (b) antibody functionality of naive mouse sera (clear bar) or combo /smq (light green bar), combo /alum (dark green bar), and m /alum (red bars) immune mouse sera was tested using a standardized in vitro opsonophagocytic assay. differentiated hl- cells were incubated in the presence of gas m , a source of complement, and mouse sera. the opsonic index was calculated as the serum dilution where % killing of bacteria occurred. opsonic indices were compared using the kruskal-wallis test corrected for multiple comparisons using dunnett's test, with p Ͻ . considered statistically significant. bars represent geometric means of opsonic indices Ϯ geometric sd. tion receptor ( ) . both of these inulin-based adjuvants induced significantly higher antibody titers for all combo antigens than were seen with pbs/adjuvant. both advax- and advax- induced high antigen-specific antibody titers; however, the rate of survival of vaccinated mice was not significantly higher than that seen with the pbs-adjuvant control groups. swe adjuvant is a squalene-in-water emulsion. the composition of swe adjuvant is similar to that of mf , an adjuvant currently used in several influenza vaccines available on the market ( ) , which has been shown to promote recruitment of immune cells to the injection site and enhance antigen uptake by antigen-presenting cells ( ) by promoting local release of atp ( ) . combo in formulation with swe generated high antibody titers against all antigens but showed no protection against gas lethal challenge compared to pbs formulated with swe. lq and lmq adjuvants are neutral liposome-based formulations containing cholesterol, , -dioleoyl-sn-glycero- -phosphocholine (dopc), and qs , a proinflammatory saponin adjuvant that activates dendritic cells (dcs) and promotes the secretion of th cytokines ( ) . lmq also contains the synthetic toll-like receptor (tlr ) agonist - -desacyl- -monophosphoryl lipid a ( d-mpl). a bacterium-derived tlr agonist has shown a synergistic adjuvant effect when combined with the saponin qs in the neutral liposome carrier as adjuvant. as is included in mosquirix (rts,s) malaria vaccine and shingrix (hz/su) herpes zoster vaccine, both of which are approved for human use ( ) . smq is a squalene-in-water emulsion containing cholesterol, synthetic d-mpl, and qs . the as adjuvant, also a combination of emulsion containing a bacterial tlr agonist and qs , has been tested in humans as part of the development of the mosquirix (rts,s) vaccine ( ) . combo formulated with lq, lmq, or smq provided significant protection against invasive lethal gas infection, with %, %, or % survival, respectively. given that the common ingredient between these three vaccine-induced th -type response against invasive gas ® adjuvant formulations is qs (table ) , we hypothesize that addition of qs is crucial for protection against gas in this invasive challenge model. given that the antibody titers against all five proteins in combo did not predict protection in the different adjuvant groups, we hypothesize that t cell immunity may be a more relevant predictor of protection in this model. we next characterized the immune response phenotype induced by one of these protective adjuvanted formulations. smq was chosen as it was associated with the highest rate of survival after gas challenge. mice immunized with combo /smq, combo /alum, and m /alum were used to study signature immune responses. igg subclasses generated with the different formulations showed significant differences, particularly in the ability to generate antigen-specific igg c. overall, immunization with combo /smq generated a combined th /th response with more-balanced igg / igg c ratios, while combo /alum-adjuvanted and m /alum-adjuvanted formulations produced a th -biased response ( ) . serum antibodies generated by all three formulations were able to recognize and bind antigens on the surface of live gas as measured by flow cytometry. however, only m /alum-immunized sera were able to promote opsonophagocytosis of gas. a pressing issue in gas vaccine development is the establishment of a correlate of protection that can be used to fast-track the licensure of a vaccine candidate ( ). opsonizing antibodies have long been associated with protection against infection for m protein-based vaccines and following natural infection ( , ) . in our previous report, opsonizing antibodies were detected in sera from combo /alum-immunized mice by the use of the classical lancefield whole-blood indirect bactericidal assay ( ) . in the present study, we were not able to detect opsonic antibodies by the use of the standardized hl- assay for combo /alum. discrepancies between these two assays in the detection of opsonic antibodies have been reported in the past ( ) . the technical complexity of the classical lancefield whole-blood assay makes it unfeasible for use in clinical trials, while the hl- assay may become more widely accepted as the standardized assay to investigate the presence of opsonizing antibodies against gas. in this study, mice immunized with combo /smq were protected against invasive lethal challenge in the absence of opsonizing antibodies. consequently, this work provides evidence that the opsonizing antibody correlates of protection paradigm do not necessarily apply for non-m protein antigens. this finding highlights one of the weaknesses that need to be addressed on the road to licensure for a gas vaccine. to begin to address this issue, cellular recall responses were investigated in antigenstimulated splenocytes from immunized mice. combo /alum-immune splenocytes mainly secreted cytokines (il- , il- , il- , il- , and il- ), indicating a th -type signature, with low levels of tnf-␣. in contrast, combo /smq-immune splenocytes secreted a wider variety of cytokines, including th -type and th -type cytokines. combo /smq immunization resulted in strong secretion of il- and il- and of th -type cytokines ifn-␥ and tnf-␣, suggesting a potential role for th responses in protection against invasive gas infection following vaccination. it has been previously reported that scpa-specific ifn-␥ responses may play an important role in age-related immunity to gas following natural exposure in humans ( ) . this observation, which is in line with our results, highlights the importance of th responses (especially that of ifn-␥) in protective immunity against gas infections. even though mouse models do not represent the best preclinical model to mimic gas infections, they have enabled us to investigate immune mechanisms to combat infection and identify protective vaccine candidates. mouse models are ideal for initial preclinical testing of vaccine candidates, and in this particular case, the humanized plasminogen mouse model allowed us to investigate the effect of different adjuvants to promote the protective efficacy of combo . to further advance combo /smq as a vaccine candidate, it is crucial to investigate its efficacy using more-representative models of infection, such as the pharyngitis model in nonhuman primates ( ) or, potentially, the experimental human challenge models that are being developed ( ) . this pathway of vaccine testing could ideally be used for other gas vaccine candidates, in order to build strong evidence of vaccine efficacy against gas infections. this work highlights the importance of adjuvant selection in order to raise protective immune responses against invasive gas infection. adjuvants that can provide a more balanced th /th -type response such as smq may be required to optimize the protection provided by gas vaccines, in particular, those based on non-m protein antigens. bacterial strains and growth conditions. for recombinant protein expression, escherichia coli bl star (de ) was grown in luria-bertani medium (lb) supplemented with ampicillin ( g/ml). streptococcus pyogenes m t strain , an invasive clinical isolate ( ) , was grown on horse blood agar plates and in todd-hewitt broth supplemented with % (wt/vol) yeast extract. for infection experiments, a master frozen stock was prepared as previously described ( ) . antigen expression and purification. m protein and streptococcal antigens contained in combo (adi, scpa, slo, spycep, and tf) were expressed in e. coli bl star (de ) cells and purified by affinity chromatography as previously described ( ) . protein preparations were subjected to filter sterilization before final formulation. the his tag was cleaved from combo antigens for enzyme-linked immunosorbent assay (elisa) as previously reported ( ) . adjuvants and formulations. the composition of adjuvants used in this study is provided (table ) . aluminum hydroxide gel (alhydrogel %; alum) was purchased from invivogen (ca, usa). advax- and advax- adjuvants with formulations based on microparticles of delta inulin were provided by vaxine pty. ltd., adelaide, australia ( ) . swe, lq, lmq, and smq adjuvant formulations were manufactured at the vaccine formulation institute. swe adjuvant (squalene-in-water emulsion) was prepared as previously described ( ) . smq adjuvant was prepared by mixing a solution of qs (desert king international, ca, usa) in pbs with squalene-in-water emulsion, containing cholesterol (merck-sigma c , usa) and the synthetic tlr agonist - -desacyl- -monophosphoryl lipid a ( d-mpl) also called d-( -acyl) phad (catalog no. p; merck-avanti, usa). smq adjuvant was mixed with the antigen and pbs without ca and mg to obtain d-mpl at g/ml and qs at g/ml. lq adjuvant was prepared by adding a solution of qs (desert king international, ca, usa) in pbs to neutral liposomes prepared by the lipid film method, using , -dioleoyl-sn-glycero- -phosphocholine (dopc) and cholesterol as lipids and rehydration in dulbecco's phosphate-buffered saline (dpbs) (ph . ) without ca and mg buffer followed by extrusion. lq was mixed with the antigen and pbs without ca and mg to obtain qs at g/ml. lmq adjuvant was prepared in the same way but with incorporation of d-mpl into the lipid film. the lmq adjuvant was mixed with the antigen and pbs without ca and mg to obtain d-mpl at g/ml and qs at g/ml. immunization and challenge. groups (n ϭ ; females and males) of mice ( to weeks old) heterozygous for the human plasminogen gene (albplg ) ( ) were immunized intramuscularly on days , , and with g of combo (total protein) adjuvanted with each of the adjuvants listed in table to reach a final volume of l per dose. negative-control groups were immunized with pbs combined with the corresponding adjuvant. purified m protein ( g) formulated with alum was used as a positive control. serum samples were taken before immunization and on day . on day , immunized mice were infected subcutaneously with gas ( l), with the infecting dose confirmed on the day of infection by enumerating cfu. mice were monitored twice daily for days and given scores based on observed clinical signs (see table s in the supplemental material) ( ) . for antibody binding, opsonophagocytic killing, and splenocyte stimulation assays, mice per group were immunized with combo /alum, combo /smq, or m /alum (days , , and ). naïve mice were used as negative controls. on day , mice were euthanized, blood was obtained via heart puncture, and spleens were removed under aseptic conditions. antigen-specific elisa. antibody titers against individual antigens were determined by elisa as described previously ( ) . antigen-specific mouse igg antibodies were detected with horseradish peroxidase (hrp)-conjugated goat anti-mouse igg antibody (calbiochem) at a : , dilution. to measure titers of different igg subclasses, secondary hrp-goat anti-mouse igg , igg b, igg c, and igg antibodies (abcam) were used at a : , dilution. antibody binding to the gas surface. antibody binding to the gas surface was done as previously described ( ) using gas strain . goat anti-mouse igg conjugated to alexa fluor (southern-biotech) ( : dilution) was used for detection. cells were washed with pbs and fixed in . % (wt/vol) paraformaldehyde-pbs. a total of , events were acquired using a bd accuri c flow cytometer (bd biosciences). opsonophagocytic killing assay. the opsonophagocytic killing assay was performed as previously described ( , ) . briefly, l of gas strain , washed and diluted to , cfu/ml in opsonization buffer ( % [vol/vol] defined fetal bovine serum [fbs; hyclone], . % [wt/vol] gelatin [sigma-aldrich company ltd.] in hanks' balanced salt solution with ca/mg), was mixed with l sera serially diluted in opsonization buffer in a round-bottomed -well plate and then incubated for min at rpm at room temperature. a -l volume of baby rabbit complement, diluted one in six in opsonization buffer, and l differentiated human promyelocytic leukemia cells (hl- ) were added to each dilution of sera at a multiplicity of infection of : (hl- :gas) and incubated for min at °c with % co with shaking at rpm. prior to the assay, hl- cells were differentiated by incubation in . % dimethyl-vaccine-induced th -type response against invasive gas ® formamide at °c with % co for to days and diluted in opsonization buffer to ϫ cells/ml. plates were then incubated on ice for min, and l from each well was spotted onto todd-hewitt yeast agar plates (todd-hewitt broth supplemented with . % [wt/vol] yeast extract and . % [wt/vol] bacteriological agar). an overlay agar, consisting of todd-hewitt broth supplement with . % (wt/vol) yeast extract and . % (wt/vol) , , -tetraphenyltetrazolium chloride (sigma-aldrich company ltd.), was poured over each plate. plates were incubated overnight at °c with % co . the number of surviving cfu was then determined using a protocol automated colony counter (synbiosis), and the dilution of sera resulting in % killing was calculated as the opsonic index. splenocyte stimulation. aseptically removed spleens were pressed against a -m-pore-size cell strainer (corning falcon) to obtain a single-cell suspension in pbs. cells were washed with pbs, and red blood cells were subsequently lysed using red blood cell lysis buffer (roche). cells were resuspended in rpmi supplemented with % (vol/vol) fetal bovine serum and . % (vol/vol) ␤-mercaptoethanol. splenocytes ( ϫ cells) were stimulated using g of combo antigen mix or m protein, and medium was added to negative controls (unstimulated). splenocytes were incubated at °c with % co for h. culture supernatants were isolated by centrifugation and stored at - °c for cytokine quantification. cytokine quantification. cytokines secreted into the culture supernatant were quantified using a legendplex mouse -plex th /th panel (biolegend) following the manufacturer's protocol. samples were acquired using a bd accuri c flow cytometer (bd biosciences), and data were analyzed using legendplex data analysis software (biolegend v . ). the concentration of cytokines due to antigen stimulation was calculated by subtracting the concentration of cytokines in unstimulated samples from the concentration in stimulated samples. statistical analyses. antigen-specific endpoint titers were analyzed using the two-tailed mann-whitney u test, with p values of Ͻ . considered statistically significant (graphpad prism ). murine survival curves were analyzed using the mantel-cox log rank test, with p values of Ͻ . considered statistically significant (graphpad prism ). flow cytometry data were analyzed using the probability binning algorithm in flowjo . . (tree star inc.), a cutoff value of t(x) of was experimentally determined, and samples having t(x) values of Ͼ were considered significant (p Ͻ . ) ( % confidence). opsonic indices were compared using the kruskal-wallis test corrected for multiple comparisons using dunnett's test, with p values of Ͻ . considered statistically significant (graph-pad prism ). the concentrations of cytokines secreted from splenocytes were compared using a two-tailed unpaired t test, with p values of Ͻ . considered statistically significant (graphpad prism ). ethics approvals. all animal procedures were conducted according to the australian code for the care and use of animals for scientific purposes ( ) . breeding and experimental procedures using humanized plasminogen albplg mice were approved by the university of queensland animal ethics committee (scmb/ / /nhmrc/breed and scmb/ / /nhmrc). supplemental material is available online only. table s , docx file, . mb. the global burden of group a streptococcal diseases status of research and development of vaccines for streptococcus pyogenes who/ivi global stakeholder consultation on group a streptococcus vaccine development: report from a meeting held on - development of group a streptococcal vaccines: an unmet global health need prospects for a group a streptococcal vaccine: rationale, feasibility, and obstacles-report of a national institute of allergy and infectious diseases workshop new -valent m protein-based vaccine evokes cross-opsonic antibodies against nonvaccine serotypes of group a streptococci physicochemical characterisation, immunogenicity and protective efficacy of a lead streptococcal vaccine: progress towards phase i trial group a streptococcus adsorbed vaccine: repeated intramuscular dose toxicity test in minipigs multi high-throughput approach for highly selective identification of vaccine candidates: the group a streptococcus case protective efficacy of group a streptococcal vaccines containing type-specific and conserved m protein epitopes protection against group a streptococcus by immunization with j -diphtheria toxoid: contribution of j -and diphtheria toxoidspecific antibodies to protection an experimental group a streptococcus vaccine that reduces pharyngitis and tonsillitis in a nonhuman primate model differing efficacies of lead group a streptococcal vaccine candidates and full-length m protein in cutaneous and invasive disease models how can vaccines contribute to solving the antimicrobial resistance problem deploy vaccines to fight superbugs atlas of group a streptococcal vaccine candidates compiled using large-scale comparative genomics plasminogen is a critical host pathogenicity factor for group a streptococcal infection trigger for group a streptococcal m t invasive disease a novel hepatitis b vaccine containing advax™, a polysaccharide adjuvant derived from delta inulin, induces robust humoral and cellular immunity with minimal reactogenicity in preclinical testing severe acute respiratory syndrome-associated coronavirus vaccines formulated with delta inulin adjuvants provide enhanced protection while ameliorating lung eosinophilic immunopathology advax-adjuvanted recombinant protective antigen provides protection against inhalational anthrax that is further enhanced by addition of murabutide adjuvant correlates of adjuvanticity: a review on adjuvants in licensed vaccines the mechanism of action of mf -an innately attractive adjuvant formulation the adjuvant mf induces atp release from muscle that potentiates response to vaccination elucidating the mechanisms of action of saponinderived adjuvants adjuvant system as : helping to overcome the challenges of modern vaccines characterization of t-cell immune responses in clinical trials of the candidate rts,s malaria vaccine regulation of antibody isotype secretion by subsets of antigen-specific helper t cells correlates of protection for m protein-based vaccines against group a streptococcus persistence of type-specific antibodies in man following infection with group a streptococci development of an opsonophagocytic killing assay using hl- cells for detection of functional antibodies against streptococcus pyogenes adaptive immunity against streptococcus pyogenes in adults involves increased ifn-gamma and igg responses compared with children controlled human infection for vaccination against streptococcus pyogenes (chivas): establishing a group a streptococcus pharyngitis human infection study genetic relatedness and superantigen expression in group a streptococcus serotype m isolates from patients with severe and nonsevere invasive diseases advax, a novel microcrystalline polysaccharide particle engineered from delta inulin, provides robust adjuvant potency together with tolerability and safety technology transfer of an oil-in-water vaccine-adjuvant for strengthening pandemic influenza preparedness in indonesia dnase sda provides selection pressure for a switch to invasive group a streptococcal infection development of an opsonophagocytic killing assay for group a streptococcus australian code for the care and use of animals for scientific purposes we thank the staff of the university of queensland biological resources facility for their support in the day-to-day care and breeding of the animals used in this study.this work was supported by grants from the australian national health and medical research council.m.j.w. has an intellectual property interest in adi and tf ( ) . n.p. is an associate of vaxine pty. ltd., which owns the advax adjuvant platform. we report that we have no other conflict of interest. key: cord- -uf jgig authors: wang, yi; liu, li title: the membrane protein of severe acute respiratory syndrome coronavirus functions as a novel cytosolic pathogen-associated molecular pattern to promote beta interferon induction via a toll-like-receptor-related traf -independent mechanism date: - - journal: mbio doi: . /mbio. - sha: doc_id: cord_uid: uf jgig most of the intracellular pattern recognition receptors (prrs) reside in either the endolysosome or the cytoplasm to sense pathogen-derived rnas, dnas, or synthetic analogs of double-stranded rna (dsrna), such as poly(i:c). however, it remains elusive whether or not a pathogen-derived protein can function as a cytosolic pathogen-associated molecular pattern (pamp). in this study, we demonstrate that delivering the membrane gene of severe acute respiratory syndrome coronavirus (sars-cov) into hek t, hek et, and immobilized murine bone marrow-derived macrophage (j -mφ) cells significantly upregulates beta interferon (ifn-β) production. both nf-κb and tbk -irf signaling cascades are activated by m gene products. m protein rather than m mrna is responsible for m-mediated ifn-β induction that is preferentially associated with the activation of the toll-like receptor (tlr) adaptor proteins myd , tirap, and ticam but not the rig-i signaling cascade. blocking the secretion of m protein by brefeldin a (bfa) failed to reverse the m-mediated ifn-β induction. the antagonist of both tlr and tlr did not impede m-mediated ifn-β induction, indicating that the driving force for the activation of ifn-β production was generated from inside the cells. inhibition of traf expression by specific small interfering rna (sirna) did not prevent m-mediated ifn-β induction. sars-cov pseudovirus could induce ifn-β production in an m rather than m(v a) dependent manner, since the valine-to-alanine alteration at residue in m protein markedly inhibited ifn-β production. overall, our study indicates for the first time that a pathogen-derived protein is able to function as a cytosolic pamp to stimulate type i interferon production by activating a noncanonical tlr signaling cascade in a traf -independent manner. in addition to the tlr, which can be defined as a membraneassociated prr, another set of prrs is localized at the cytoplasm and mainly includes rig-like receptors (rlrs) and nod-like receptors (nlrs) to sense viral dsrnas and bacterial cell wall components, respectively ( , ) . the rlrs consist of at least three members, including rig-i, mda , and lgp . rig-i recognizes =-triphosphate rna and short dsrna ( , ) , while mda senses long dsrna ( ) . an adaptor protein, mavs, is required for the activation of the rig-i/mda signaling pathway. the association of viral nucleic acids with mavs promotes the aggregation of mavs on the mitochondrial membrane ( ) . the "ligation" of traf with the aggregated mavs may promote the phosphorylation of irf that is required for ifn-␤ production ( ) . a recent study also shows that an endoplasmic reticulum (er)-derived adaptor protein, sting, could also function downstream of mavs to promote irf phosphorylation and the subsequent ifn-␤ response ( ) . pathogen-derived proteins such as virus-encoded proteins are frequently documented as negative regulators in subverting type i interferon (ifn-i) induction by interfering with a certain key component(s) of ifn-i activation signaling cascades. viral evolution may develop a unique strategy to inhibit host innate immunity by generating virus-derived antagonists to some key signaling molecules. the vaccinia virus encodes two toll/interleukin- (il- ) receptor (tir) domains containing proteins a r and a r, which can negatively regulate tlr signaling by two distinct mechanisms ( ) . the vaccinia virus a r inhibits tlr signaling by physically interacting with the bb loop of tir containing adaptor proteins such as myd adaptor-like (mal) and trif-related adaptor molecule (tram) to disrupt receptor-adaptor (e.g., tlr -mal and tlr -tram) interactions ( , ) . differently, the a r protein may function as a dominant negative myd to directly interact with traf and irak ( , ) . on the other hand, the vaccinia virus n l protein, another protein homologous to a r, employs a different anti-ifn-i strategy by targeting both the tbk /ib kinase (ikk) and ikk␣/ikk␤ complexes to inhibit irf and nf-b signaling, respectively ( ) . alternatively, virus may invade the cells to target the retinoic acid-inducible gene i (rig-i)-like receptor signaling pathways for the prevention of ifn-i induction. for example, the influenza virus nonstructural protein ns can sequester either the dsrna or =triphosphate rna products of viral infection which can be sensed by or directly bound to the rna helicase sensor rig-i to inhibit rig-i-mediated ifn-␤ production ( , , ) . the paramyxovirus v protein inhibits ifn-␤ induction through the blockage of mda , another rig-i-homologous cytosolic dsrna sensor ( ) . a recent study revealed that the transcriptional factor irf might be alternatively targeted and inhibited by the paramyxovirus v protein to impede ifn-␤ gene transcription ( ) . it has been demonstrated in some cases that viral proteins may function as extracellular pamps to activate the ifn-i immune response, most often through tlr (such as tlr and tlr ) signaling pathways ( ) ( ) ( ) . however, evidence is lacking in regard to whether or not a virus-derived protein can function as a cytosolic pamp. our initial study indicates that delivering the membrane gene into hek cells markedly induces type i interferon (ifn-i) production ( ) . to our knowledge, there are limited reports regarding the induction of ifn-␤ expression directly by viral structural genes. therefore, it is intriguing to know which mechanism is responsible for the severe acute respiratory syndrome coronavirus (sars-cov) m gene-mediated ifn-␤ response. in this study, we demonstrate that sars-cov m protein, rather than its mrna, activates ifn-␤ and nf-b responses through tlr-related traf -independent signaling cascades. the driving force for m-mediated ifn-␤ induction was most likely generated from the inside of the cells. using sars-cov pseudovirus as an infectious agent, we further show that single point mutation at the valine residue of m protein markedly inhibits virus-induced ifn-␤ production. overall, sars-cov m protein may stand out as a novel cytosolic pamp in mediating the ifn-␤ immune response. the sars-cov m gene stimulates beta interferon gene expression in the human embryonic kidney t cell line. the overexpression of the sars-cov m gene has been shown to upregulate the transcriptional level of ifn-␤ ( ) . to further confirm the result, using either enhanced green fluorescent protein (egfp) or poly(i:c) as a negative or positive control, respectively, we demonstrated that m gene products specifically promoted ifn-␤ production, since cotransfection with m small interfering rna (sirna) completely abolished m-mediated ifn-␤ induction at both protein (fig. a , comparing lanes and ) and mrna ( fig. b ) levels. moreover, after a -h transfection, cell supernatants were collected and assayed for the presence of ifn-␤ by enzyme-linked immunosorbent assay (elisa). figure c clearly demonstrated that delivering pcmv-myc-m into hek t cells specifically and significantly promoted the secretion of ifn-␤ into cell culture medium. in addition, the promoter sequence of ifn-␤ was placed upstream of the firefly luciferase reporter to generate the pgl -ifn-␤-luc construct. to test the specificity of m-mediated ifn-␤ induction, other viral envelope-associated genes such as the spike (s) and envelope (e) protein genes as well as the m mutant [m(v a)] from the gz isolate were also included ( ) . the result of a dual-luciferase assay using the renilla luciferase gene as a transfection control demonstrated that the sars-cov m gene rather than the s and e genes markedly increased ifn-␤ promoter activity (fig. d) , whereas the valineto-alanine alteration at residue of m protein completely abolished this induction, indicating that the specificity of m gene products played a role in this process. consistent with these results, western blotting and elisa further validated the above observation ( fig. e and f). to detect if the sars-cov m gene has a direct effect on nf-b activation, pcmv-myc-m was cotransfected with pnfb-luc, which contained five copies of nf-b recognition sites, into hek t cells. the results of the dualluciferase assay revealed that the sars-cov m gene specifically and dramatically upregulated nf-b activity compared with the controls (fig. g) . moreover, m could mediate ifn-␤ induction in both dose-and time-dependent manner ( fig. h and i) . overall, the data strongly indicated that the sars-cov m gene product was sufficient to promote ifn-␤ gene expression. the sars-cov m gene product activates the ifn-␤ signaling pathway at or upstream of tbk . to further confirm the above results, increased doses of the pcmv-myc-m gene were transiently transfected into hek et cells. the cell lysates prepared from the transfection were examined for the activation of the downstream modulator and/or effector molecules, such as tbk , irf , and nf-b. figure a clearly demonstrates that sars-cov m gene products not only enhanced the phosphorylation level of tbk but also promoted the activation of both nf-b p and irf , indicating that m gene products may stimulate ifn-␤ activation by promoting its enhanceosome activity. to further define the activation level of m-mediated ifn-␤ induction, specific sir-nas that selectively targeted either tbk ( fig. b and c) or irf ( fig. e and f) mrnas were generated. individually diminishing either tbk or irf mrna expression by sitbk or siirf sig-nificantly reversed m-mediated ifn-␤ induction ( fig. d and g, respectively), indicating that m-mediated ifn-␤ induction functions at a level at or above the signaling molecule tbk . the sars-cov m gene product preferentially activates ifn-␤ production through toll-like-receptor-related signaling pathways in hek et cells. rlr and tlr are two main prrs hek t cells. after , , , and h of transfection, dual-luciferase assays were performed to detect ifn-␤ expression. each value represents the mean Ϯ standard deviation from three independent tests. *, p Յ . ; **, p Յ . . in all data presented above, the relative luciferase activity was determined as firefly luciferase activity divided by renilla luciferase activity. the effect of tbk sirna (sitbk ) on the expression of endogenous tbk by semiquantitative rt-pcr. the increased doses of pbs/u -sitbk plasmid dnas ( , . , and g) were transiently transfected into hek et cells. total rnas or whole-cell lysates were isolated or harvested at h posttransfection. one-step rt-pcr (rt) was conducted to detect the tbk mrna expression with specific primers (upper panel), while western blotting (wb) was performed to detect tbk protein expression using specific anti-tbk antibody (lower panel). the expression of ␤-actin served as a loading control. the relative band intensity was quantitated with the image j program in comparison with the ␤-actin control. the result is representative of at least identical experiments. (c) effect of sitbk on the expression of tbk mrnas by real-time qrt-pcr analysis. total rnas isolated in panel b were subjected to qrt-pcr analysis using specific tbk primers. each value represents the mean Ϯ standard deviation from three reactions. the result is representative of at least identical experiments. (d) effect of sitbk on m-mediated ifn-␤ induction. plasmid pgl -ifn-␤-luc reporter was cotransfected with g of pcmv-myc-m or g of pcmv-myc-m plus increased doses of sitbk ( , . , and g) into hek et cells grown on a -well plate. at h posttransfection, the dual-luciferase assay was conducted to assay fold induction of m-mediated ifn-␤ expression. each value represents the mean Ϯ standard deviation from three independent tests. (e) effect of irf sirna (siirf ) on expression of endogenous ifr by semiquantitative rt-pcr. the increased doses of pbs/u -siirf plasmid dnas ( , . , and g) were transiently transfected into hek t cells. total rnas or whole-cell lysates were isolated or harvested at h posttransfection. one-step rt-pcr was conducted to detect irf expression with specific primers (upper panel), while western blotting was performed to detect irf protein expression (lower panel). the expression of ␤-actin served as a loading control. the result is representative of at least identical experiments. (f) effect of siirf on m-mediated ifn-␤ mrna expression by real-time qrt-pcr analysis. real-time qrt-pcr was performed to detect the irf mrna (isolated in panel e) expression. each value represents the mean Ϯ standard deviation from three reactions. the result is representative of at least identical experiments. (g) effect of siirf on m-mediated ifn-␤ induction by luciferase assay. plasmid pgl -ifn-␤-luc reporter was cotransfected with g of pcmv-myc-m or g of pcmv-myc-m plus increased doses of siirf ( , . , and g) into hek et cells grown on a -well plate. at h posttransfection, the dual-luciferase assay was conducted to assay fold induction of m-mediated ifn-␤ expression. each value represents the mean Ϯ standard deviation from three independent tests. recognizing the majority of extracellular and intracellular pamps. upon the ligation of a prr with its specific pamp, both rlr and tlr pathways transmit the signal to a common class of adaptors called tumor necrosis factor (tnf) receptor-associated factors (trafs) including traf /traf , traf , and traf ( , ) . to test the effect of m on rlr and tlr signaling as well as traf expression, an increased dose of pcmv-myc-m constructs was first transiently transfected into hek et cells. the results in fig. a demonstrate that the increased delivery of pcmv-myc-m into hek et cells markedly enhanced traf but not traf and traf expression, indicating that traf -mediated signaling transduction might contribute to the upregulation of ifn-␤ production. to further address how traf expression is associated with rlr and/or tlr signaling pathways, the m genetransfected hek et cells were also assayed for the expression of upstream sensors and/or signaling molecules of trafs. figure b demonstrates that no significant alteration was observed in the expression of rig-i, mda , and mavs after exogenously delivering m genes into hek et cells, indicating that the rlr signaling pathway might not be targeted by m gene products. in contrast, three adaptor proteins (myd , tram/ticam , and tirap) associated with tlrs were all upregulated (fig. c) , while the adaptor protein trif failed to be upregulated in responding to m gene overexpression (fig. d) . overall, the results indicate that tlr signaling pathways are mainly targeted by the sars-cov m gene product for the induction of ifn-␤ expression. the sars-cov m gene product promotes ifn-␤ production through toll-like-receptor-related signaling pathways in immortalized murine bone marrow-derived macrophage cells. to further confirm the above results, the pcmv-myc-m construct was also transiently transfected into j -m cells, an immortalized murine bone marrow-derived macrophage cell line established with j virus ( , ) . the delivery of the m gene product is effec-tive in stimulating the activation of both ifn-␤ and nf-b in murine j -m cells (fig. a , b, and c). figure d demonstrates that increased delivery of m gene product into j -m cells indeed promotes ifn-␤ induction through the phosphorylation of irf , nf-b p , and tbk . in accord with the results in hek et cells, the increased delivery of pcmv-myc-m into j -m cells markedly enhanced traf but not traf and traf expression (fig. e) . moreover, the m gene product did not significantly increase the protein levels of rig-i, mavs, sting, and mda ( fig. f) , indicating that the rig-i signaling pathway might not be activated in responding to exogenous delivery of the m gene product into j -m cells. in contrast, the increased delivery of the m gene into j -m cells markedly enhanced myd and tram/ ticam but not trif expression (fig. g ), indicating that tlrrelated signaling pathways might be mainly associated with m-mediated ifn-␤ induction. overall, our data in both hek et and j -m cells strongly indicate that m-mediated induction of ifn-␤ expression is likely associated with the activation of tlr-related signaling pathways. the sars-cov m gene product functions at the protein level to induce ifn-␤ production. the next question that we tried to ask was at which level (mrna or protein) the m-mediated ifn-␤ induction occurred. to address this issue directly, we created an m-stop construct by replacing the start codon aug with three in-frame tandem stop codons at the = end of the m gene (fig. a ). this expression construct can generate only mrna and no protein due to the translation failure of the mrna substrates. western blot analysis shows that the m protein synthesis was completely blocked in the m-stop construct but not the wild-type m construct (fig. b ). real-time quantitative reverse transcription pcr (qrt-pcr) analysis shows that the m-stop construct did not induce ifn-␤ production in hek t cells, indicating that m-mediated ifn-␤ production is dependent on m protein rather than m mrna (fig. c) . to directly compare m-and m-stop-induced ifn-␤ production levels, the increased doses of either m or m-stop constructs were cotransfected with ifn-␤ luciferase reporter into hek t cells. figure d clearly demonstrates that m-stop does not induce ifn-␤ production, indicating that protein translation is necessary for m-induced ifn-␤ production. to confirm this result further, the chemical inhibitor cycloheximide (chx) was used to block the protein biosynthesis in transfected hek t cells. figure e shows that addition of chx significantly inhibited and completely blocked m protein synthesis at g/ml and above g/ ml, respectively, which are directly correlated with the marked reduction and complete inhibition in ifn-␤ expression, indicating that m protein may function as a pamp to induce ifn-␤ production. if m protein indeed functions as a pamp, blocking m protein synthesis should prevent the m-mediated activation of the ifn-␤ signaling pathway. figure f clearly shows that m-stop reverses the m-mediated upregulation of the adaptor proteins myd and ticam /tram in the initiating phase of tlr signaling pathways. moreover, m-stop prevents the activation of the downstream modulator and key effectors of the ifn-␤ signaling pathway, such as tbk , irf , and nf-b p , in a dose-dependent manner (fig. f ). thus, blocking m protein translation could prevent m-mediated ifn-␤ induction by inactivating the tlrrelated signaling pathway. sars-cov m protein may function as a novel intracellular pamp to induce ifn-␤ production. one critical question remaining to be answered is whether the driving force for m protein-mediated ifn-␤ induction is generated intracellularly or extracellularly. to answer this question directly, the trap␥ gene, an endoplasmic reticulum (er)-associated gene, was cotransfected with m into hela cells. brefeldin a (bfa) was employed in the assay system to block m protein transport from the rough endoplasmic reticulum to the cell surface. indeed, the addition of bfa effectively increased the retention of m proteins in the er compartment (fig. a) . figure b shows that addition of bfa also effectively inhibited the secretion of ifn-␤ into the cell culture medium (right panel) but did not inhibit m-mediated ifn-␤ induction at either the mrna level or the protein level (left panel), indicating that the driving force for m-mediated ifn-␤ induction was indeed derived from intracellular stimulation by m proteins. reports indicate that sars-cov m protein alone can form virus-like particles (vlps) that can be secreted extracellularly. therefore, the secreted m protein might be sensed by its own or other cell prrs on the cell surface to activate ifn-i responses. to rule out this possibility, oxpapc (a tlr and tlr inhibitor) was used to block the function of the accessory proteins cd , lbp, and md that are required for tlr and tlr signaling ( ) . figure c shows that addition of oxpapc could effectively inhibit lipopolysaccharide (lps)-mediated ifn-␤ production (right panel) but did not reverse the m-mediated ifn-␤ induction (left panel), indicating that the extracellular stimulation by m pro- sars-cov m protein promotes ifn-␤ induction independently of traf . it has been well known that traf plays a critical role in tlr-mediated ifn-␤ induction, especially through tlr and tlr pathways ( , , ) . since m protein could activate the tlr pathway from inside the cells, it remained unclear whether or not this activation is in a traf -dependent or -independent manner. to address this issue directly, a specific sirna against traf was successfully constructed (fig. a) . a dual-luciferase assay was conducted to assay the effect of sitraf on m-mediated ifn-␤ induction in hek t cells. figure b clearly shows that the increased delivery of sitraf did not reverse the m-mediated ifn-␤ induction, indicating that traf might not be essential for this activation. to further confirm the above result, western blot analysis was performed to detect ifn-␤ expression in responding to the increased delivery of sitraf into hek t cells. the increased delivery of sitraf into hek t co-ip was conducted as shown in the left panel. about % input from each lysate preparation was subjected to western blot analysis using anti-ha, anti-flag, and anti-myc antibodies as probes. the rest of the lysate was first immunoprecipitated with anti-flag antibody conjugated with an affinity gel. then, the reaction products were probed with anti-ha and anti-myc antibodies. a reverse co-ip experiment was also conducted as shown in the right panel. the lysates were first immunoprecipitated with anti-myc antibody. then, the reaction products were individually probed with anti-myc, anti-flag, and anti-ha antibodies and subsequently subjected to western blotting. the result is representative of at least identical experiments. ib, immunoblotting; ns, nonspecific. (h) the m gene product does not suppress poly(i:c)-mediated ifn-␤ induction. about g/ml of poly(i:c) was cotransfected with g of either m or m(v a) plasmid along with pgl -ifn-␤luciferase reporter ( ng) plus prl-tk ( ng) into hek t cells. after h of transfection, dual-luciferase assays were performed to detect ifn-␤ expression (left panel). each value represents the mean Ϯ standard deviation from three independent tests. the statistical difference was considered to be significant at a p value of Յ . . the ifn-␤ expression was also subjected to western blotting (right panel). the relative band intensity was quantitated with the image j program in comparison with the ␤-actin control. cells failed to inactivate the activation of irf and nf-b p and did not affect the m-mediated ifn-␤ induction (fig. c) , while a result was obtained consistent with the increased delivery of si-traf into j -m cells (fig. d) . in addition, a stable hek cell line with traf knocked down by sitraf was also established (fig. e) . a dose-dependent increase in ifn-␤ production was observed with the increased delivery of m gene into si-traf stable cells (fig. f) . thus, the above data strongly indicate that traf is not required for m-mediated ifn-␤ induction. sars-cov m protein has been shown to destabilize the functional traf -tbk complex formation ( ) . however, it remains unknown whether or not m protein is able to associate with the key components of this complex. to clarify this issue directly, a coimmunoprecipitation (co-ip) experiment was conducted. plasmid tbk -flag, traf -ha, and myc-m dnas were transiently cotransfected into hek t cells. the cell lysates were first immunoprecipitated with anti-flag antibody conjugated with an affinity gel and then subsequently probed with antihemagglutinin (anti-ha) and anti-myc antibodies. the left panel of fig. g indicates that m protein was able to disrupt the physical interaction between tbk and traf , but m protein itself could not form a complex with tbk . in the reverse immunoprecipitation (ip) experiment as shown in the right panel of fig. g , m protein was unable to interact with both tbk and traf directly, indicating that m protein may modulate the tbk -traf complex formation indirectly. interestingly, in contrast to the inhibitory effect of m(v a) on poly(i:c)-mediated ifn-␤ induction, the m gene product did not affect the ifn-␤ induction stimulated by poly(i:c) (fig. h) , indicating that m has no effect on poly(i:c)induced ifn-␤ production while retaining the ability to destabilize the complex formation. one critical question remaining to be answered is whether or not m-mediated ifn-␤ induction could occur in real virus infection. it has been shown that codelivering the m, n, and s genes of sars-cov into hek cells readily produced sars-cov pseudovirus with the corona-like halo ( ) . earlier results demonstrate that valine-toalanine mutation at residue [abbreviated as m(v a)] inhibited ifn-␤ induction ( ) . we employed a sars-cov pseudovirus system to mimic the real sars-cov infection. cell lysate supernatants isolated from either m, n, and s cotransfection or m(v a), n, and s cotransfection were first incubated with -ace stable cells (fig. a) for h. then, the cell culture medium was replaced with fresh medium and further incubated at °c for another h before harvesting. the sars-cov pseudovirus vlp(s-m-n) markedly upregulated ifn-␤ expression at both rna and protein levels (fig. b) , whereas the point mutation at the valine residue of m protein completely diminished sars-cov pseudovirus-mediated ifn-␤ induction, strongly indicating that the m protein residing on sars-cov virion could specifically induce ifn-␤ production upon infecting the targeted cells. taken together, our data indicate for the first time that sars-cov m protein may function as a novel cytosolic pamp to activate ifn-␤ induction through an intracellular tlr-related signaling pathway in a traf -independent manner. pathogen-associated molecular patterns (pamps) are pathogenborne components that can be sensed by either transmembrane, endolysosomal, or cytosolic sensors known as pattern recognition receptors (prrs) ( ) . in this study, we demonstrate that the membrane protein of sars-cov significantly upregulates ifn-␤ production by activating both nf-b and tbk -irf signaling cascades. our data show that m-mediated ifn-␤ production is induced by m protein rather than m mrna, indicating that pathogen-derived protein might be able to serve as a cytosolic pamp. in addition, a mechanism study indicates that m proteinmediated ifn-␤ induction may be mainly due to the selective activation of tlr-related signaling pathways rather than the rlr signaling pathway in a traf -independent manner. overall, the current study indicates for the first time that pathogen-derived protein may function as a novel cytosolic pamp to initiate a tlrrelated traf -independent signaling pathway that subsequently promotes type i interferon (ifn-i) production. the membrane-associated prrs, such as tlr and tlr , can sense not only bacterial components but also viral coat proteins ( ) . kurt-jones and colleagues first demonstrated that the innate immune response to the fusion (f) protein of respiratory syncytial virus (rsv) is mediated by tlr plus its cofactor cd on the plasma membrane, indicating that nonbacterial components can serve as extracellular pamps ( ) . although some viral structural proteins, such as the nucleocapsid (n) from measles virus ( ) and viral ribonucleoprotein from vesicular stomatitis virus ( ) , can activate irf and tbk /ikk␦, respectively, it remains to be determined how these viral products can function as pamps and where the driving forces for their activation come from. it has been shown that the sars-cov m protein alone not only can form virus-like particles (vlps) in a viral rna-independent manner ( ) but also can induce cell apoptosis by inhibiting some key survival signal pathways, such as the akt signaling pathway ( ) . thus, the m proteins derived from these infected and apoptotic cells might be eventually secreted and released into the extracellular compartments where the tlrs (such as tlr and tlr ) on the cell surface can be potentially recognized and subsequently activated by these extracellular pamps for the induction of the ifn-i response. to clarify this possibility, we employed several approaches to test whether the driving force for m proteinmediated ifn-␤ production is generated from inside or outside the cells. we used two types of inhibitors, bfa and oxpapc, to block either the intracellular transport of m protein or the extracellular binding of m protein on tlr and tlr , respectively. our results reveal that m-mediated ifn-␤ induction is independent of m protein secretion as well as the extracellular stimulation of tlr /tlr signaling, indicating that the driving force for the m-mediated ifn-␤ induction is likely generated from inside the cells. our results may contradict some previous reports related to m-mediated ifn-i response. siu et al. demonstrated that the m protein of sars-cov negatively regulated the dsrna-induced and signaling molecule-induced ifn-i production. they also showed that m protein alone was unable to activate the ifn-i promoter activity ( ) . one study even showed that m protein negatively regulates the nf-b signaling pathway ( ) . a possible explanation for these discrepancies might be related to the m gene itself. early study has shown that m protein possesses a higher substitution rate than other structural proteins in sars-cov, and the outcome of these substitutions alters the biochemical and immunological properties of m proteins ( , ) . one amino acid alteration from valine to alanine at residue is indeed found in the m protein from the gz isolate studied by siu et al. compared with the isolate used in the current study. our functional analysis provides strong evidence to demonstrate that the valine-toalanine change at residue of m protein is sufficient to abolish m-mediated ifn-␤ induction at both the transient-transfection level and the viral infection level (fig. d and e and b and c) . therefore, this amino acid substitution in m proteins indeed affects the interaction between m protein and the prr for the subsequent ifn-i induction. it still remains elusive which cytosolic prr is responsible for m-mediated ifn-␤ induction. the m protein might be a multifaceted molecule that physically interacts with diverse intracellular sensors and signaling factors ( , ) . our data indicate that the tlr-related signaling pathway rather than the rig-i signaling cascade is responsible for m-mediated ifn-␤ induction. interestingly, we observed consistent traf reductions in both hek t and j -m cells as tested by the transiently intracellular overexpression of the m gene. traf is one of the key signaling molecules specifically responsible for ifn-i induction ( ) . data from the work of siu and colleagues have shown that m(v a) excludes the traf inclusion in the traf .tank.tbk /ikk complex ( ) . although m(v a)-mediated traf exclusion inhibits ifn-␤ induction, our study showed that m-mediated traf exclusion is independent of ligand stimulation and the disassociated traf and tbk could not complex with m protein, indicating that m protein might modulate the functions of tbk complex indirectly. interestingly, we demonstrated that m-mediated ifn-␤ induction was associated with the upregulation of the adaptor proteins myd , tirap, and ticam but not trif, which are all involved in the initiating phase of tlr signaling pathways. trif is required for both tlr -and tlr mediated ifn-␤ induction. in the activation of tlr , trif is directly recruited to the tir domain of dimerized tlr , while in the activation of tlr , trif is recruited to dimerized tlr indirectly through another tir-containing adaptor protein, ticam (also called tram). if trif is not required for m-mediated ifn-␤ induction, m-mediated ifn-␤ induction might be activated via a noncanonical tlr -related signaling cascade independently of trif and traf . in summary, the current study demonstrates for the first time that the m protein of sars-cov is able to function as a cytosolic pamp to promote ifn-␤ production by activating a tlr-related traf -independent pathway. the driving force for m-mediated ifn-␤ induction is likely generated from inside the cells rather than the extracellular binding of m proteins with the defined cell surface prrs, such as tlr . plasmid construction. plasmids pcmv-myc-m and pbs-u -sim were constructed previously ( ) . plasmids pcmv-myc-s and pcmv-myc-e were constructed by inserting s and e into the ecori and kpni sites of pcmv-myc. the mutant of pcmv-myc-m(v a) was generated by using the site-directed mutagenesis kit (takara, dalian, china). one copy of the ifn-␤ promoter sequence ( =-ctaaaatgtaaatgacata ggaaaactgaaagggagaagtgaaagtgggaaattcctctgaat agagagaggaccatctcatataaataggccatacccatggagaa aggacattctaactgcaacctttcga- =) was pcr amplified and subcloned into the kpni and xhoi sites of the luciferase reporter pgl basic (promega, madison, wi, usa) to generate the pgl -ifn-␤-luc construct. all primers were synthesized by sangon (shanghai, china). for the construction of pbs/u sitbk , the sense strand = tcgagttgcg aagccggaagtgtcctaagcttaggacacttccggcttcgcaat ttttg = and antisense strand = acgcttcggccttcacaggattc gaatcctgtgaaggccgaagcgttaaaaacttaa = were annealed and then subcloned into the ecori and xhoi sites of pbs/u . for the construction of pbs/u siirf , the sense strand = tcgagcatcggct tttgggtctgttaaagctttaacagacccaaaagccgatgtttt tg = and antisense strand = cgtagccgaaaacccagacaatttcg aaattgtctgggttttcggctacaaaaacttaa = were annealed and then subcloned into the ecori and xhoi sites of pbs/u . for construction of psilencer-sitraf , the single-strand oligonucleotides = gatccgcgagaactcctctttccctcgagggaaagagg agttctcgcaga = and = agcttctgcgagaactcctctttccc tcgagggaaagaggagttctcgcg = were annealed to double strands before being subcloned into the hindiii and bamhi sites of psilencer . -cmv neo to form the psilencer-sitraf construct. transient transfection. cells (hek et, hek t, and j -m) were transiently transfected with the plasmid dnas (pcmv-myc-m, pcmv-myc, pgl -ifn-␤-luc, or pnf-b-luc) using vigofect (vigorous biotechnology, beijing, china) according to the manufacturer's instructions. for example, about g plasmid dnas was first added to l of . % nacl. then, . l vigofect was resuspended into another l of . % nacl solution. then, the dna-nacl mixture was added to the vigofect-nacl mixture drop by drop with gentle vortexing. after a min incubation at room temperature, the reaction product was evenly distributed onto the cell culture surface of either -well plates or -mm dishes and then continuously incubated for h before harvesting. construction of the sitraf stable cells. plasmid psilencer-sitraf dnas ( g) were transfected into hek cells. after a -h transfection, the cell culture medium was replaced with fresh medium containing , g/ml g . after weeks of culture, cell colonies were picked up and expanded in a -well tissue culture plate. finally, western blot analysis was performed to detect traf expression. reverse transcription-pcr (rt-pcr) and qrt-pcr. total rnas were extracted from the cultured cells with trizol (invitrogen, carlsbad, ca, usa). the purified total rnas were treated with dnase i (qiagen, düsseldorf, germany). all primers used in the rt-pcrs are listed in table . the rt-pcr was carried out with the primescript one-step rt-pcr kit (takara biotechnology, dalian, china) according to the manufacturer's instructions. the rt-pcr was carried out in a dna thermal cycler (applied biosystems, carlsbad, ca) under the following conditions: °c for min for reverse transcription and °c for min for denaturation. the pcr conditions were °c for s, °c for s, and °c for s, repeated for to cycles; the reaction was extended at °c for min before the reaction product was stored at °c. one-step real-time quantitative rt-pcr (qrt-pcr) (takara biotechnology, dalian, china) was also performed to monitor the targeted gene expression. the primers used in qrt-pcr were also listed in table . real-time qrt-pcr was carried out with the cfx real-time pcr detection system (bio-rad laboratories, hercules, ca, usa) under the following conditions: °c for min and °c for s, and then °c for s and °c for s, repeated for cycles. the dissociation of the reaction products was conducted from °c to °c as the temperature rose at . °c per s. viral evasion and subversion of pattern-recognition receptor signalling induction and function of ifnbeta during viral and bacterial infection innate immunity to virus infection role of adaptor trif in the myd -independent toll-like receptor signaling pathway innate antiviral responses by means of tlr -mediated recognition of singlestranded rna tlr signals after translocating from the er to cpg dna in the lysosome the interferon inducing pathways and the hepatitis c virus =-triphosphate rna is the ligand for rig-i differential roles of mda and rig-i helicases in the recognition of rna viruses mavs forms functional prion-like aggregates to activate and propagate antiviral innate immune response regulation of antiviral responses by a direct and specific interaction between traf and cardif the adaptor protein mita links virus-sensing receptors to irf transcription factor activation vaccinia virus protein a r targets multiple toll-like-interleukin- receptor adaptors and contributes to virulence poxviral protein a antagonizes toll-like receptor signaling by targeting bb loop motifs in toll-il- receptor adaptor proteins to disrupt receptor:adaptor interactions viral inhibitory peptide of tlr , a peptide derived from vaccinia protein a , specifically inhibits tlr by directly targeting myd adaptor-like and trif-related adaptor molecule the poxvirus protein a r targets toll-like receptor signaling complexes to suppress host defense a r and a r from vaccinia virus are antagonists of host il- and toll-like receptor signaling poxvirus protein n l targets the i-kappab kinase complex, inhibits signaling to nf-kappab by the tumor necrosis factor superfamily of receptors, and inhibits nf-kappab and irf signaling by toll-like receptors inhibition of retinoic acid-inducible gene i-mediated induction of beta interferon by the ns protein of influenza a virus influenza c virus ns protein counteracts rig-i-mediated ifn signalling the v proteins of paramyxoviruses bind the ifninducible rna helicase, mda- , and inhibit its activation of the ifn-beta promoter inhibition of interferon regulatory factor activation by paramyxovirus v protein murine retroviruses activate b cells via interaction with toll-like receptor pattern recognition receptors tlr and cd mediate response to respiratory syncytial virus reading the viral signature by toll-like receptors and other pattern recognition receptors small interfering rna effectively inhibits the expression of sars coronavirus membrane gene at two novel targeting sites the family of five: tir-domain-containing adaptors in toll-like receptor signalling traf molecules in cell signaling and in human diseases expanding traf function: traf as a tri-faced immune regulator tumoricidal alveolar macrophage and tumor infiltrating macrophage cell lines interaction between raf and myc oncogenes in transformation in vivo and in vitro oxidized phospholipid inhibition of toll-like receptor (tlr) signaling is restricted to tlr and tlr : roles for cd , lps-binding protein, and md as targets for specificity of inhibition assembly and localization of toll-like receptor signalling complexes severe acute respiratory syndrome coronavirus m protein inhibits type i interferon production by impeding the formation of traf .tank.tbk /ikkepsilon complex generation of synthetic severe acute respiratory syndrome coronavirus pseudoparticles: implications for assembly and vaccine production pattern recognition receptors and inflammation pathogen recognition by the innate immune system recognition of the measles virus nucleocapsid as a mechanism of irf- activation activation of tbk and ikkvarepsilon kinases by vesicular stomatitis virus infection and the role of viral ribonucleoprotein in the development of interferon antiviral immunity self-assembly of severe acute respiratory syndrome coronavirus membrane protein the sars-coronavirus membrane protein induces apoptosis through modulating the akt survival pathway the membrane protein of sars-cov suppresses nf-kappab activation the m protein of sars-cov: basic structural and immunological properties persistent replication of severe acute respiratory syndrome coronavirus in human tubular kidney cells selects for adaptive mutations in the membrane protein the orphan nuclear receptor tr /nur regulates er stress and induces apoptosis via interaction with trap␥ we thank genhong cheng and hang-zi chen for providing the j -m cells and trap␥, respectively. western blot analysis. the transfected cells were lysed with a lysis buffer containing % np- , mm tris-hcl (ph . ), mm nacl, m navo , g/ml leupeptin, g/ml aprotinin, and m phenylmethylsulfonyl fluoride (pmsf). about g of cell lysate for each sample was resolved by % sds-page. after separation, the reaction products were transferred onto a hybond nitrocellulose membrane (pharmacia, st. louis, mo, usa). the transferred membrane was first probed with a primary antibody. then, a secondary antibody labeled with horseradish peroxidase was added to the reaction product and was finally visualized with an enhanced chemiluminescence (ecl) kit (santa cruz biotechnology, santa cruz, ca, usa).dual-luciferase assay. the dual-luciferase kit was purchased from promega (promega corporation, usa). cells were transfected with pgl -ifn-␤-luc or pnf-b-luc plus prl-tk at the ratio of : or : as suggested by the manual. the detection was done by following the assay protocol in the kit. the firefly and renilla luciferase activities were read with a modulus microplate multimode reader (turner biosystems, sunnyvale, ca, usa).coimmunoprecipitation (co-ip) assay. the transfected cells were lysed with a lysis buffer containing mm tris-hcl (ph . ), mm nacl, mm edta, . % triton, g/ml leupeptin, g/ml aprotinin, and mm pmsf. about % of the lysate was subjected to input analysis. flag affinity gel (sigma, usa) or anti-myc and protein g plus agarose (santa cruz biotechnology, santa cruz, ca, usa) were added to the rest of the lysate ( %) and rotated overnight at °c. the reaction products were washed times with a wash buffer and then centrifuged at , rpm for min. the ip products were resuspended in the loading buffer and subjected to western blot analysis.elisa. cell culture supernatants were harvested h after transfection. the human ifn-␤ elisa kit was purchased from bluegene (shanghai, china). the reaction was carried out by following the manufacturer's instructions. the reaction products were detected with synogen (biotek, seattle, wa, usa) under nm.generation of sars-cov pseudovirus and assay of virus-induced ifn-␤ production. the generation of sars-cov pseudovirus followed the procedure described by huang et al. ( ) . briefly, hek cells were cotransfected with either the s, m, and n genes or the s, m mutant m(v a), and n of sars-cov. after h of transfection, the culture medium and the transfected cells were collected and subjected to freezing and thawing cycles at least four times. the reaction products were centrifuged at , rpm for min. the supernatant containing vlps was then applied to hek -ace stable cells and incubated for h. then, the cell culture medium was replaced with fresh medium. after h, the infected cells were subjected to total rna isolation and preparation of whole-cell lysates as described elsewhere. sars-cov-mediated ifn-␤ expression was monitored by standard western blotting, rt-pcr, and qrt-pcr.immunostaining assay. hela cells were transiently transfected with myc-m and flag-trap␥ ( ) . after h of transfection, the cells were fixed with % formaldehyde for min on ice and then incubated in ϫ phosphate-buffered saline (pbs) containing % bovine serum albumin (bsa) for h. the cells were first incubated with primary antibody rabbit anti-flag or mouse anti-myc for h. then, the secondary antibody fluorescein isothiocyanate (fitc)-labeled goat anti-mouse or tetramethyl rhodamine isocyanate (tritc)-labeled goat anti-rabbit antibody was added, and the mixture was incubated for another h. finally, the cells were incubated with =, -diamidino- -phenylindole (dapi) and sealed with glycerol. the results were observed under an olympus fv confocal microscope.statistical analysis. all values were calculated as means Ϯ standard deviations (sds) from three independent experiments. the statistical difference between the assayed group and the standard group was subjected to student's t test. the calculated difference was considered significant at the p value of Ͻ . . key: cord- -vnmc nqd authors: pfeiffer, julie k. title: is the debate and “pause” on experiments that alter pathogens with pandemic potential influencing future plans of graduate students and postdoctoral fellows? date: - - journal: mbio doi: . /mbio. - sha: doc_id: cord_uid: vnmc nqd nan m uch has been said and written about the risks and benefits of certain experiments that alter pathogens with pandemic potential (ppp) (see recent references to and references within). the experiments in question alter transmissibility, virulence, host range, drug susceptibility, immune responses, and/or infectivity or stability. a debate and research "pause" for certain experiments using certain respiratory viruses (influenza, middle east respiratory syndrome [mers] , and severe acute respiratory syndrome [sars] viruses) has electrified the scientific community, with strong "propause" advocates, strong "antipause" advocates, and everything in between. this letter is not about whether the experiments, debate, or pause is good or bad. this letter is about the potential impact of the debate and pause on graduate students and postdoctoral fellows and how their future plans may be affected. policy makers, ethicists, cognitive scientists, epidemiologists, public health experts, biosafety experts, bioterror experts, mathematical modelers, principal investigators, and others have expressed their opinions in multiple forums, including at a recent meeting hosted by the national academy of sciences ( to december [http://bit.ly/ o j y]). however, graduate students and postdoctoral fellows have not had a voice in this debate. furthermore, virtually nothing is known about how this debate and research pause has influenced trainees and their future plans. the lack of information is a concern, considering that graduate students and postdoctoral fellows are the ones physically performing many of the relevant experiments. furthermore, trainees are the scientists that will populate these fields in the future. to gain initial insight into how the debate and research pause have affected trainees, i created an informal survey days before the national academy of sciences meeting. my goal was simple: begin to gather data on trainee plans to stimulate discussion at the meeting and encourage future study on this topic. the poll is unofficial, relies on self-reporting, and may have a limited respondent pool, and it can be argued that the questions/wording/tone/ advertising need improvement. for full disclosure, i report that this survey was conceptualized and implemented over a -h period on a saturday while a beer was consumed. however, the results from this preliminary unofficial poll are worthy of discussion and future study. trainees were asked to complete a surveymonkey poll. as a "reward," i included a link to my "how to find a postdoctoral fellowship" tutorial, which students at ut southwestern have found helpful (http://bit.ly/ gsk x ). the poll was advertised via twitter (http://bit.ly/ bycimf and http://bit.ly/ qzyyya), the american society for virology facebook page (http://on.fb.me/ tkkau), and forwarded e-mails. the twitter announcement reached up to , people, counting followers of the people who retweeted the advertisement, making it reasonably publicly available. the poll (https://www.surveymonkey.com/s/b xjp b) had five questions about the respondent's background and research interests, with one question assessing knowledge level/ awareness about the debate. a paragraph (reproduced below) summarized the debate, and links to four articles ( - ) were given for additional information, followed by two questions assessing the potential impact of the debate and the research pause on future plans. scientists and policy makers are currently debating the pros and cons of performing certain types of experiments using certain pathogens. this debate largely focuses on pathogens with pandemic potential (or ppp), and the most relevant viruses are influenza, mers coronavirus, and sars coronavirus. in october of , the u.s. government implemented a "pause" for certain research projects involving influenza (biosafety level and strains), mers (biosafety level ), and sars (biosafety level ). these projects involve a subset of "gain of function" experiments designed to create mouse adapted viral strains, generate drug resistant viruses to understand drug mechanisms of action, understand host immunity by analyzing viruses with resistance to certain host immune pathways, and to study factors that influence transmission by the respiratory route (which was made famous by work from the kawaoka and fouchier labs in ). principal investigators of federally funded research projects were told to stop experiments specifically related to the "gain of function" work described above while risks and benefits could be examined. the future of these particular projects is uncertain. work continues on other projects using influenza, mers, and sars. complete poll results from december to december are available at the following urls: http://bit.ly/ gus px (summary of responses) and http://bit.ly/ c trmg (all individual responses/comments). there was interest in the poll; respondents completed the survey in the first days, with responding over the weekend. of respondents, % were ph.d. students and % were postdoctoral fellows. a majority, % of respondents, said that virology was their field of study, and % said that they currently work on a respiratory virus. other viral systems included enteric viruses ( %), "other" mammalian viruses (such as arboviruses, hiv, herpesviruses, and poxvirus [ %]), urogenital viruses ( %), eukaryotic/nonmammalian viruses ( . %), bacteriophages ( . %), and plant viruses ( . %). therefore, respondents study a diverse array of viral systems. importantly, % of respondents said that they want to work on a respiratory virus in the future, making this an area of potential growth. career goals among respondents were diverse: academic research faculty ( %), government research ( %), academic research scientist ( %), industry (large company [ %] and small company [ %]), policy or science writing ( . %), foundation ( . %), teaching ( . %), clinical laboratory ( . %), state public health laboratory ( . %), consulting or entrepreneurship ( . %), and technology transfer ( . %). respondents were knowledgeable about the debate; % said that they know a fair amount of information about the debate, % said that they have heard of the debate but do not know the details, and % said that they had not heard about the debate. after learning more about the debate, % said that they were more likely to work on influenza, sars, or mers virus in the future, % said that they were less likely to work on influenza, sars, or mers virus in the future, and % said that they were equally likely to work on influenza, sars, or mers virus in the future. an additional % of respondents said that their opinion was unchanged because they are not planning to work on a virus in the future. finally, for those interested in virology, % said that the debate and research pause have changed their future plans/research direction, % said that their plans have not changed, and % said that the debate and research pause have made them consider other factors in choosing their future plans/research direction. i have a few thoughts about the poll results. first, this was an informal poll developed by a poll-making novice (me) with a limited sample size, quasi-limited advertising, and a self-reporting format. it can be argued that some of the questions/answers/text could have been phrased differently. therefore, there is room for improvement in future surveys that would be developed ideally by trained individuals using something more sophisticated than sur-veymonkey. that said, the data generated by this poll suggest that this is an important topic worthy of follow-up and consideration. i invite others to improve upon this initial effort (see reference for an example). second, trainees are aware of the debate and research pause; % had heard about the debate. this was impressive to me, since many of my microbiology faculty colleagues were unaware of the debate until it was mentioned at a faculty meeting this month. perhaps i should not be surprised that trainees are well informed. after all, the millennial generation is the most connected, technologically savvy generation in history. third, the debate and research pause are influencing future plans of virology trainees. twenty-eight percent of respondents ( % of virologists [ trainees]) said that they are less likely to work on influenza, sars, or mers virus in the future. respiratory viruses are a press-ing global concern, and a potential loss of future investigators is a serious threat. i encourage policy makers to consider trainee impact and potential damage due to lost researchers in the influenza, mers, and sars virus fields. the potential effect of lost future investigators should be factored into current risk-benefit analyses undertaken during the pause. it would be worthwhile for those with expertise in surveys and forecasting to examine this topic with wellaccepted and -established methods to generate data on the potential future impact to the field of virology, particularly for respiratory viruses. for example, factoring in retirement rates and potential loss of future investigators, what will the influenza/ mers/sars virus fields look like in , , or ? additionally, trainees are stakeholders in this debate and should have representation in the discussions. finally, i remind trainees and others that this debate and research pause are affecting only a very small subset of experiments with a few respiratory viruses. there are many, many interesting projects and experiments that can and should be done. people on both sides of the debate agree that influenza, sars, and mers viruses are incredibly important human pathogens and should be studied using a variety of approaches. we need talented and devoted scientists studying a diverse array of viruses, including influenza, sars, mers, and other emerging viruses. many thanks go to the trainees that responded so quickly to this survey. good luck with your experiments and best wishes for your exciting careers! gain-of-function experiments: time for a real debate vagueness and costs of the pause on gain-of-function (gof) experiments on pathogens with pandemic potential, including influenza virus moratorium on research intended to create novel potential pandemic pathogens mbio addresses the pause in gain-of-function (gof) experiments involving pathogens with pandemic potential (ppp) a survey of attitudes and actions on dual use research in the life sciences: a collaborative effort of the national research council and the american association for the advancement of science my laboratory studies viral pathogenesis but not respiratory viruses. work in my laboratory has not been impacted by the funding pause or debate. i have funding from the national institutes of health, the american cancer society, and the burroughs wellcome fund. key: cord- -s s wb authors: noga, marek j.; büke, ferhat; van den broek, niels j. f.; imholz, nicole c. e.; scherer, nicole; yang, flora; bokinsky, gregory title: posttranslational control of plsb is sufficient to coordinate membrane synthesis with growth in escherichia coli date: - - journal: mbio doi: . /mbio. - sha: doc_id: cord_uid: s s wb every cell must produce enough membrane to contain itself. however, the mechanisms by which the rate of membrane synthesis is coupled with the rate of cell growth remain unresolved. by comparing substrate and enzyme concentrations of the fatty acid and phospholipid synthesis pathways of escherichia coli across a -fold range of carbon-limited growth rates, we show that the rate of membrane phospholipid synthesis during steady-state growth is determined principally through allosteric control of a single enzyme, plsb. due to feedback regulation of the fatty acid pathway, plsb activity also indirectly controls synthesis of lipopolysaccharide, a major component of the outer membrane synthesized from a fatty acid synthesis intermediate. surprisingly, concentrations of the enzyme that catalyzes the committed step of lipopolysaccharide synthesis (lpxc) do not differ across steady-state growth conditions, suggesting that steady-state lipopolysaccharide synthesis is modulated primarily via indirect control by plsb. in contrast to steady-state regulation, we found that responses to environmental perturbations are triggered directly via changes in acetyl coenzyme a (acetyl-coa) concentrations, which enable rapid adaptation. adaptations are further modulated by ppgpp, which regulates plsb activity during slow growth and growth arrest. the strong reliance of the membrane synthesis pathway upon posttranslational regulation ensures both the reliability and the responsiveness of membrane synthesis. activities have been proposed or identified. however, how each of these individual elements is used to regulate membrane synthesis during steady-state growth remains unclear ( ) . the issue of how membrane construction is coordinated with growth can be approached by considering how the metabolic pathways that synthesize membrane building blocks (pl and lps) are regulated. biosynthetic fluxes are regulated either by control of enzyme concentrations or by direct control of enzyme activity. examples of both forms of regulation can be readily found: in escherichia coli, the steady-state protein synthesis rate is controlled by ribosome concentration, which is transcriptionally regulated to balance amino acid supply with translation demand ( ) . in contrast, fluxes through central carbon metabolism are controlled by concentrations of substrates and inhibitors (i.e., posttranslational control) rather than by changes in enzyme concentration ( , ) . posttranslational control is also known to contribute to membrane synthesis regulation ( ) ( ) ( ) ( ) ( ) ( ) ; however, how cells use transcriptional and posttranslational control to coordinate membrane synthesis with growth has never been defined well. in order to understand how the model gram-negative species escherichia coli coordinates membrane synthesis with growth, we quantified substrates and enzymes of the fatty acid and pl synthesis pathways under both steady-state and dynamic conditions. we found that posttranslational control of the first enzyme in the pl synthesis pathway, plsb, is sufficient to ensure steady-state regulation of pl synthesis. in contrast, transcriptional regulation maintains stable enzyme concentrations regardless of growth rate. furthermore, due to feedback regulation of the fatty acid pathway, plsb exerts strong control over lps synthesis via its effects on concentrations of an lps fatty acid precursor. ( fig. d) . we also found no significant positive correlation between pl flux and plsb substrate c : -acp or c : -acp (pearson correlation coefficient r ϭ Ͻ . , two-tailed test of significance p ϭ Ͼ . ), while c : -acp concentrations correlated negatively with pl flux (r ϭ Ϫ . , p Ͻ . ) ( fig. e ; see also table ). although g p concentrations were found to correlate with pl flux (r ϭ Ͼ . , p ϭ Ͻ . ), growth in glycerol medium increased g p by -fold without increasing pl abundance or flux. the absence of any significant positive correlation between acp substrate concentrations and pl flux indicates that steady-state pl synthesis is not controlled by substrate concentrations. in contrast to pl precursors, concentrations of the fatty acid initiation and elongation substrate malonyl-acp correlated with pl flux (fig. e ; r ϭ . , p ϭ Ͻ . ) (table ), while the concentrations of most saturated acp species remained relatively constant (see fig. s in the supplemental material), suggesting that the fatty acid initiation and elongation reactions are controlled primarily by malonyl-acp concentrations and thus by the rate of malonyl-acp synthesis by acc and fabd. concentrations of c : -oh-acp, the lipid precursor of lps, also correlated with pl flux (r ϭ . , p ϭ Ͻ Ϫ ), suggesting that pl synthesis is naturally coupled with lps synthesis via concentrations of c : -oh-acp (fig. e ). concentrations of holo-acp, the most abundant acp species, declined with increasing pl flux (r ϭ Ϫ . , p ϭ Ͻ . ) (table ) , as levels of fatty acid synthesis intermediates increased due to increased fatty acid flux. as with substrates, positive correlations between enzyme concentrations and reaction flux may indicate that a reaction rate is controlled by enzyme concentrations, i.e., via transcriptional control. we measured concentrations of fatty acid synthesis pathway enzymes using lc/ms to determine whether fatty acid flux is coupled with by transcription-based regulation. while concentrations of one of the four acc subunits (acca) and several of the fatty acid synthesis pathway enzymes correlated with pl flux (table ; see also fig. s ), the increases were small ( %) relative to the pl flux increase ( -fold). we infer that the rate of malonyl-acp synthesis and thus the rate of fatty acid synthesis are determined primarily by posttranslational control of acc. the negative correlation between c : -acp and pl flux is consistent with long-chain acp inhibiting malonyl-coa synthesis by acc via negative-feedback inhibition ( ) . unlike long-chain acp, the concentrations of all pl synthesis intermediates downstream of plsb were found to correlate positively with pl flux (fig. f ; r ϭ . to . ) ( table ; see also fig. s ). the close match between the increase in the concentration and the level of pl flux is consistent with the rates of these reactions being under substrate control. the contrast between the trends in concentrations of the substrates and the products of plsb implies that plsb activity alone controls pl synthesis. plsb activity might be regulated either via transcriptional control of plsb concentration or via posttranslational control of plsb activity. transcription of plsb is controlled by the membrane stress-activated sigma factor rpoe ( ) and by the -sensitive regulator guanosine tetraphosphate (ppgpp) ( ) . concentrations of ppgpp are inversely correlated with (fig. d) , implying that pl flux may be coupled to by transcriptional control of the plsb gene by ppgpp. however, we found no correlation between pl flux and plsb concentration (table , p ϭ Ͼ . ). instead, plsb concentrations were nearly constant across all conditions studied (fig. g ). the invariance of plsb concentrations over a -fold range of pl flux indicates that plsb activity is regulated mainly via posttranslational control. unexpectedly, concentrations of lpxc, which catalyzes the committed step in the lps pathway ( ), also did not correlate with pl flux (table , p ϭ Ͼ . ) and also remained stable (fig. g) , indicating that changes in lps flux between steady-state growth conditions are not driven by changes in lpxc concentrations. can be modulated independently of nutrient conditions by artificially altering levels of ppgpp. titrating ppgpp above basal concentrations, but below the concentrations that occur during starvation, reduces steady-state by reducing rrna synthesis ( ) . furthermore, plsb activity is inhibited at the high ppgpp concentrations observed during amino acid starvation ( ) . we titrated by expressing the catalytic domain of the ppgpp synthesis enzyme rela (rela*) from an inducible promoter (p tet ) in glucose medium. as previously observed ( ) , rela*-titrated cultures exhibited ppgpp concentrations that were elevated -fold above those seen with wild-type cultures growing at similar rates (fig. d ). trends in pl abundance, pl flux, acp species, pl intermediates, and pl synthesis enzyme concentrations in ppgpp-limited cultures closely followed the trends observed when was adjusted by carbon source (fig. b , c, and e to g) (see also fig. s , s , and s ). notably, despite ppgpp regulation of plsb transcription, concentrations of plsb also did not substantially vary with increasing concentrations of steady-state ppgpp. interestingly, lpxc concentrations decreased with increasing pl flux under these conditions (fig. g) . the trends in substrate, enzyme, and intermediate concentrations observed in ppgpp-limited cultures are consistent with growth regulating pl flux via posttranslational control-not transcriptional control-of plsb. mathematical modeling supports plsb control of steady-state pl and lps synthesis. to test whether regulation of plsb activity is sufficient to control steady-state pl synthesis, we constructed a simplified differential equation model that describes fatty acid, lps initiation, and pl biosynthesis ( fig. a ; see also text s and table s ). the model includes competitive inhibition of acc by both c : -acp and c : -acp as the sole regulatory interactions. the model also includes a branch point at c : -oh-acp into lps synthesis. lps initiation is represented in the model by a single step that combines reactions catalyzed by lpxa and lpxc, as lpxc catalyzes the first irreversible reaction in the lps pathway. concentrations of g p and c : -acp are fixed in the model to reflect experimental observations of plsb saturation by g p and invariance of c : -acp concentrations, respectively (see text s in the supplemental material for model details, parameter sets, and sensitivity analyses). we used the model to identify enzymes and substrates that exert control over pl and lps synthesis. increasing either acc or plsb v max by -fold increased simulated pl and lps fluxes in parallel by -fold (fig. b) , while changing v max of all other enzymes in the simulated pathway had little or no effect on simulated pl flux. the insensitivity of pl flux to v max of all fatty acid and pl synthesis enzymes aside from acc and plsb suggests that the experimentally observed positive correlations between enzyme concentrations and pl flux did not actually result in increased pl flux. the unexpectedly strong control of lps flux by plsb is due to the natural coupling between pl flux and concentrations of the lps synthesis substrate c : -oh-acp. changes in c : -oh-acp dehydration/reduction v max (catalyzed in e. coli by fabz and fabi, respectively) and lps initiation v max (catalyzed by lpxa and lpxc) exert strong and opposing forms of control over lps flux. however, lpxc and fabz do not exert significant control over pl synthesis, indicating that substantial flux can be diverted into the lps pathway without depleting that required for pl synthesis. of the two substrates considered in the model (acetyl-coa and c : -acp), only acetyl-coa affects pl and lps flux (fig. b) . fig. ). line plots are offset to prevent overlap. differential equations and parameters of the simulation are provided in text s and table s . to determine which of the two enzymes with nonnegligible levels of pl flux control-acc or plsb-actually controls pl flux during steady-state growth, we compared the predicted trends in substrate concentrations caused by simulated v max variations against experimentally observed trends. the predicted trends driven by acc and plsb v max variation closely reproduce most experimentally observed trends in both fatty acid and pl intermediate concentrations (fig. c ). however, variations in acc and plsb v max cause opposing trends in predicted concentrations of plsb substrates c : -acp and c : -acp, allowing acc regulation to be distinguished from plsb-based regulation. increasing acc v max causes c : -acp and c : -acp concentrations to increase with pl flux, which contradicts our experimental observations. however, increasing plsb v max is predicted to decrease c : -acp and c : -acp concentrations, a trend that better follows the experimentally observed trends (fig. c) . we therefore conclude that pl synthesis is primarily regulated by plsb during steady-state growth. our model also suggests that plsb control over steady-state lps flux (achieved by its tight control over c : -oh-acp concentrations) may be sufficient to couple lps synthesis with growth as well. translation inhibition causes carbon overflow into fatty acid synthesis. we set out to evaluate whether a known regulator of pl synthesis, ppgpp, is able to directly regulate plsb activity during steady-state growth. high concentrations of ppgpp lead to plsb inhibition in vivo ( ) , although it remains unclear whether this inhibition acts via a ppgpp-plsb interaction or via an indirect route. low concentrations of ppgpp correlated inversely with (fig. d ). the notion that ppgpp might directly control pl synthesis even at the low basal concentrations present during steady-state growth was proposed previously but never tested ( ) . levels of acyl-acp substrates of plsb increased as ppgpp was titrated upwards with rela* whereas the level of the product of plsb (lpa) decreased, consistent with ppgpp inhibiting flux into the pl pathway ( fig. e and f) and closely following simulated variations in plsb v max (fig. c) . therefore, changes in driven by substringent concentrations of ppgpp must somehow influence steady-state plsb activity. however, the mode of control by ppgpp (transcriptional control of plsb, posttranslational inhibition of plsb, or indirect control via an unknown regulator of plsb) cannot be determined from steady-state data alone. we first wished to observe the effects of high concentrations of ppgpp on the fatty acid and pl synthesis pathways. synthesis of high concentrations of ppgpp by rela (the stringent response) is triggered by any stress or starvation conditions that lead to the specific biochemical cue of uncharged trna bound to the ribosome. during the stringent response, ppgpp accumulates by more than -fold over basal concentrations and pl synthesis rates are reduced by approximately half ( , ) , likely due to plsb inhibition ( ) . we triggered ppgpp synthesis by adding the trna aminoacylation inhibitor mupirocin to glucose cultures of wild-type e. coli. to distinguish effects of ppgpp from effects of translation inhibition, mupirocin was also added to a culture of Δrela e. coli. mupirocin caused a -fold accumulation of ppgpp in the wild-type strain within min to over pmol/od, reaching Ͼ pmol/od after min (fig. s ) . unexpectedly, mupirocin treatment also transiently increased concentrations of malonyl-acp and all hydroxyl-acp species at the expense of holo-acp in both strains ( c : -acp accumulation was followed in turn by an increase in holo-acp and a decrease in malonyl-acp levels, consistent with acc inhibition (fig. a) . pl intermediates phosphatidic acid (pa), ps, and pgp were rapidly depleted in the wild-type strain after briefly increasing in abundance (fig. b ). the transient carbon influx briefly shifts the population of the acyl chains incorporated into pl toward longer-chain fatty acids, likely due to increased malonyl-acp concentrations favoring fatty acid elongation over membrane incorporation by plsb and plsc (fig. s ). while suppression of fatty acid synthesis in the wild-type strain can be attributed to ppgpp, it is unclear what had attenuated the carbon influx in the Δrela strain; however, c : -acp accumulation after min likely contributed to acc inhibition. inhibition of fatty acid elongation also depleted the lps precursor c : -oh-acp, which is expected to decrease lps synthesis in parallel with pl synthesis (fig. a) . as malonyl-acp levels increased transiently after mupirocin addition in both the wild-type and Δrela strains, we hypothesized that translation inhibition somehow diverts carbon into lipid synthesis. we added the ribosome inhibitor chloramphenicol and the transcription initiation inhibitor rifampin to glycerol cultures of wild-type e. coli. both compounds inhibit translation via mechanisms that suppress ppgpp synthesis. as with mupirocin treatment of the Δrela strain, chloramphenicol triggered a rapid decrease in holo-acp and an increase in long-chain unsaturated acp species c : -acp and c : -acp levels (fig. c ). both antibiotics triggered an increase in the levels of pl synthesis intermediates pa and ps that resembled the response of the Δrela strain to mupirocin (fig. d) . what might have caused the transient increase in fatty acid synthesis observed after translation inhibition? interestingly, addition of both rifampin and chloramphenicol increased acetyl-coa concentrations in glycerol cultures by -fold (fig. e) , suggesting a possible cause. acetyl-coa concentrations also increased in both wild-type and Δrela strains after mupirocin treatment in glucose medium, before decreasing ϳ % in the wild-type strain (fig. s ) . while it is unclear why translation inhibition would increase acetyl-coa concentrations, the observed response of the fatty acid pathway is consistent with our mathematical model, which predicts fatty acid flux to be highly sensitive to changes in acetyl-coa concentrations (fig. b) . we confirmed the sensitivity of the fatty acid and pl synthesis pathways to environmental changes using a fast nutritional upshift. addition of glucose and amino acids to a glycerol culture also caused rapid accumulation of pa and ps species that resembled the increases observed following translation inhibition (fig. s ) . moderate to high concentrations of ppgpp regulate pl synthesis via posttranslational control. in order to clearly discern the effects of ppgpp on the fatty acid and pl synthesis pathways without complications introduced by translation inhibition, we monitored the fatty acid and pl synthesis pathways immediately after inducing rela*. the responses of the fatty acid and pl synthesis pathways were again consistent with plsb inhibition causing long-chain acp to accumulate, which depletes malonyl-acp by inhibiting acc (fig. a) . pl intermediates also responded in a manner consistent with plsb inhibition: levels of lpa species steadily decreased, followed by pa, ps, and pgp (fig. b) . addition of chloramphenicol min following rela* induction caused an increase in unsaturated long-chain acp levels, though ppgpp appears to attenuate the response of the pl pathway to translation inhibition. we next sought to evaluate whether ppgpp concentrations far below those occurring during the stringent response would also be capable of regulating plsb via posttranslational control. as the effects of posttranslational control can be distinguished from those of transcriptional regulation by response time, we closely followed the dynamics of the pl pathway after mildly inducing ppgpp synthesis with a low doxycycline concentration. ppgpp concentrations increased within min of rela* induction and reached the elevated steady-state concentration (ϳ pmol/od) by min. concentrations of ps begin to decrease within min (fig. c) , consistent with posttranslational inhibition of pl synthesis. to verify that the immediate ps depletion had occurred too quickly to be explained by transcriptional regulation, we compared the ps response kinetics with the kinetics of a ppgpp-driven response known to be mediated by transcriptional control. cyclopropyl pl is produced from unsaturated fatty acids of membrane pl by the activity of the enzyme cfa, expression of which is induced via transcriptional control by ppgpp ( ) . cyclopropyl pl began to accumulate min economy of membrane synthesis in e. coli ® after rela* induction, well after the ps decrease was established. we conclude that ppgpp concentrations well below stringent response concentrations inhibit plsb via a posttranslational mechanism. over several decades, biochemical and genetic research has extensively characterized the pathways and the enzymes that produce the building blocks of bacterial membranes ( , , ) . many control mechanisms acting on both the transcriptional and posttranslational levels have been proposed to maintain membrane homeostasis. for instance, each of the enzymes acc ( ), fabh ( ), fabz ( ), fabi ( ), lpxc ( ) , and plsb ( ) have been suggested to contribute to membrane synthesis regulation. however, the existence of a control mechanism does not necessarily indicate that it is used to regulate total membrane synthesis flux during steady-state growth. furthermore, models derived from biochemical and genetic studies can overlook selfregulating mechanisms that occur automatically in a metabolic pathway, such as the coupling of c : -oh-acp concentrations to pl flux. the primary value of our work is that it reveals the regulatory mechanisms that are actually used by growing e. coli cells to regulate membrane biosynthesis. both our data and our modeling indicate that only two of the many control mechanisms previously identified are sufficient to regulate total pl biosynthesis during steady-state growth. specifically, we found that cellular demand for membrane synthesis is communicated to plsb via an undiscovered posttranslational mechanism. in turn, plsb activity regulates fatty acid synthesis by consuming long-chain acp species, relieving feedback inhibition of acc ( ) and increasing fatty acid flux. this demand-level flux regulation is consistent with metabolic control theory ( ) . our measurements of enzyme concentrations indicate that pl flux is regulated primarily via posttranslational control of enzyme activity, rather than via transcriptional regulation. the concentrations of the pathway enzymes with the greatest control over pl flux (acc and plsb) are maintained at nearly invariant levels across a -fold range of . although it has been long accepted that plsb activity is adjusted via posttranslational control and not via transcriptional control ( ), the strong flux control of acc ( ) and the growth-regulated transcription of the accbc, acca, and accd genes ( ) inspired the suggestion that pl flux might be coupled with via transcriptional regulation. instead, we found that transcriptional regulation of fatty acid and pl synthesis genes stabilizes the concentrations of fatty acid and pl synthesis enzymes across . the correlations between concentrations of several enzymes (e.g., fabf) and pl flux that we observed are unlikely to increase total pl flux but rather likely regulate aspects of membrane metabolism aside from pl flux, such as membrane fluidity. maintaining a stable membrane synthesis capacity enables the cell to quickly respond to any change in membrane demand at the cost of expressing enzymes that remain less active at low or moderate ( ) . our data are also essential for evaluating proposed models of lps synthesis regulation. most importantly, both our model and our experiments indicate that pl fluxand thus, plsb activity-controls concentrations of lps precursor c : -oh-acp. this tightly links fluxes into the pl and lps synthesis pathways, as varying pl synthesis by adjusting either plsb or acc activity would naturally vary lps synthesis in parallel. surprisingly, we found that flux into the lps synthesis pathway is not adjusted by variations in lpxc concentrations, as lpxc concentrations remain constant despite a -fold change in . lps flux may be varied instead by fabz, concentrations, which increased by % over the -fold range of sampled (see fig. s in the supplemental material). our model predicts that this would attenuate the effects of increasing c : -oh-acp concentrations caused by elevated fatty acid synthesis and divert flux away from lps synthesis without affecting pl flux. lpxc concentrations may be stabilized across by degradation by the ftsh protease ( ) . degradation of lpxc by ftsh might be used as an emergency brake to rapidly halt lps synthesis during growth arrest or growth transitions to prevent accumulation of toxic lps intermediates. com-prehensive characterization and modeling ( ) of lps substrates and enzymes across several steady-state conditions, as we have done for pl synthesis, will be necessary to determine how lps synthesis is varied. although plsb determines pl flux during steady-state growth, this control is not absolute, as fatty acid and pl flux (and likely lps flux) are also sensitive to acetyl-coa concentrations and acc activity. the sensitivity of membrane synthesis to protein synthesis arrest demonstrates the responsiveness of membrane synthesis to carbon metabolism. while this sensitivity facilitates rapid adaptation of pl flux to environmental changes, the tight connection between protein synthesis, central carbon metabolism, and membrane biogenesis demands additional regulation (including inhibition by ppgpp) to rebalance the pathway with and prevent pl overflow. if protein synthesis were inhibited in a manner that does not decrease carbon flow into the fatty acid pathway (e.g., by nitrogen starvation), continued pl and lps synthesis would outpace synthesis of the lipoproteins that tether the outer membrane to the peptidoglycan layer. inhibition of pl and lps pathways by ppgpp would thus prevent production of excess membrane, enforcing the coupling of pl and lipoprotein synthesis. consistent with this notion, Δrela strains generate higher quantities of extracellular pl and lps, likely as outer membrane vesicles ( ) . identifying the specific signal that controls plsb will reveal how membrane synthesis is synchronized with growth. but what controls plsb? posttranslational control of plsb by moderately high concentrations of ppgpp (Ն pmol/od) at least partly contributes to steady-state membrane synthesis regulation, but likely only during nutritional downshifts, stress, or very slow steady-state growth ( Ͻ . h Ϫ ) when such concentrations are achieved. whether basal ppgpp concentrations observed during fast growth ( Ͼ . h Ϫ , ppgpp Ͻ pmol/od) also regulates pl synthesis via plsb control remains unclear. in vivo experiments such as ours cannot determine the mechanism by which ppgpp inhibits plsb. although it was demonstrated previously that high concentrations of ppgpp directly inhibit plsb ( ) , multiple groups were unable to observe ppgpp inhibition of plsb in vitro when acyl-acp was used as a substrate ( , ) . ppgpp may therefore inhibit plsb indirectly via a regulator or a cellular process that interacts with plsb. immunoprecipitation experiments revealed several proteins that interact with plsb, including acp and pssa, as well as some (plsx and ybgc) whose roles are unclear ( ) , suggesting that plsb forms part of a pl synthesis complex that may couple plsb activity with . as pl synthesis flux has been observed to oscillate with the cell division cycle in e. coli and other bacteria ( ) , plsb may also be regulated by the divisome or by septum formation. degradation of pl by the activity of phospholipases may also play an important role in membrane homeostasis during steady-state growth ( ) , as is known to occur during growth and division in eukaryotes ( ) . in addition to identifying the allosteric regulators of plsb, studies that integrate connections between pl catabolism and transport with pl synthesis are needed for a comprehensive understanding of membrane homeostasis. culture conditions. cultures were grown in -ml to , -ml erlenmeyer flasks filled to % of nominal volume with mops (morpholinepropanesulfonic acid) minimal medium ( ) with . mm nh cl or nh cl and . % (wt/vol) carbon source (acetate, succinate, malate, glycerol, glucose, u- c-glucose [cambridge isotope laboratories], or glucose supplemented with . % cas-amino acids). culture flasks were placed in a grant instruments sub aqua pro dual water bath at °c and agitated by stirring with a -mm-long magnetic stir bar (vwr), coupled to a magnetic stir plate ( mag mixdrive eco and mixcontrol ) set at , rpm. growth was monitored by optical density measurement at nm using an ultrospec cell density meter (ge healthcare). samples for acp, lipid analysis, and proteomics were collected using cultures without isotopic labeling. samples for nucleotide phosphate measurements were collected from u- n-labeled cultures, and samples for g p measurements were collected from u- c-labeled cultures. strains and plasmid prela*. all experiments were performed using escherichia coli k- strain ncm (cgsc catalog no. ) and its derivatives. ncm rela::kan was constructed by p phage transduction using strain cf [mg Δlac (rph ϩ ) rela ::kan] as a donor. plasmid prela* was created by cloning dna encoding residues to from the e. coli rela protein into bglbrick plasmid economy of membrane synthesis in e. coli ® pbbs k ( ) (sc * origin of replication, p tet promoter, kanamycin resistance). the fluorescent protein mvenus was fused by restriction-digestion to the c terminus of rela via a glycine-serine linker. metabolite sampling. samples for acp, proteomics, and lipid analysis were acquired by fast quenching of ml of culture sample into l of ice-cold % trichloroacetic acid. after min incubation at °c, cells were pelleted by centrifugation and stored at Ϫ °c until analysis. for analysis of nucleotide phosphates and polar metabolites, samples were acquired by the use of a modified fast vacuum filtration method ( ) . a -ml volume of culture was collected by vacuum on a prewetted . -cm-diameter . -m-pore-size hv durapore membrane filter. after rapid collection, the filter was immediately placed upside down in quenching solution. for measurement of the nucleotide phosphates, ml of ice-cold m formic acid was used with l internal standards (is) mix as a quenching solution, which was subsequently neutralized by the use of l of % ammonium hydroxide. for g p measurements, ml of a : : (vol/vol/vol) mixture of methanol, acetonitrile, and water with . % formic acid with l internal standard solution (cooled on dry ice) was used as a quenching solution. after min of incubation, cells were washed from the filter, transferred to a tube, and stored at Ϫ °c until analysis. preparation of internal standards. isotopically labeled internal standards (is) were used to control for sampling and measurement variation. for acp and proteomics assays, u- n e. coli whole-cell extracts were prepared using a mops minimal medium culture with nh cl as the sole nitrogen source. at od of ϳ . , % trichloroacetic acid (tca) was added at a : ratio to the culture to facilitate quenching of metabolism. after min of incubation on ice, -ml single-use is aliquots were collected by centrifugation and stored at Ϫ °c until the sample preparation step. for the phospholipid measurement, u- c lipid extract was prepared using a culture grown in minimal mops medium with . % u- c glucose as the sole carbon source. at od of ϳ . , % tca was added at a : ratio to the culture and insoluble cell material was collected by centrifugation after min of incubation on ice. pellets were resuspended in a mixture consisting of l meoh, l mm citric acid- mm dipotassium phosphate buffer, and l of methyl-t-butyl ether per ml of initial culture volume. after vortex mixing and min of sonication, phase separation was induced by addition of l/ml of mm citric acid/ mm dipotassium phosphate buffer. after further vortex mixing, sonication and min of incubation at room temperature, the phases were separated by min of centrifugation at , rpm at room temperature. the upper phase was collected, placed in a glass vial, and stored at Ϫ °c until the sample preparation step. instrumentation. all lc/ms runs were performed using a agilent lc/ms system consisting of a binary pump (g b), an autosampler (g a), a temperature-controlled column compartment (g a), and a triple-quadrupole (qqq) mass spectrometer (g c) equipped with a standard electrospray ionization (esi) source, all operated using masshunter data acquisition software (version . ). a mass spectrometer was operated in dynamic multiple-reaction monitoring (mrm) mode using transitions generated in silico by the use of a script written in python, an rdkit library, and chemical structures of the target compound as the input. transitions for targeted proteomics assays were developed using skyline ( ) based on protein sequences from the uniprot database. lc/ms quantification of acp intermediates. acyl-acp levels were measured using a published method ( ) with minor modifications. lysis buffer was prepared by suspending an appropriate number of frozen u- n-labeled e. coli pellets in ml of a mixture consisting of mm potassium phosphate buffer (ph . ), m urea, mm n-ethyl-maleimide, mm edta, and mm ascorbic acid. a -ml volume of lysis buffer was added to each of the preparations of tca-quenched and pelleted cells, and proteins were isolated by chloroform/methanol precipitation as described previously. protein pellets were resuspended in l of digestion buffer ( % -octyl-glucoside- mm potassium phosphate buffer, ph . ) and, after addition of l of . mg/ml gluc protease (promega), incubated overnight at °c. after quenching was performed by addition of l meoh, samples were centrifuged and l was injected in an lc/ms system. separation was performed on a csh c column (waters) ( . mm by mm, . -m pore size) held at °c using the following binary gradient: % b, -min ramp to %, -min increase to %, and -min hold at % b followed by min of reequilibration under the starting conditions (a, mm formic acid; b, mm formic acid) at a flow rate of . ml/min. lc/ms quantification of phospholipids. the sample preparation procedure used for the phospholipids consisted of a combination of an methyl tert-butyl ether (mtbe) extraction method ( ) and an established lc/ms method ( ) . pelleted e. coli cells were resuspended in a mixture containing l of meoh, l of u- c e. coli extract prepared as described above, and l of mtbe. after vigorous vortex mixing and sonication, l of mm citric acid- mm dipotassium phosphate buffer was added to homogenized pellets. following further vortex mixing and min of incubation at room temperature, liquid phases were separated by centrifugation for min at , ϫ g. a -l volume of the upper phase was moved to a new tube and dried in a vacuum centrifuge (labconco). dried lipid films were resuspended in l of : : (vol/vol/vol) isopropanol/acetonitrile/h o, supplemented with mm acetylacetone. after resuspension, l h o was added to reduce the organic content of the buffer and l of the resulting mixture was injected into the lc/ms system. separation was performed on a csh c column (waters) ( . mm by mm, . -m pore size) at °c with a flow rate of . ml/min using the following binary gradient: % b, ramp to % b in min followed by a linear increase to % b in min, -min hold at % b, and min reequilibration (a, . % nh oh-water; b, . % nh oh- % isopropanol- % acetonitrile [acn]). lc/ms quantification of nucleotides. frozen cell extracts were defrosted by to min of incubation in a °c water bath and sonicated for min in a water-ice slurry. after min of centrifugation at , ϫ g, samples were loaded on a ml/ mg oasis wax cartridge (waters) preconditioned with ml of meoh and ml mm ammonium acetate buffer (ph . ). after washing with ml ammonium acetate buffer was performed, analytes were eluted with l of . % ammonium hydroxide-meoh/acn/h o : : (vol/vol/vol). after addition of l of % trehalose and brief vortex mixing, the samples were dried in a vacuum centrifuge (labconco). dried trehalose-stabilized extracts were redissolved in l of meoh/acn/h o ( : : [vol/vol/vol]) and moved to an autosampler vial for analysis. separation was performed on an ihilic column (hilicon) ( . mm by mm, . -m pore size) or a sequant zic-chilic column (merck) ( . mm by mm, -m pore size) at . ml/min using the following binary gradient: % b, ramp to % b in . min followed by min of isocratic hold at % b and a linear decrease to % b in min with a -min hold at % b and min reequilibration under the initial conditions (a, . mm ammonium acetate- . mm acetic acid- mm acetylacetone-milli-q water, b, . mm ammonium acetate- . mm acetic acid- mm acetylacetone- % acn). the injection volume was l. lc/ms quantification of g p. stored metabolite extracts were dried down in a vacuum centrifuge (labconco), redissolved in l of meoh:acn:h o ( : : [vol/vol/vol]), and moved to an autosampler vial for analysis. separation was performed on an ihilic column (hilicon) ( . mm by mm, . -m pore size) at . ml/min using the following binary gradient: % b, ramp to % b in min followed by linear decrease to % b in min, -min hold at % b, and min reequilibration. the injection volume was l. lc/ms targeted protein quantification. relative concentrations of enzymes were measured by targeted proteomics using a modified version of the acp assay. lysis buffer was prepared by suspending an appropriate number of frozen u- n-labeled e. coli pellets in ml of mm potassium phosphate buffer (ph . )- m urea. to improve the detection of peptides from plsb and lpxc, u- n-labeled e. coli ncm strains overexpressing plsb and lpxc were used as internal standards. lpxc was overexpressed using the corresponding plasmid from the aska library ( ) (national bioresource project [nig, japan]). a -ml volume of lysis buffer was added to each of tca-quenched and pelleted cells, and proteins were isolated by a chloroform/methanol precipitation as described previously. protein pellets were resuspended in l of digestion buffer [ % -octyl-glucoside- mm tris buffer (ph . ) supplemented with mm cacl and mm tris( -carboxyethyl)phosphine hydrochloride (tcep)]. alkylation of cysteine residues was performed by adding l of mm iodoacetamide followed by min of incubation in darkness. digestion was performed by adding l of . mg/ml trypsin gold (promega) followed by overnight incubation at °c. samples were centrifuged, and l of the reaction volume was injected in an lc/ms system. separation was performed on a csh c column (waters) ( . mm by mm, . -m pore size) held at °c using a binary gradient: % b, min ramp to % b, min increase to % b, . ramp to %, and -min hold at % b before min of reequilibration under the starting conditions (a, mm formic acid; b, mm formic acid) at a flow rate of . ml/min. data analysis. all lc-ms data files were processed in versions of skyline ( .x) using a target list chosen on the basis of an in silico-generated transition list. each target compound had a matching isotopically labeled internal standard (is). processed data were exported as target compounds and is peak areas and processed further using a set of python scripts. growth rates were obtained from linear fits to log-transformed growth curves. od-corrected data were obtained by dividing the signal by the od value interpolated from the growth curve at the time of sampling. pe-corrected results were produced by dividing the signal by the sum of all of the signals for all phosphatidyl-ethanolamine species from the same measurement (in the case of the phospholipids) or matching sample (in the case of the other assays). in the nucleotide phosphate and g p assays, absolute concentrations were estimated based on amounts of internal standards in is-spike solution by assuming that rr ϭ implies equimolar amounts of target compound and is at the moment of quenching. correlations in table were calculated from log( )-normalized concentrations and pl fluxes using the descriptive statistics function (originpro v. ). mathematical modeling. the computational model was constructed and tested using copasi version . ( ) . full details of the model are described in text s in the supplemental material. results from sensitivity analysis are included in fig s . data availability. mass spectrometry data have been deposited in the embl-ebi metabolights database (https://academic.oup.com/nar/article/ /d /d / ; pmid ) with the identifier mtbls . the complete data set can be accessed here: https://www.ebi.ac.uk/metabolights/ mtbls . (the authors could not make this metabolights record available at the time of this paper's publication due to circumstances related to the covid- pandemic, but it will be made accessible as soon as possible after publication.) supplemental material is available online only. text s , docx file, . mb. outer membrane of salmonella typhimurium: accessibility of phospholipid head groups to phospholipase c and cyanogen bromide activated dextran in the external medium asymmetric localization of lipopolysaccharides on the outer membrane of salmonella typhimurium murein-lipoprotein of escherichia coli: a protein involved in the stabilization of bacterial cell envelope a retrospective: use of escherichia coli as a vehicle to study phospholipid synthesis and function interdependence of cell growth and gene expression: origins and consequences transcriptional regulation is insufficient to explain substrate-induced flux changes in bacillus subtilis systems-level analysis of mechanisms regulating 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(plsb) regulation of phospholipid synthesis in escherichia coli by guanosine tetraphosphate regulation of membrane phospholipid synthesis by the rela gene: dependence on ppgpp levels effect of ppgpp on escherichia coli cyclopropane fatty acid synthesis is mediated through the rpos sigma factor (sigmas) enzymology, genetics, and regulation of membrane phospholipid synthesis in escherichia coli bacterial membrane lipids: where do we stand? overproduction of acetyl-coa carboxylase activity increases the rate of fatty acid biosynthesis in escherichia coli inhibition of ␤-ketoacyl-acyl carrier protein synthase iii (fabh) by acyl-acyl carrier protein in escherichia coli mutants resistant to lpxc inhibitors by rebalancing cellular homeostasis enoyl-acyl carrier protein reductase (fabi) plays a determinant role in completing cycles of fatty acid elongation in escherichia coli ftsh-mediated coordination of lipopolysaccharide biosynthesis in escherichia coli correlates with the growth rate and the alarmone (p) molecular biology of bacterial membrane lipids regulating the cellular economy of supply and demand growth rate regulation of escherichia coli acetyl coenzyme a carboxylase, which catalyzes the first committed step of lipid biosynthesis lessons on enzyme kinetics from quantitative proteomics balanced biosynthesis of major membrane components through regulated degradation of the committed enzyme of lipid a biosynthesis by the aaa protease ftsh (hflb) in escherichia coli crosstalk between the lipopolysaccharide and phospholipid pathways during outer membrane biogenesis in escherichia coli relation between protein synthesis and phospholipid synthesis and turnover in escherichia coli the involvement of guanosine -diphosphate- -diphosphate in the regulation of phospholipid biosynthesis in escherichia coli. lack of ppgpp inhibition of acyltransfer from acyl-acp to sn-glycerol -phosphate regulation of fatty acid synthesis during the cessation of phospholipid biosynthesis in escherichia coli a protein network for phospholipid synthesis uncovered by a variant of the tandem affinity purification method in escherichia coli lipid synthesis during the escherichia coli cell cycle the escherichia coli phospholipase plda regulates outer membrane homeostasis via lipid signaling mechanisms of glycerophospholipid homeostasis in mammalian cells culture medium for enterobacteria bglbrick vectors and datasheets: a synthetic biology platform for gene expression systematic identification of allosteric protein-metabolite interactions that control enzyme activity in vivo skyline: an open source document editor for creating and analyzing targeted proteomics experiments lipid extraction by methyl-tert-butyl ether for high-throughput lipidomics lpp mediates self-generation of chemotactic lpa gradients by melanoma cells complete set of orf clones of escherichia coli aska library (a complete set of e. coli k- orf archive): unique resources for biological research copasi -a complex pathway simulator we thank michael cashel for providing e. coli strain cf . we thank christophe danelon, bertus beaumont, and frank bruggeman for invaluable suggestions for improving the manuscript. we thank the national bioresource project (nig, japan) for sharing the lpxc overexpression plasmid. this research was funded by a grant to g.b. from the netherlands organisation for scientific research (alw open . . ) and an award from the frontiers of nanoscience program.we key: cord- -x wqtlxh authors: fedson, david s.; opal, steven m.; rordam, ole martin title: hiding in plain sight: an approach to treating patients with severe covid- infection date: - - journal: mbio doi: . /mbio. - sha: doc_id: cord_uid: x wqtlxh patients with covid- infection are at risk of acute respiratory disease syndrome (ards) and death. the tissue receptor for covid- is ace , and higher levels of ace can protect against ards. angiotensin receptor blockers and statins upregulate ace . clinical trials are needed to determine whether this drug combination might be used to treat patients with severe covid- infection. t he severe respiratory disease that has recently emerged in china is caused by a novel coronavirus (covid- ) ( ). the virus is similar to the sars coronavirus that spread internationally in , infected more than , people, and killed almost . infection with covid- has now spread throughout the world, causing widespread social and economic disruption. to control its spread, chinese officials have imposed extensive travel bans and quarantined large areas. accelerated development of new vaccines and treatments is already under way. it is too early to know whether any of these efforts will contain the outbreak. thus far, patients hospitalized with severe covid- infection have had pneumonia ( ) . of , laboratory-confirmed patients, almost % have had critical illness and almost % of critically ill patients have died ( ) . the overall case fatality rate ( . %) has been higher than that seen with seasonal influenza. most deaths have involved older adults, many of whom have had underlying chronic illnesses. although there is no known treatment for any coronavirus infection, investigators in china have undertaken several clinical trials. except for corticosteroids, all of the drugs being tested target coronavirus replication. unfortunately, very few of these antiviral drugs will be available to people who have been (or will be) infected with covid- . yet, for those who develop severe disease, only one question matters: "will i live or die?" this is the question that clinical investigators should address. could they discover a treatment that might reduce the severity of covid- infection and improve patient survival? in , one of us suggested that statins might be used to treat patients with ebola virus disease ( ) . a supply of a generic statin and a generic angiotensin receptor blocker (arb) was sent to sierra leone. experimental studies had shown that both drugs improved outcomes in experimental acute lung injury/acute respiratory disease syndrome (ards) ( ) ( ) ( ) ( ) ( ) . in sierra leone, local physicians treated approximately ebola patients with a combination of the two drugs. they noted "remarkable improvement" in survival. although there was no support for a proper clinical trial, the findings from this unconventional and poorly documented treatment experience were published ( , ) . during the current ebola outbreak in the democratic republic of the congo (drc), expensive vaccines are being used. investigational monoclonal antibody preparations ( ), but not inexpensive generic drug treatments ( ), have been tested. there are preliminary signs that the drc outbreak is coming under control, although the case fatality rate is still %. an approach to treating patients with severe covid- infection might be hiding in plain sight. the tissue receptor for the virus is ace , which is also the receptor for the sars coronavirus ( ) . several years ago, investigators in the netherlands and elsewhere showed that arbs and statins upregulate the activity of ace ( , ) , and higher levels of ace are associated with a reduced severity of ards ( ) . both statins and arbs target the host response to infection, not the virus ( ). they act largely (although not exclusively) on endothelial dysfunction, which is a common feature of many virus infections ( ) . both drugs counter endothelial dysfunction by affecting the ace / angiotensin-( - )/mas and angiopoietin/tie- signaling axes ( ) . combination treatment with these two drugs appears to accelerate a return to homeostasis, allowing patients to recover on their own. the host response is a major determinant of the pathogenesis of infectious diseases ( ) . we believe that investigators in china and elsewhere should undertake studies of patients with severe covid- infection to determine whether targeting the host response with widely available and inexpensive generic drugs, like arbs and statins, will improve their chances of survival. the studies would not have to be large; a successful clinical trial might require only patients ( ) . convincing evidence of the effectiveness of this treatment would suggest a syndromic approach to treating patients with other emerging infectious diseases, like ebola and pandemic influenza, as well as everyday illnesses, like sepsis and pneumonia ( ) . the long-term benefits of these findings for global public health could be immense. emerging coronaviruses: genome structure, replication, and pathogenesis clinical characteristics of hospitalized patients with novel coronavirusinfected pneumonia in wuhan the epidemiological characteristics of an outbreak of novel coronavirus diseases (covid- )-china a practical treatment for patients with ebola virus disease simvastatin decreases lipopoly-saccharide-induced pulmonary inflammation in healthy volunteers role of claudin- in the attenuation of murine acute lung injury by simvastatin losartan prevents sepsis-induced acute lung injury and decreases activation of nuclear factor kappab and mitogen-activated protein kinases angiotensin-converting enzyme /angiotensin-( - )/mas axis prevents lipopolysaccharide-induced apoptosis of pulmonary microvascular endothelial cells by inhibiting jnk/nf-b pathways treating the host response to emerging virus diseases: lessons learned from sepsis, pneumonia, influenza and ebola treating the host response to ebola virus disease with generic statins and angiotensin receptor blockers treating ebola patients: a 'bottom up' approach using generic statins and angiotensin receptor blockers palm consortium study team. . a randomized, controlled trial of ebola virus disease therapeutics treating ebola in eastern drc acute respiratory distress syndrome leads to reduced ratio of ace/ace activities and is prevented by angiotensin-( - ) or an angiotensin ii receptor antagonist tissue specific up regulation of ace in rabbit model of atherosclerosis by atorvastatin: role of epigenetic histone modifications imbalance between pulmonary angiotensindistress syndrome do viral infections mimic bacterial sepsis? the role of microvascular permeability: a review of mechanisms and methods the damage-response framework as a tool for the physician-scientist to understand the pathogenesis of infectious diseases clinician-initiated research on treating the host response to pandemic influenza we declare no conflicts of interest. no funding was provided to support the preparation of this article. key: cord- - f doo authors: fouchier, ron a. m. title: studies on influenza virus transmission between ferrets: the public health risks revisited date: - - journal: mbio doi: . /mbio. - sha: doc_id: cord_uid: f doo nan l ipsitch and inglesby recently estimated the potential public health risks associated with research on influenza virus transmission via respiratory droplets or aerosols between ferrets, leading them to conclude that such research is too risky to be conducted ( ) . the authors of that and other publications ( ) ( ) ( ) estimated the probability of laboratory-acquired infections (lais) and onward transmission of the viruses under investigation, as well as the potential consequence to public health if such events were to occur. given the weight assigned to these risk estimates, it is important that potential pitfalls in the underlying assumptions in these analyses be rigorously scrutinized. importantly, the published estimates were based on historical data and did not take into account the numerous risk reduction measures that are in place in the laboratories where the research is conducted. here, i provide a critical appraisal of the published work, discussing, challenging, and modifying the estimates based on the specific conditions under which the work is performed and the properties of the viruses under investigation. by doing so, the outcome of the risk assessment changes from serious risks to negligible risks for humans and the environment. as a consequence, a more balanced debate about the research on influenza virus transmission via respiratory droplets or aerosols between ferrets is warranted, in particular given the substantial public health benefits assigned to this type of research ( , ) . initial calculations of the potential risks associated with research on influenza virus transmission via respiratory droplets or aerosols between ferrets ( - ) used reports on select agent theft, loss, and release collected by the u.s. centers for disease control and prevention (cdc) from to ( ) to calculate the probability of occurrence of lais. although these reports have limitations ( , , ) , they provide the most recent account of lais in the united states and probably reflect the current state of the art in biosafety and biosecurity practices better than older studies on laboratory incidents ( , ) , e.g., as a consequence of the implementation of the u.s. select agent program and best practices developed in biosafety and biosecurity in general over the last decades. from to , lais in total were reported to the u.s. cdc, of which occurred in biosafety level (bsl ) facilities. during this -year period, on average , individuals per year had access to select agents in an average of laboratories per year, thus totaling , laboratory-years and , personyears of follow-up ( ) . from these data, the probability of occurrence of lais under bsl conditions was calculated as / , (or ϫ Ϫ ) per laboratory-year, or / , (or . ϫ Ϫ ) per person-year ( ) ( ) ( ) ( ) . these estimates, however, do not take into account specific pathogen types or research settings. this is crucial, because working practices in, e.g., virology and microbiology laboratories are different and because each biosafety laboratory is unique ( , ) . research facilities and the experiments that are conducted are therefore appraised through targeted risk assessments, in which the planned studies are scrutinized before any experiment is started. on this note, it is important that none of the lais reported to the u.s. cdc from to involved viruses ( ) , and the risks of lais associated with work on viral pathogens should thus be estimated as less than per , (Ͻ ϫ Ϫ per laboratory-year), or less than per , (Ͻ . ϫ Ϫ per person-year). unfortunately, the report by henkel et al. ( ) does not specify how many of the , laboratory-years and , person-years were related to bsl facilities versus bsl and bsl facilities. thus, using , and , as the denominators yields an underestimation of the true probability of lais under bsl conditions, as discussed previously ( , ) . research on influenza virus transmission via respiratory droplets or aerosols between ferrets is performed in facilities and under conditions that are specifically designed for the purpose of such studies ( ) ( ) ( ) ( ) ( ) . in ordinary bsl laboratories, including diagnostic laboratories, work is performed in open-front class biosafety cabinets with directional airflow, aimed at protecting the environment from release of pathogens and protecting laboratory workers from exposure. contrary to ordinary bsl conditions for work with viruses, all in vivo and in vitro experimental work on influenza virus transmission in the erasmus mc facility is carried out in class isolators or class biosafety cabinets, which are airtight boxes with negative pressure (ϽϪ pa), to ensure inward flow in case of leakage ( , ) . handling is done through airtight gloves fitted to the front of these cabinets. air released from the class units is filtered by high efficiency particulate air (hepa) filters and then leaves directly via the facility ventilation system, again via hepa filters. only authorized and experienced personnel that have received extensive training can access the facility. for animal handling, personnel always work in pairs to reduce the chance of human error. although the laboratory is considered "clean" because all experiments are conducted in closed class cabinets and isolators, special personal protective equipment, including laboratory suits, gloves, and ffp (class filtering face piece) facemasks, are used, and all personnel are vaccinated with the homologous a/h n vaccine. all equipment in the fa-cilities is monitored electronically, and alarm systems are employed to ensure that workers do not enter the facilities if equipment is malfunctioning. all personnel have been instructed and trained how to act in case of incidents, which are handled upon consultation between a senior staff member, a clinical microbiologist, the institutional biosafety officers, and the facility management. antiviral drugs (oseltamivir or zanamivir) are used immediately in the event that an incident should occur. every incident in the laboratory is followed up by actions to prevent such incidents from happening again. the facilities, personnel, and procedures are inspected by the u.s. cdc every years, in agreement with the u.s. select agent regulations for overseas laboratories and by the dutch government (inspectie leefomgeving en transport [ilt] inspection) ( , ) . the biosafety conditions in the erasmus mc facility thus extend well beyond "normal" bsl conditions for working with viruses, and a number of these biosafety measures should be considered when the probability of lais is inferred from the u.s. cdc report. unfortunately, an exact number for the effectiveness of individual biosafety measures is not available ( ) . however, it is reasonable to assume that the effectiveness of the physical separation of personnel from the viruses they work with through the use of class isolator units and class biosafety cabinets, the use of personnel protective equipment, the extensive training program, the use of experienced personnel only, and the application of a two-person rule to reduce human error during animal experiments would yield a decrease in the probability of lais. although the magnitude of this increase in safety is not known, i assume that it is at least a factor of . using the risk analysis done by others and this assumption of reduced risk, the probability of a lai in the erasmus mc facility would be reduced to below . ϫ ( . ϫ Ϫ ), or Ͻ . ϫ Ϫ per person-year. the quantitative risk assessment to be performed upon request of the u.s. government ( ) will have the challenging task of a better quantitative assessment of the effectiveness for each of the biosafety measures in individual laboratories, to yield exact numbers instead of the conservative estimates used here. the vaccination of laboratory personnel against the homologous a/h n virus under investigation produces another layer of safety that results in further risk mitigation. given the generally accepted efficacy of influenza vaccine of~ % for laboratoryconfirmed influenza in healthy adults ( ) , vaccination reduces the probability of an lai that results in viral escape to below . ϫ ( . ϫ Ϫ ), or Ͻ ϫ Ϫ per person-year. the % value is almost certainly an underestimate, because this number is taken from general population studies that include individuals with impaired immunity and nonexact matches between the vaccine antigen and the circulating viruses. it is important to note that the antibody titers in our vaccinated laboratory workers are high (geometric mean titer, ; range, to , ) compared to the titers generally accepted as protective against seasonal influenza (Ն ) ( ) and that individuals are revaccinated if and when their antibody titers decrease ( ) . as a consequence of the monitoring of equipment both electronically and by visual inspection, potential exposures to virus are unlikely to go unnoticed. upon any potential exposures, personnel receive oseltamivir treatment upon consultation with various specialists as indicated above. such early treatment with drugs has been reported to have~ % efficacy against human influenza virus infection ( ) and avian influenza virus infection ( ) . here, it is important to note that viruses under study in the erasmus mc facility are evaluated for their sensitivity to oseltamivir ( ) . the immediate treatment of laboratory personnel with oseltamivir upon any potential exposure to virus is thus expected to reduce the probability of lai further, to below . ϫ ( ϫ Ϫ ), or Ͻ ϫ Ϫ per person-year, given the average % efficacy in preventing laboratory-confirmed influenza. from this analysis, the conservative estimate is that when research is performed on transmission of influenza viruses via respiratory droplets or aerosols between ferrets in the erasmus mc facility, to which persons have access, lai would be expected to occur less frequently than once every million years. the second factor in the equation of the previous risk assessments is the probability of onward transmission from each case of lai. previous studies used to % as the probability of onward transmission ( , ), which is based on the unlimited spread of a pandemic influenza virus in the general population. it is important to note that onward transmission from lais has so far been uncommon ( ) ( ) ( ) . in the case of research on influenza virus transmission via respiratory droplets or aerosols between ferrets, a substantial, scientifically justified reduction from the probability of . to . of onward transmission from an lai can be made, based on the above-mentioned biosafety measures and risk mitigation strategies that are in place ( ) . the first factor that needs to be considered is that laboratory personnel that acquired the lai were vaccinated against the homologous a/h n virus and treated with oseltamivir upon any incident with potential exposure to the virus. although the vaccination and treatment may have been insufficient to prevent infection altogether (hence the occurrence of the lai at a frequency of less than once every million years), the virus shedding in h vaccinated and oseltamivir-treated individuals is likely to be reduced substantially, compared to the onward transmission in times of spread during an influenza pandemic from untreated immunologically naive individuals. if we assume a conservative -log reduction in virus excretion in immunized and treated individuals ( - ) compared to untreated immunologically naive individuals, the range of probability of onward transmission from a case of lai would be reduced to Ͻ ϫ Ϫ to ϫ Ϫ . as an important risk mitigation strategy to reduce onward transmission upon any potential lai, erasmus mc policy dictates enforcement of quarantine of any laboratory personnel that are potentially virus exposed. this policy would reduce the exposure of nonlaboratory personnel to one (the partner of the laboratory worker) or nil, rather than the~ contacts human adults would ordinarily have during a -day time frame ( ) . as a consequence, the transmission probability can be further reduced~ -fold, yielding a probability of onward transmission from the case of lai of Ͻ ϫ Ϫ to ϫ Ϫ . a final factor to consider in the calculation of the probability of onward transmission from each case of lai is the basic reproduction number (r ) of the influenza virus under investigation. as indicated above, the previous risk assessments were based on r of pandemic influenza virus. however, laboratory experiments have shown that the efficiency of transmission of the laboratoryderived influenza viruses was lower than that of the transmission of pandemic and seasonal influenza viruses in ferrets, as could be letter to the editor expected ( , , ) . moreover, given that the viruses are ferret adapted rather than human adapted, even an extremely conservative adjustment of the transmissibility parameter by a factor of would yield a "final" estimation of the probability of onward transmission from a case of lai of Ͻ . ϫ Ϫ to ϫ Ϫ . multiplying the probability of occurrence of an lai by the probability of onward transmission from each case of lai, one can estimate that the probability of an lai resulting in onward transmission would range between ( ϫ Ϫ ) ϫ ( . ϫ Ϫ ) (or . ϫ Ϫ ) and ( ϫ Ϫ ) ϫ ( ϫ Ϫ ) (or ϫ Ϫ ). from this analysis, the estimate is that when research is performed on transmission of influenza viruses via respiratory droplets or aerosols between ferrets in the erasmus mc facility, to which persons have access, lai with onward transmission would be expected to occur far less frequently than once every billion years. this probability could be assigned the term "negligible," given that the age of our planet is only billion years. in previous work ( ) ( ) ( ) ( ) , it was assumed that if a ferret-adapted avian influenza virus caused an lai and onward transmission, it could cause a pandemic with an attack rate of to %, as deduced from previous pandemics, and a case fatality rate ranging from to % in a population of billion people, thus leading to millions of, or more than a billion, fatalities. however, i consider an attack rate and case fatality rate of this magnitude to be unrealistic. given that the avian influenza viruses under investigation are ferret adapted rather than human adapted, it is unlikely that these viruses would spread as efficiently between humans. of note, this does not mean that the ferret model is therefore useless for studies to increase our fundamental knowledge about airborne virus transmission; it simply means that-just like when the mouse model is used to address fundamentals in immunology-we need to carefully validate any results obtained in animals before extrapolation to humans. throughout the history of virology, scientists have adapted viruses to cells, chicken embryos, or animal species in order to yield viruses that have increased replication properties in these specific hosts or cells but at the same time lose replication capacity and virulence in others ( ) . examples are the passaging of vaccinia virus in chicken embryo fibroblasts to yield modified vaccinia virus ankara (mva), which is now in use as a safe vaccine vector ( ) , the passaging of measles virus, mumps virus, and rubella virus in various cells to yield the live-attenuated mmr vaccine ( ) , and the passaging of influenza viruses in mice, ferrets, and eggs to yield the vaccine strain a/pr/ / ( ) , all of which are highly attenuated in humans. the higher bound of the range of case fatality rates of % stems from the number of deaths recorded among laboratoryconfirmed cases of a/h n influenza reported to the who ( ). since mild cases of infection-those individuals that do not consult a physician or remain untested-are not recorded, the true case fatality rate of a/h n virus infections in humans is unknown. due to intrinsic difficulties associated with serology data to estimate the numbers of previously infected individuals, there is no consensus on the incidence of a/h n infections in southeast asia ( , ) , but case fatality rates orders of magnitude lower than % have been inferred ( ) . in addition, it is important to note that fatalities in ferrets infected with a/h n virus via respiratory droplets or aerosols have not occurred, contrary to when ferrets received large dosages of a/h n virus directly in the (lower) airways ( , , ) . on the topic of intentional or accidental releases of viruses from laboratories involved in influenza virus transmission studies, it is important to note that during a decade of transmission studies on pandemic and epidemic strains derived from the , , , and pandemics and on various wild-type and laboratoryadapted zoonotic viruses of subtypes h , h , h , h , and h (summarized in reference ), no lais have been recorded. there have also been no recorded intentional or accidental releases during more than a century of research with human and animal influenza viruses, including highly pathogenic avian influenza viruses, even at times when biocontainment measures were largely nonexistent. some have argued that the russian influenza epidemic was the result of a laboratory accident ( ) , but in , influenza research was done under conditions of limited biocontainment, and attenuated and wild type strains were tested in humans. we do not know what happened in , but we cannot conclude that the virus escaped a bsl (ϩ) laboratory. since natural influenza pandemics have occurred on average every years over the last century, the probability that the next pandemic will emerge in nature is orders of magnitude larger than emergence from a laboratory. given the recently summarized immediate and short-term benefits of research on influenza viruses that are transmitted via respiratory droplets or aerosols between ferrets ( , ) and the longer-term aims to fully understand and predict and prevent pandemics, combined with the extremely low risk to humans and the environment associated with properly conducted experiments, i conclude that it is sensible and acceptable to restart the research, provided that any laboratory participating in this research adopt biosafety and biosecurity conditions comparable to those that are currently in place ( ) ( ) ( ) ( ) ( ) . i am very grateful for the help of friends and colleagues that provided valuable input and support for this letter. i am active in the field of research on influenza virus transmission via respiratory droplets or aerosols between ferrets, funded by the european union and niaid/nih. i am an inventor on a patent related to influenza virus reverse genetics. moratorium on research intended to create novel potential pandemic pathogens ethical alternatives to experiments with novel potential pandemic pathogens containing the accidental laboratory escape of potential pandemic influenza viruses the consequences of a lab escape of a potential pandemic pathogen. front public health : influenza gain-of-function experiments: their role in vaccine virus recommendation and pandemic preparedness use of highly pathogenic avian influenza a(h n ) gain-of-function studies for molecularbased surveillance and pandemic preparedness monitoring select agent theft, loss and release reports in the united states- - national bio and agrodefense facility: final environmental impact statement, appendix b. us department of homeland security evidence-based biosafety: a review of the principles and effectiveness of microbiological containment measures biorisk management laboratory biosecurity guidance challenges and practices in building and implementing biosafety and biosecurity programs to enable basic and translational research with select agents airborne transmission of influenza a/h n virus between ferrets experimental adaptation of an influenza h ha confers respiratory droplet transmission to a reassortant h ha/h n virus in ferrets airborne transmission of highly pathogenic h n influenza virus in ferrets h n hybrid viruses bearing /h n virus genes transmit in guinea pigs by respiratory droplet identification, characterization, and natural selection of mutations driving airborne transmission of a/h n virus u.s. government gain-of-function deliberative process and research funding pause on selected gain-offunction research involving influenza, mers, and sars viruses effectiveness and harms of seasonal and pandemic influenza vaccines in children, adults and elderly: a critical review and re-analysis of metaanalyses seasonal inactivated influenza virus vaccines use of neuraminidase inhibitors for rapid containment of influenza: a systematic review and meta-analysis of individual and household transmission studies effectiveness of personal protective equipment and oseltamivir prophylaxis during avian influenza a (h n ) epidemic, the netherlands use of the oral neuraminidase inhibitor oseltamivir in experimental human influenza: randomized controlled trials for prevention and treatment immunization with reverse-genetics-produced h n influenza vaccine protects ferrets against homologous and heterologous challenge efficacy of oseltamivir therapy in ferrets inoculated with different clades of h n influenza virus measured dynamic social contact patterns explain the spread of h n v influenza limited airborne transmission of h n influenza a virus between ferrets h n influenza viruses: facts, not fear modified vaccinia virus ankara (mva) as production platform for vaccines against influenza and other viral respiratory diseases trivalent combined measles-mumps-rubella vaccine. findings in clinical-laboratory studies trials in man with live recombinants made from a/pr/ / (h n ) and wild h n influenza viruses cumulative number of confirmed human cases of avian influenza a(h n ) reported to who comment on "seroevidence for h n influenza infections in humans: meta-analysis seroevidence for h n influenza infections in humans: meta-analysis key: cord- -qeltdch authors: graepel, kevin w.; lu, xiaotao; case, james brett; sexton, nicole r.; smith, everett clinton; denison, mark r. title: proofreading-deficient coronaviruses adapt for increased fitness over long-term passage without reversion of exoribonuclease-inactivating mutations date: - - journal: mbio doi: . /mbio. - sha: doc_id: cord_uid: qeltdch the coronavirus (cov) rna genome is the largest among the single-stranded positive-sense rna viruses. covs encode a proofreading ′-to- ′ exoribonuclease within nonstructural protein (nsp -exon) that is responsible for cov high-fidelity replication. alanine substitution of exon catalytic residues [exon(-)] in severe acute respiratory syndrome-associated coronavirus (sars-cov) and murine hepatitis virus (mhv) disrupts exon activity, yielding viable mutant viruses with defective replication, up to -fold-decreased fidelity, and increased susceptibility to nucleoside analogues. to test the stability of the exon(-) genotype and phenotype, we passaged mhv-exon(-) times in cultured cells (p ), in parallel with wild-type mhv (wt-mhv). compared to mhv-exon(-) p , mhv-exon(-) p demonstrated enhanced replication and increased competitive fitness without reversion at the exon(-) active site. furthermore, mhv-exon(-) p was less susceptible than mhv-exon(-) p to multiple nucleoside analogues, suggesting that mhv-exon(-) was under selection for increased replication fidelity. we subsequently identified novel amino acid changes within the rna-dependent rna polymerase and nsp of mhv-exon(-) p that partially accounted for the reduced susceptibility to nucleoside analogues. our results suggest that increased replication fidelity is selected in exon(-) covs and that there may be a significant barrier to exon(-) reversion. these results also support the hypothesis that high-fidelity replication is linked to cov fitness and indicate that multiple replicase proteins could compensate for exon functions during replication. a paradigm of rna virus biology is error-prone genomic replication due to the lack of proofreading or postreplicative rna repair mechanisms ( ) ( ) ( ) . decreased replication fidelity may constrain rna genome size and complexity and risks the accumulation of deleterious mutations leading to population extinction ( ) ( ) ( ) ( ) . while genetic diversity allows viral populations to adapt rapidly under selective pressure, many mutations are neutral or detrimental to viral fitness ( ) ( ) ( ) ( ) ( ) . research performed with many rna viruses supports the hypothesis that the mutation rate of rna virus replicases has evolved to balance multiple characteristics of the viral population such as genetic diversity, genomic integrity, and virulence. high-or low-fidelity variants are described for many rna viruses infecting animals, including the coronaviruses (covs) murine hepatitis virus (mhv-a ) and severe acute respiratory syndrome-associated coronavirus (sars-cov) ( ) ( ) ( ) ( ) ( ) , as well as foot-and-mouth disease virus ( ) ( ) ( ) ( ) ( ) , poliovirus ( ) ( ) ( ) ( ) ( ) ( ) ( ) , chikungunya virus ( , ) , influenza virus ( ) , coxsackievirus b ( , ) , and human enterovirus ( ) ( ) ( ) . most altered-fidelity variants described to date harbor mutations within the viral rna-dependent rna polymerase (rdrp), are attenuated in vivo, and protect against reinfection, highlighting their potential utility as live attenuated vaccines ( , , , , ) . those studies underscored the importance of understanding the molecular mechanisms by which rna viruses regulate their replication fidelity. viruses in the coronavirinae subfamily have large single-stranded positive-sense rna genomes [(ϩ)ssrna] ( ) , ranging between and kb in length ( ) . covs encode a =-to- = exoribonuclease (exon) in the n-terminal half of nonstructural protein (nsp -exon) ( , ) . cov exon activity depends on conserved magnesiumcoordinating acidic amino acids in three motifs (de-e-d) that together constitute the active site ( fig. ) ( ) . the cov exon is grouped with the de-d-dh superfamily of exonucleases involved in proofreading during prokaryotic and eukaryotic dna replication ( ) ( ) ( ) ( ) ( ) . alanine substitution of cov motif i de residues (de-to-aa) reduces biochemical exon activity in sars-cov ( , ) and human coronavirus e ( ) . mhv-a and sars-cov lacking exon activity [exon(-)] have mutation frequencies -fold to -fold greater than are seen with wt viruses and are highly susceptible to the activity of nucleoside analogues ( ) ( ) ( ) ( ) ( ) ) . thus, all available data to date support the hypothesis that nsp -exon is the first known proofreading enzyme encoded by an rna virus. despite the critical role of exon in virus replication, fidelity, fitness, and virulence, reversion of the exon-inactiviting substitutions ( fig. ) has not been detected following passages in culture, acute passages of sars-cov-exon(-) in aged balb/c mice, and days of persistent sars-cov-exon(-) infection in immunodeficient rag Ϫ/Ϫ mice ( , , , , ) . in this study, we sought to determine whether long-term passage of mhv-a -exon(-) ( passages over year [p ])-here mhv-exon(-)-would result in virus extinction, exon(-) reversion, or compensation for the loss of proofreading. we demonstrate that mhv-exon(-) did not extinguish during passage and adapted for increased replication. mhv-exon(-) concurrently evolved reduced susceptibility to multiple nucleoside and base analogues, consistent with selection for increased replication fidelity. importantly, the exon-inactivating substitutions did not revert. the evolved mutations in mhv-exon(-) nsp and nsp , which encodes the rdrp, accounted for only part of the increased nucleoside analogue resistance of mhv-exon(-) p , implicating multiple replicase proteins in adaptation for viral fitness. the results of this study support the proposed link between cov fidelity and fitness, demonstrate the surprising stability of the exon-inactivating substitutions, and identify additional proteins outside nsp and nsp that may contribute to cov fidelity regulation. long-term passage of wt-mhv and mhv-exon(-). we serially passaged wt-mhv and mhv-exon(-) in delayed brain tumor (dbt) cells times (p ). virus from each passage was harvested once % to % of the monolayer was involved in syncytia, which occurred between and hours postinfection (hpi). passage conditions varied increased replication of mhv-exon(-) p was affected by the presence of potential defective viral genomes or by some other population-based phenomenon, both wt-mhv p and mhv-exon(-) p were plaque purified three times. the plaque-purified viruses replicated indistinguishably from the parent populations (fig. c ). together, these data demonstrate that wt-mhv and mhv-exon(-) populations had adapted for increased replication and that either individual genomes or those derived from a single virus plaque encoded the adaptive changes required by the total population. mhv-exon(-) accumulated -fold-more mutations than wt-mhv but did not revert exon-inactivating substitutions. to determine whether the increased replication of mhv-exon(-) p resulted from primary reversion of exon(-) motif i, we sequenced nsp from infected-cell total rna. mhv-exon(-) p retained the motif i de-to-aa substitutions, demonstrating that primary reversion of exon(-) motif i did not occur. to identify potentially adaptive consensus mutations, we performed full-genome di-deoxy sequencing of mhv-exon(-) p and wt-mhv p . within wt-mhv p , we identified mutations, of which were nonsynonymous (ns) (fig. a) . in contrast, mhv-exon(-) p had total mutations ( ns) (fig. b) . the full-genome sequences have been deposited in genbank, and the mutations for both viruses are listed in tables s and s in the supplemental material. we identified only one mutation shared by both viruses (nsp a t), though it was present in approximately % of the wt-mhv p population by di-deoxy sequencing. both viruses deleted most of the hemagglutinin esterase (he). in mhv-a , he mrna is not transcribed in vitro ( ) ( ) ( ) , and he protein expression is detrimental to mhv-a fitness in cell culture ( ) . wt-mhv p also deleted open reading frame a (orf a), which is dispensable for mhv replication in cell culture ( ) . the c-terminal region of ns within mhv-exon(-) p was truncated and fused to he with a Ϫ frameshift. ns is a phosphodiesterase (pde) that protects viral rna by degrading =-to- = oligoadenylate, the activating factor for cellular rnase l ( ) ( ) ( ) . the portion of ns deleted in mhv-exon(-) p lies outside the pde catalytic domain, in a region of unknown function. c-terminally truncated ns retains enzymatic activity ( ) , but whether these specific deletions and fusions disrupt pde activity remains to be tested. nevertheless, ns is dispensable for mhv replication in immortalized cells ( , ) . details about the deletion sites are provided in fig. s in the supplemental material. within proteins predicted to be part of the replicasetranscriptase complex (nsp - and nucleocapsid) ( ), wt-mhv p had only one ns change, located in the nsp -helicase ( fig. a and table s ). in contrast, mhv-exon(-) p had ns changes within this region ( fig. b and table s ). mhv-exon(-) p displays increased genomic rna accumulation and increased resistance to -fluorouracil. coronaviruses lacking exon consistently display defects in rna synthesis relative to wt strains ( , , ) . to determine whether the increased replication of mhv-exon(-) p was associated with restored genomic rna (grna) production, we measured grna accumulation over time using two-step realtime quantitative pcr ( , ) . mhv-exon(-) p accumulated levels of grna similar to those accumulated by wt-mhv p and wt-mhv p at early time points, while grna levels for mhv-exon(-) p were~ log lower (fig. a ). mhv-exon(-) p grna levels fell below those of wt-mhv and wt-mhv p after h and were similar to those of mhv-exon(-) p at hpi. normalizing to the grna abundance at h for each virus demonstrated that the rates of grna accumulation were similar for all four viruses (fig. b) . these data suggest that the increased replication of p viruses relative to wt-mhv is not fully accounted for by increased rna synthesis. in addition to rna synthesis defects, exon(-) covs have up to -fold-increased mutation frequencies and profoundly increased sensitivity to nucleoside and base analogues relative to wt covs ( , , , , ) . to determine whether the nucleoside analogue sensitivity of mhv-exon(-) was altered by long-term passage, we treated cells infected with parental and passaged viruses with the base analog, -fluorouracil ( -fu). -fu is converted intracellularly into a nucleoside analogue that incorporates into growing rna strands and causes a:g and u:c mutations. for simplicity, we will hereafter refer to -fu as a nucleoside analogue. incorporation of -fu is increased in the absence of exon activity ( ) . all viruses displayed a concentration-dependent decrease in viral titer but differed greatly in their levels of susceptibility to -fu (fig. c ). at m, wt-mhv p titers were reduced by~ log , while mhv-exon(-) p titers were undetectable (Ͼ log fold reduction). wt-mhv -fu sensitivity was not altered by passage. mhv-exon(-) p was less susceptible than mhv-exon(-) p to -fu treatment, with a decrease in titer of only~ . log at m. mhv-exon(-) p remained more sensitive to -fu than wt-mhv, suggesting that wt-like resistance requires an intact exon. these data demonstrate that mhv-exon(-) p evolved resistance to -fu through mutations outside exon(-) motif i. spike mutations in mhv-exon(-) p do not increase resistance to -fu. bacteriophage x acquired resistance to -fu by delaying cell lysis, thereby reducing the number of replication cycles in which -fu can be incorporated ( ) . mhv-exon(-) p had multiple mutations in the spike glycoprotein, including one in the spike furin cleavage site that reduced syncytium formation. to test whether the spike mutations manifested in resistance to -fu, we cloned the spike gene from mhv-exon(-) p into the isogenic mhv-exon(-) background. the recombinant virus demonstrated intermediate replication kinetics between mhv-exon(-) p and mhv-exon(-) p (fig. a ) and did not form syncytia. spike-p also increased the specific infectivity of viral particles (fig. b) . however, the mhv-exon(-) p spike did not affect the sensitivity of the recombinant virus to -fu (fig. c ). thus, any adaptive increase in -fu resistance must be located elsewhere in the genome. mhv-exon(-) passage resulted in unique mutations in nsp and nsp . to date, three proteins have been shown to alter cov sensitivity to -fu: nsp -rdrp, nsp -exon, and nsp (which stimulates exon activity) ( , , ) . neither wt-mhv nor mhv-exon(-) p contained an ns mutation in nsp , and wt-mhv p had no mutations within either nsp or nsp . in contrast, mhv-exon(-) p had ns mutations in nsp and ns mutations in nsp ( fig. and ) , none of which have been described previously in vitro or in viable viruses. within nsp , six mutations were in the predicted rdrp finger, palm, and thumb domains ( fig. a ) ( ) . four residues (h , f , s , and m ) can be visualized on a phyre -modeled structure of the mhv-nsp rdrp, while the remaining residues lie outside the modeled core rdrp (fig. a ) ( ) . one mutation, m t, lies in the cov-specific domain, which is conserved among nidoviruses. this domain has been implicated in membrane targeting in mhv-a ( ) and performs an essential nucleotidylation activity in the arterivirus equine arteritis virus ( ) . however, m t is not predicted to catalyze nucleotidylation. within nsp , ns mutations were identified in the exon domain, and ns mutations were in the c-terminal n -methyltransferase domain (fig. b ). we next modeled the structure of mhv nsp using phyre software ( ), resulting in highest-probability similarity to the sars-cov nsp -nsp complex (pdb c s) ( ) with high confidence (i.e., the calculated probability of true homology between the structures) of % for residues to of mhv-nsp . the model predicts that five mutations are located close to surface of the protein (fig. b ). all three modeled zinc finger domains contain one ns mutation (f y, y h, and l i). two mutations, d e and f y, are located near the interface between nsp and nsp , though neither site has previously been implicated in nsp -nsp interactions ( , , ) . one ns mutation resulted in a d e substitution in exon motif iii, a metal-coordinating active site residue. we previously reported that alanine substitution of d results in an exon(-) phenotype ( ) , but the viability or phenotype of a d e substitution was not tested in that study. these data suggest that a network of residues evolved to regulate nsp and nsp activity or stability in the exon(-) background. fixed mutations in nsp and nsp in mhv-exon(-) p directly correlate with increased resistance to multiple nucleoside analogues. to determine approximately when the mutations in nsp and nsp arose, we performed di-deoxy sequencing across these protein-coding regions roughly every passages (p , p , p , p , p , p , p , p , p , p , p , p , and ). by this method, we detected consensus ns mutations at p , p , and p for nsp and at p and p for nsp (fig. ). both nsp and nsp carried their full complement of p consensus mutations by p , except for a minority variant (d e) in nsp that was maintained at Ͻ % of the population between p and p . these passage levels correlated with increased replication of mhv-exon(-) (fig. b ) and with decreasing sensitivity to -fu (fig. c ). neither replication nor -fu sensitivity of mhv-exon(-) changed substantially between p and p . to determine whether mhv-exon(-) adaptation of proofreading-deficient coronaviruses ® evolved increased resistance to multiple nucleoside analogues, we treated virusinfected cells with three additional analogues that are substrates for viral rdrps: ribavirin (rbv), a guanine analogue that inhibits viral replication through multiple mechanisms, including mutagenesis and inhibition of purine biosynthesis ( ); -azacytidine (azc), an rna mutagen ( ) ; and =-c-methyladenosine (cmea), which is proposed to incorporate in viral rna and terminate nascent transcripts ( ) . as with -fu, we observed dose-dependent sensitivity to rbv, azc, and cmea in all mhv-exon(-) viruses that decreased with increasing passage number (fig. d to f). except for azc, mhv-exon(-) sensitivity did not change between p and p . together, these data demonstrate that mhv-exon(-) evolved increased resistance to multiple nucleoside analogues that correlated with the length of passage and the acquisition of mutations in nsp and nsp . importantly, this occurred in the absence of specific mutagenic selection and without reversion of exon motif i. this increased general selectivity toward all four classes of nucleotide strongly supports the idea of an overall increase in fidelity in mhv-exon(-) p . mutations in nsp partially account for increased resistance of mhv-exon(-) p to multiple nucleoside analogues. we hypothesized that mutations in mhv-exon(-) p nsp and nsp were most likely to impact replication and nucleoside analogue sensitivity based on their enzymatic activities and temporal association with phenotypic changes. to test this hypothesis, we engineered recombinant mhv-exon(-) to encode the p nsp and nsp sequences, alone and together. expression of nsp -p and nsp -p , alone or in combination, altered replication kinetics of mhv-exon(-) without affecting peak titers (fig. a) and increased grna levels above those of mhv-exon(-) p (fig. b ). nsp -p had a greater effect than nsp -p on the sensitivity of mhv-exon(-) to all analogues tested, and the combination of nsp and nsp -p did not increase resistance above that seen with nsp -p alone ( fig. c to e) . none of the recombinant viruses recapitulated the resistance phenotypes of the mhv-exon(-) p population. together, these data demonstrate that nsp -p mutations account only partially for the nucleoside analogue resistance of mhv-exon(-) p and that adaptations in nsp -p mask those in nsp -p . we also can conclude that the nsp -p d e active site mutation does not correct the defect caused by the motif i de-to-aa substitutions. resistance to nucleoside analogues correlates with mhv-exon(-) fitness. we hypothesized that mutations in nsp and nsp provided a fitness advantage to mhv-exon(-) p . we competed the recombinant viruses with a reference mhv-exon(-) virus (p stock) containing silent mutations in the nsp coding region. mutant and reference viruses were detected in the mixed infection by real-time quantitative pcr using dually labeled probes specific for each virus. mhv-exon(-) p showed a modest fitness advantage over the reference p mhv-exon(-) silent strain (fig. f, solid green) . mhv-exon(-) p profoundly outcompeted mhv-exon(-) silent, with Ͼ , -fold more mhv-exon(-) p genomes present at the end of passage (fig. f, dotted green line) . mhv-exon(-) nsp -p had greater relative fitness than mhv-exon(-) nsp -p , and mhv-exon(-) nsp / -p was intermediate between the single recombinants, implicating a complex evolutionary interaction between these two proteins. the measured fitness correlated with the patterns of nucleoside analogue . for panels c to e, the statistical significance of changes in the titer of swapped viruses relative to mhv-exon(-) p at the highest drug concentration tolerated was determined using the mann-whitney test (*, p Ͻ . ; **, p Ͻ . ; ***, p Ͻ . ; ns, not significant). for panel f, the statistical significance for the indicated comparisons was determined using the mann-whitney test. boxed points have the same p value. adaptation of proofreading-deficient coronaviruses ® resistance and rna synthesis associated with mutations in nsp and nsp , suggesting a link between the evolutions of these phenotypes. the result also confirms that nsp and nsp are important but not sufficient to account for the significantly increased fitness of mhv-exon(-) p relative to mhv-exon(-) p . in this report, we describe experimental adaptive evolution of wt-mhv and mhv-exon(-) during long-term passage in cell culture. wt-mhv evolved increased replication kinetics over passages, with few consensus mutations arising in the wt-mhv p genome. in contrast, mhv-exon(-) accumulated -fold-more mutations than wt-mhv, none of which occurred at the exon-inactivating substitutions. nevertheless, mhv-exon(-) p demonstrated increased replication kinetics and fitness compared to mhv-exon(-) p ( fig. and ) . our previous studies demonstrated that exon-mediated proofreading is required for cov fitness ( , , ) . thus, mhv-exon(-) was likely under selective pressure for restoration of high-fidelity replication or for tolerance of the increased mutational load. consistent with this hypothesis, mhv-exon(-) p exhibited increased resistance to multiple nucleoside analogues, a phenotype strongly associated with high-fidelity viruses ( , , , ) . our results raise several important questions. in the face of selection for increased fidelity, why did mhv-exon(-) not revert? can mhv replicase proteins mediate high-fidelity replication without exon proofreading? which mechanisms other than increased fidelity can compensate for the loss of proofreading? in the face of selective pressure for increased fidelity, why did mhv-exon(-) not revert? although our data suggest that mhv-exon(-) was under selective pressure for increased fidelity, we detected no primary reversion at the de-to-aa substitutions in mhv-exon(-) at any passage tested. these data are consistent with and significantly extend previous studies reporting genotypic stability of exon(-) motif i in mhv and sars-cov ( , , , , ) . complete reversion to de within exon(-) motif i would require four nucleotide changes. this likely represents a high genetic barrier to reversion, especially given that fitness can be increased by mutations outside nsp -exon (fig. f) ( ) . single and double nucleotide changes within motif i could restore an acidic charge to individual residues (e.g., motif i ea, ad, ed, etc.). however, the active site compositions of deddh exonucleases, such as the klenow fragment, are so stringent that even conservative mutations (d to e or e to d) reduce exon activity by Ͼ % ( ). thus, intermediate amino acid changes may not have a selective advantage compared to motif i aa, limiting the evolutionary pathways to reversion. however, nsp -p had detectable effects on rbv and azc resistance as well as on the competitive fitness of mhv-exon(-) (fig. f) , demonstrating a modest capacity for fitness adaptation in nsp outside the catalytic residues. whether these mutations resulted from genetic drift or positive selection remains unclear. nevertheless, our data show that mhv-exon(-) can adapt for increased fitness without fully restoring exoribonuclease activity. while some mutations in mhv-exon(-) p likely confer dbt cellspecific selective advantages, others may represent generalizable strategies for overcoming exon(-) defects in other cell types and in other coronaviruses. thus, understanding the mechanisms by which mhv-exon(-) p compensated for exon activity could allow recovery of exon(-) variants of other covs, such as transmissible gastroenteritis virus and human cov e, which to date have been nonviable as exon(-) recombinants ( , ) . can mhv replicase proteins mediate high-fidelity replication without exon proofreading? mhv-exon(-) p exhibits increased resistance to four nucleoside analogues after passage (fig. ) . although resistance to a single nucleoside analogue can evolve without increasing overall fidelity, resistance to multiple nucleoside analogues strongly suggests a broadly increased capacity to discriminate nucleotides ( , , , ) . increased-fidelity variants in rna viruses have most frequently been mapped to rdrps ( , , , ) . thus, if increased fidelity contributes to nucleoside analogue resistance in mhv-exon(-) p , the most likely protein involved would be nsp -p . three findings are consistent with the hypothesis that mutations within nsp -p increase rdrp fidelity. first, nonsynonymous mutations to nsp arose in the low-fidelity mhv-exon(-) strain but not in the presence of proofreading (wt-mhv). second, five of the mutations lie in or near structural motifs important for fidelity regulation in other rdrps. amino acid substitutions in the finger and palm domains have been repeatedly shown to affect viral rdrp fidelity ( , ) , and we have recently reported a finger mutation (nsp -v i) that likely increases the fidelity of the mhv rdrp ( ) . our modeled structure predicts that nsp -p contains three mutations in the palm domain and one in the finger domain, with the m k thumb domain mutation lying near the palm (fig. a) . third, exchange of nsp -p alone into the background of mhv-exon(-) reduced the susceptibility of mhv-exon(-) to three different nucleoside analogues (fig. ) . thus, all data support the hypothesis that nsp -p is a high-fidelity rdrp. we are actively developing biochemical, phenotypic, and deep sequencing assays to quantify the fidelity of nsp -p . importantly, nsp -p accounts only partially for the mhv-exon(-) p nucleoside analogue resistance phenotype (fig. ) , suggesting a possible limit to the compensation achievable by mutating the rdrp alone. further, the effects of mutations in nsp -p and nsp -p are not additive and may be antagonistic when they are isolated from the whole passaged virus (fig. ) , indicating that the relationships between nsp -and nsp -p mutations are likely evolutionarily linked with those in other mhv proteins. in fact, a substantial component of the evolved resistance to nucleoside analogues cannot be explained by the presence of nsp -p and nsp -p , alone or together. in support of this hypothesis, we identified several nonsynonymous mutations in other replicase proteins, such as nsp , nsp , nsp , and nsp . sars-cov nsp and nsp have functional interactions with nsp , acting as a primase/ processivity factor ( , ) and a helicase/ntpase, respectively ( ) . processivity factors in herpes simplex virus and mycobacterium tuberculosis regulate dna polymerase fidelity by balancing polymerase extension and exonuclease activity ( , ) , and helicases in chikungunya virus and foot-and-mouth disease virus can evolve to increase fidelity ( ) and alter the frequency of ribavirin-induced mutations ( ), respectively. sars-cov nsp has rna-binding activities and is proposed to participate in the multiprotein replicase complex ( , ) , and mhv nsp is a uridylate-specific endoribonuclease ( ) . both could plausibly be involved in modulating polymerase activity. additionally, it remains possible that evolution for increased fidelity could involve proteins outside the canonical replication complex (nsp to nsp ), including those in the structural and accessory cassette. thus, while the immediate studies will focus on testing whether replicase proteins nsp , nsp , nsp , and nsp regulate fidelity, it is exciting to consider the possibility that this virus-directed discovery approach will reveal novel interactions between multiple mhv proteins. which mechanisms other than increased fidelity might account for mhv-exon(-) p nucleoside analog resistance? genomic mutations in rna viruses are most commonly detrimental or lethal ( ) ( ) ( ) ( ) ( ) . one strategy to prevent extinction by mutational load is to increase replication fidelity, as discussed above. an alternative strategy is to increase mutational robustness. mutational robustness describes the capacity of a virus to buffer the fitness effects of mutations. in the setting of low-fidelity replication, as in mhv-exon(-), increased mutational robustness could have provided a selective advantage ( ) ( ) ( ) . selection for increased robustness could explain the~ synonymous changes in mhv-exon(-) p . synonymous changes can alter codons to reduce the probability of nonconservative amino acid changes ( ) . alternatively, the increased population size of mhv-exon(-) p could promote robustness by a "safetyin-numbers" effect, allowing efficient purging of low-fitness mutants while maintaining population fitness ( ) . large populations also increase the likelihood of coinfection, allowing complementation between viral genomes. although increased replication conferred by mutations in spike did not alter -fu resistance (fig. ) , results of a recent study performed with poliovirus suggest that mutagenized populations have elevated coinfection frequencies ( ) . thus, complementation may contribute to mhv-exon(-) p nucleoside analogue resistance. conflicting evidence exists regarding whether mutational robustness itself affects the sensitivity to rna mutagens ( , , ) ; nevertheless, the robustness of mhv-exon(-) p merits further investigation. conclusions. the proofreading activity of the nsp exoribonuclease is a critical determinant of cov replication, fidelity, and fitness. we showed that covs have the capacity to compensate for loss of exon activity through a network of mutations in nsp and nsp and elsewhere in the genome. thus, while nsp -exon appears to play a dominant role in cov replication fidelity, its activity is likely closely tied to a highly evolved network of proteins. the demonstrated coadaptation for replication, competitive fitness, and likely increased fidelity within mhv-exon(-) supports the hypothesis that these roles are linked functionally and evolutionarily. it will be interesting to test whether evolution in other cell types derived from different species or with different innate immune environments would result in similar adaptive strategies. genetic and biochemical testing of the rich mutational resource revealed in mhv-exon(-) p will likely inform the design of countermeasures for endemic and emerging covs by defining novel common targets for stable virus attenuation or direct inhibition. cell culture. dbt- (delayed brain tumor, murine astrocytoma clone ) cells were maintained as described previously ( ) . baby hamster kidney (bhk) cells stably expressing the mhv-a receptor ceacam (bhk-r; ) were maintained under conditions of selection with . mg/ml of g (mediatech) as described previously ( ). long-term passage of virus and stock generation. the infectious cdna clone for mhv-a and the recovery of mhv-exon(-) were described previously ( , ) . long-term passage was initiated by infecting subconfluent monolayers of dbt- cells in -cm flasks with either wild-type mhv-a or mhv-exon(-) at an moi of approximately . pfu/cell. one lineage of each virus was subjected to a total of passages (p ). supernatant was harvested at each passage and stored at Ϫ °c. total rna was harvested for most passages using ml of trizol reagent (ambion) per -cm flask and stored at Ϫ °c. virus stocks of select intermediate passages were generated by infecting a subconfluent -cm flask of dbt- cells at an moi of . pfu/cell. at approximately hpi, the flask was frozen at Ϫ °c and the supernatant was clarified by centrifugation at , ϫ g (sorvall rc- b plus; ha- a rotor) for min at °c. the virus titer of each stock was determined by plaque assay using dbt- cells as described previously ( , ) . for plaque assays of viruses containing the spike protein from mhv-exon(-) p , which does not form syncytia, plaques were visualized with neutral red (sigma catalog no. n ) (dilution at : in phosphate-buffered saline [pbs] containing calcium and magnesium). neutral red was added h after plating, and the reaction mixture was incubated for an additional to h before formaldehyde fixation. plaque purification was performed by infecting dbt cells with serial dilutions of virus and overlaying the cultures with agar. single plaques were isolated, resuspended in pbs containing calcium and magnesium, and inoculated onto fresh dbts. this process was completed times before experimental stocks were generated as described above. sequencing of virus stocks. following p , rna was purified from the harvested trizol samples according to the manufacturer's protocol and reverse transcribed (rt) using superscript iii (invitrogen) as described previously ( ) . full-genome di-deoxy sequencing was performed for both wt-mhv p and mhv-exon(-) p using overlapping amplicons approximately kb in length. all coding regions were sequenced fully, and, of , nucleotides, Ͼ % were sequenced for each virus [for wt-mhv p , to , ; for mhv-exon(-) p , to , ]. two microliters of rt product was used for each pcr ( ) . di-deoxy sequencing was performed by genhunter corporation (nashville, tn) and genewiz (south plainfield, nj). sequence analysis was performed using macvector version (macvector, inc., apex, nc) and the mhv-a infectious clone reference genome (genbank accession number ay ). the nucleotide sequences of the amplicon and sequencing primers are available upon request. sequencing of nsp and nsp from intermediate passages was performed using trizol-purified rna from infected monolayers and the primers listed below. primers m f ( =-ttttggcgagatggtagc- =) and m r ( =-ggtaagacagttttaggtgag- =) were used to generate a , nucleotide amplicon containing all of nsp . primers m f ( =-atgcttaccaactatgagc- =) and m r ( =-ccgatttgaatggcgta g- =) were used to generate a , nucleotide amplicon containing all of nsp . the pcr conditions for these reactions were the same as those used to generate the amplicons used for full-genome sequencing ( ) . replication and rna synthesis kinetics. viral replication kinetics in dbt- cells were determined at an moi of pfu/cell or an moi of . pfu/cell as described previously ( ) . supernatant ( l) was harvested at the indicated time points, and the virus titer was determined by plaque assay. the accumulation of genomic rna at an moi of pfu/cell was measured by two-step real-time quantitative rt-pcr. intracellular rna was harvested using trizol and reverse transcribed using superscript iii (invitrogen). the levels of cdna derived from intracellular positive-sense viral rna were measured using primers directed to nsp . values were normalized to levels of the endogenous control glyceraldehyde- -phosphate dehydrogenase (gapdh). no mutations within the primer binding sites emerged in either p population. the primers and amplification conditions were the same as reported previously ( ), except that the rt product was diluted : prior to use. samples were plated in technical duplicate to minimize well-to-well variation. data are presented as ϪΔct , where Δct denotes the threshold cycle (c t ) value for the target (nsp ) minus the c t value for the reference (gapdh). determination of specific infectivity. subconfluent monolayers of dbt- cells in -well plates were infected with the indicated virus at an moi of pfu/cell, and supernatant was harvested at hpi. the levels of genomic rna in supernatant were measured using one-step real-time quantitative rt-pcr (rt-qpcr) of trizol-extracted rna as described previously ( ) . briefly, genomic rna was detected with a = -carboxyfluorescein (fam)-labeled and = black hole quencher (bhq- )-labeled probe targeting nsp (biosearch technologies, petaluma, ca), and rna copy numbers were calculated by reference to an rna standard derived from the mhv a fragment. samples were plated in technical duplicate to minimize well-to-well variation. titers were determined by plaque assay in dbt- cells, and specific infectivity was calculated as pfu per supernatant genomic rna copy. nucleoside and base analogue sensitivity assays. -azacytidine (azc), -fluorouracil ( -fu), and ribavirin (rbv) were purchased from sigma (product numbers a , f , and r , respectively), and stock solutions were prepared in dimethyl sulfoxide (dmso). cmea ( =-c-methyladenosine) was received from gilead sciences (foster city, ca). sensitivity assays were performed as described previously ( ) , except that -well plates were used at an moi of . pfu/cell. supernatants were harvested at hpi, and titers were determined by plaque assay. phyre modeling of mhv-nsp . the mhv nsp structure was modeled with the phyre online program ( ) using nsp residues to , corresponding to residues , to , of the orf ab polyprotein. the model was analyzed using the pymol molecular graphics system, version . (schrödinger, llc). generation of nsp and nsp swaps. viruses containing nsp -p or nsp -p or both were generated using the mhv-a reverse genetics system ( ) . rna from the mhv-exon(-) p virus was reversed transcribed with superscript iii (invitrogen) and used to generate amplicons containing either nsp or nsp . each amplicon was flanked by bp that overlapped an amplicon generated from the backbone plasmid. amplicons were inserted into mhv-a fragments using an infusion hd cloning kit (takara bio usa, inc., mountain view, ca). nsp is split across mhv e and f, while nsp is contained within mhv f. reaction mixtures contained ng of vector, ng of insertion, and l of enzyme and were incubated for min at °c. errors were corrected by site-directed mutagenesis using pfu turbo polymerase (agilent, santa clara, ca). the nsp / -p swap was generated through restriction digestion of the individual swaps using bsmbi and stui followed by gel purification and assembly using t dna ligase (neb, ipswich, ma). viable viruses were constructed and rescued as described previously ( ) . competitive fitness assays. competitor viruses were competed with an mhv-exon(-) virus harboring silent mutations in the probe-binding region within nsp . subconfluent dbt- monolayers in -well plates were coinfected at a total moi of . pfu/cell with competitor and reference viruses at a : ratio and passaged times. for each passage, supernatants were harvested at h. rna was extracted from l of supernatant using l of trizol reagent and purelink rna minikit columns (thermo scientific, waltham, ma), and l of supernatant was used to infect fresh cells in a -well plate (total moi estimated at pfu/cell). the proportion of each virus was determined by real-time rt-qpcr from the infection supernatant using two taqman probes with different fluorescent dyes in separate reactions. competitor viruses were detected with the same probe used in specific infectivity analyses ( ) . reference viruses were detected by a probe targeting the same region but with silent mutations ( =-tccgaactactgcaaccccaagtg- =) and labeled with = quasar and = black hole quencher (bhq- ) (biosearch technologies, petaluma, ca). rna copy numbers were calculated by reference to an rna standard generated by in vitro transcription of the corresponding mhv a fragment, and relative rna abundances were calculated as ratios of competitor genomes to reference genomes. statistical analysis. graphpad prism (la jolla, ca) was used to perform statistical tests. only the comparisons shown (with statistical significance indicated as ЉnsЉ [nonsignificance] or asterisk[s]) within each figure or described in each legend were performed. in many cases, the data were normalized to the results obtained with untreated controls. this was performed using graphpad prism . the number of replicate samples is denoted within each figure legend. accession numbers. full-length genome sequences for wt-mhv p and mhv-exon(-) p have been deposited in genbank (accession numbers mf and mf , respectively). supplemental material for this article may be found at https://doi.org/ . /mbio . - . fig s , tif file, . mb. lack of evidence for proofreading mechanisms associated with an rna virus polymerase viral mutation rates mutation rates among rna viruses theory of lethal mutagenesis for viruses viral quasispecies evolution correlation between mutation rate and genome size in riboviruses: mutation rate of bacteriophage q␤ extremely high mutation rate of a hammerhead viroid distribution of fitness effects caused by single-nucleotide substitutions in bacteriophage f the rate and character of spontaneous mutation in an rna virus rate of deleterious mutation and the distribution of its effects on fitness in vesicular stomatitis virus the mutational robustness of influenza a virus the distribution of fitness effects caused by single-nucleotide substitutions in an rna virus infidelity of sars-cov nsp -exonuclease mutant virus replication is revealed by 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oas-rnase l pathway by murine coronavirus ns protein is required for virus replication and liver pathology antagonism of rnase l is required for murine coronavirus replication in kupffer cells and liver sinusoidal endothelial cells but not in hepatocytes homologous ', '-phosphodiesterases from disparate rna viruses antagonize antiviral innate immunity crystal structure of the mouse hepatitis virus ns phosphodiesterase domain that antagonizes rnase l activation murine coronavirus nonstructural protein ns is not essential for virus replication in transformed cells cell-type-specific type i interferon antagonism influences organ tropism of murine coronavirus delayed lysis confers resistance to the nucleoside analogue -fluorouracil and alleviates mutation accumulation in the single-stranded dna bacteriophage x structure-function relationships among rna-dependent rna polymerases characterization of the expression, intracellular localization, and replication complex association of the putative mouse hepatitis virus rna-dependent rna polymerase discovery of an essential nucleotidylating activity associated with a newly delineated conserved domain in the rna polymerase-containing protein of all nidoviruses the phyre web portal for protein modeling, prediction and analysis coronavirus nsp , a critical co-factor for activation of multiple replicative enzymes murine hepatitis virus replicase protein nsp is a critical regulator of viral rna synthesis ribavirin's antiviral mechanism of action: lethal mutagenesis? -azacytidine and rna secondary structure increase the retrovirus mutation rate inhibition of hepatitis c virus rna replication by =-modified nucleoside analogs the the '- ' exonuclease of dna polymerase i of escherichia coli: contribution of each amino acid at the active site to the reaction mutagenesis of coronavirus nsp reveals its potential role in modulation of the innate immune response fidelity of nucleotide incorporation by the rna-dependent rna polymerase from poliovirus dna replication across taxa one severe acute respiratory syndrome coronavirus protein complex integrates processive rna polymerase and exonuclease activities a second, non-canonical rna-dependent rna polymerase in sars coronavirus mechanism of nucleic acid unwinding by sars-cov helicase the herpes simplex virus type dna polymerase processivity factor increases fidelity without altering pre-steady-state rate constants for polymerization or excision the ␤ clamp in the mycobacterium tuberculosis dna polymerase iii ␣␤ replicase promotes polymerization and reduces exonuclease activity viral polymerase-helicase complexes regulate replication fidelity to overcome intracellular nucleotide depletion involvement of a joker mutation in a polymerase-independent lethal mutagenesis escape mechanism the severe acute respiratory syndrome-coronavirus replicative protein nsp is a single-stranded rna-binding subunit unique in the rna virus world the severe acute respiratory syndrome coronavirus nsp protein is an endoribonuclease that prefers manganese as a cofactor selection for robustness in mutagenized rna viruses evolvability and robustness in populations of rna virus ⌽ evolution of mutational robustness in an rna virus codon usage determines the mutational robustness, evolutionary capacity, and virulence of an rna virus the role of mutational robustness in rna virus evolution plaques formed by mutagenized viral populations have elevated coinfection frequencies mutational robustness of an rna virus influences sensitivity to lethal mutagenesis does mutational robustness inhibit extinction by lethal mutagenesis in viral populations? systematic assembly of a full-length infectious cdna of mouse hepatitis virus strain a we thank members of the denison laboratory for valuable discussions. t -ai (n.r.s.), t -ai (e.c.s.), and f -ai (e.c.s.), all from the national institutes of health.the content is solely the responsibility of the authors and does not necessarily represent the official views of the national institutes of health.we declare that we have no conflicts of interest. key: cord- - rhoffx authors: yu, changqing; li, sunan; zhang, xianfeng; khan, ilyas; ahmad, iqbal; zhou, yulong; li, shuo; shi, jing; wang, yu; zheng, yong-hui title: march inhibits ebola virus glycoprotein, human immunodeficiency virus type envelope glycoprotein, and avian influenza virus h n hemagglutinin maturation date: - - journal: mbio doi: . /mbio. - sha: doc_id: cord_uid: rhoffx membrane-associated ring-ch-type (march ) strongly blocks human immunodeficiency virus type (hiv- ) envelope glycoprotein (env) incorporation into virions by downregulating its cell surface expression, but the mechanism is still unclear. we now report that march also blocks the ebola virus (ebov) glycoprotein (gp) incorporation via surface downregulation. to understand how these viral fusion proteins are downregulated, we investigated the effects of march on ebov gp maturation and externalization via the conventional secretion pathway. march interacted with ebov gp and furin when detected by immunoprecipitation and retained the gp/furin complex in the golgi when their location was tracked by a bimolecular fluorescence complementation (bifc) assay. march did not reduce the gp expression or affect the gp modification by high-mannose n-glycans in the endoplasmic reticulum (er), but it inhibited the formation of complex n-glycans on the gp in the golgi. additionally, the gp o-glycosylation and furin-mediated proteolytic cleavage were also inhibited. moreover, we identified a novel furin cleavage site on ebov gp and found that only those fully glycosylated gps were processed by furin and incorporated into virions. furthermore, the gp shedding and secretion were all blocked by march . march also blocked the furin-mediated cleavage of hiv- env (gp ) and the highly pathogenic avian influenza virus h n hemagglutinin (ha). we conclude that march has a very broad antiviral activity by prohibiting different viral fusion proteins from glycosylation and proteolytic cleavage in the golgi, which inhibits their transport from the golgi to the plasma membrane and incorporation into virions. required for viral entry ( , ) . ebov also expresses several different mature and soluble gp forms that are secreted or shed extracellularly, which is easily detectable by western blotting (wb) ( ) . thus, we used ebov gp and its mutant bearing mld deletion (Δmld) to study the march activity. march is expressed in terminally differentiated myeloid cells ( ) . we could not detect march expression in commonly used human cell lines including t, vero, thp , huh , hep g , hela, and hela-derived tzm-bi cells (fig. a) . thus, we investigated the march antiviral activity via ectopic expression. initially, we tested how march proteins from human (h), cow (bos taurus, b), and mouse (m) affect ebov entry using pseudotyped hiv- , which is a faithful system for studying the ebov gp function ( ) ( ) ( ) ( ) . all these march proteins restricted the pseudotyped virus replication as effectively as the retroviral restriction factor apobec g (fig. b) . to explore the march antiviral mechanism, ebov virus-like particles (vlps) and hiv- vlps were produced by expressing ebov vp or hiv- gag proteins in the presence of march and gp or gpΔmld. march strongly reduced the gp incorporation into these virions ( fig. c and d) . in contrast, the inactive ring domain mutant w a did not have such in addition, hiv- pseudoviruses containing the full-length ebov gp were produced and purified similarly. the levels of gp expression in these vlps were analyzed by wb using anti-flag, and the levels of vlps were determined by anti-hiv- gag (p ). protein expressions in t cells are shown in fig. d . (d) ebov virus-like particles (vlps) were produced from t cells after expressing ebov vp , human march or its w a mutant, and ebov gp or gpΔmld. after purification via ultracentrifugation, these vlps were analyzed by wb. protein expressions in viral producer cells are shown in fig. c . (e) ebov gpΔmld that has a gfp tag was expressed in hela cells in the presence or absence of human march . after staining with dapi, the gp localization was detected by confocal microscopy. (f) ebov gpΔmld was expressed with increasing amounts of human march in t cells. the gp expression on cell surface was detected by flow cytometry using anti-flag followed by alexa fluor -conjugated goat anti-mouse igg. activity (fig. d ). when gp subcellular localization was determined by confocal microscopy, although gp alone was detected predominantly on the plasma membrane, it was retargeted into the cytoplasm when march was expressed (fig. e) . consistently, gp was strongly downregulated from the cell surface by march in a dose-dependent manner when detected by flow cytometry (fig. f) . march retains ebov gp in the golgi. to understand where gp is targeted by march , we studied gp and march subcellular localization. first, we tested their interactions by immunoprecipitation. gpΔmld could specifically pull down march ( fig. a, lanes to ) , and furin could pull down gpΔmld and march ( fig. a , lanes to ). these results demonstrate that gp, furin, and march interact with each other in cells. second, we set up a bimolecular fluorescence complementation (bifc) assay to track their interactions in live cells ( ) . the c termini of gp, march , and furin were fused to the n-terminal to amino acids (aa) of a green fluorescent protein venus (vn) or its c-terminal to aa (vc). as a control, serine incorporator (serinc ) protein in addition, flag-tagged furin was expressed with ha-tagged human march and gpΔmld in t cells. proteins were immunoprecipitated with anti-flag and analyzed by wb. march , furin, and gp were detected by anti-ha, anti-flag, or anti-ebov gp, respectively. (b) gfp, gp-vc, furin-vn, or human march -vn proteins were individually expressed in hela cells. after staining with dapi, fluorescent signals were observed by confocal microscopy. (c) the march -vn-ha/gp-vc or furin-vn-ha/gp-vc bifc pair was expressed in hela cells. cells were stained with fluorescent anti-ha to detect march or furin. in addition, furin-vn that does not express the ha tag was expressed with gp-vc and ha-tagged march in hela cells. cells were stained with fluorescent anti-ha to detect march . fluorescent signals were observed by confocal microscopy. (d) indicated bifc fusion proteins were expressed individually or pairwise, and the levels of bifc signals were determined by flow cytometry. serinc , serine incorporator . experiments were repeated three times, and identical results were obtained. was also fused to vc. when gp-vc, furin-vn, and march-vn were expressed individually, no green bifc signal was detected by confocal microscopy (fig. b) . when gp-vc was expressed with march -vn or furin-vn, strong green fluorescent signals were produced that overlapped march or furin (fig. c) , confirming the interactions between gp and march , or gp and furin. unlike gp alone that was found on the plasma membrane (fig. e) , the gp-march bifc complex was found in intracellular compartments, confirming that march downregulates gp from the cell surface. in addition, when the furin-vn/gp-vc pair was expressed with march , their colocalization was also detected (fig. c ), confirming that these three molecules form a complex in live cells. when these interactions were quantified by flow cytometry, both march /gp and furin/gp pairs generated ϳ % bifc-positive cells, whereas positive cells produced from the march /serinc pair were only around % (fig. d ). thus, we detected specific march -gp and furin-gp interactions via bifc. third, we determined how march affects the gp and furin subcellular localization via confocal microscopy. the furin-vn/gp-vc bifc pair was expressed with an er marker, calnexin (cnx), or a tgn marker that was fused with mcherry. although the gp-furin complex colocalized with both cnx and tgn, its colocalization with tgn was much stronger than that with cnx when march was not expressed (fig. ). in the presence of march , its colocalization with tgn was strongly increased, whereas that with cnx was decreased. these results suggest that march retains ebov gp in the golgi. march blocks ebov gp proteolytic processing. the full-length gp was expressed with march from different species or its w a mutant, and gp processing was analyzed by comparing gp and gp expression levels via wb. as reported previously ( ) , because of the heavy o-and n-glycosylation of mld in the golgi, gp exhibited a higher molecular weight than gp (fig. a ). when march proteins were expressed, gp proteins were almost undetectable, indicating that march blocks the gp processing. the w a mutant was inactive, indicating that the ring domain is required for this activity. to understand whether this activity is cell type specific, similar experiments were conducted in hela, hep g , and huh cells. again, the gp expression in these cells was selectively inhibited by march (fig. b) . to confirm the specificity of this march effect, huh cells were also transfected with small interfering rnas (sirnas) to silence the march expression. when the march expression was decreased, the gp expression was effectively recovered (fig. b, lanes to ) . thus, the march effect is not cell type specific. to understand whether this march activity was affected by the mucin-like domain, the full-length gp and gpΔmld processing were compared side-by-side in the presence of wild-type (wt) march or its w a mutant (fig. c ), or march proteins from different species (fig. d ). unlike gp from the full-length gp, gp Δmld exhibited a lower molecular weight than gp Δmld. in addition, unlike similar levels of gp and gp from the full-length gp, the gp levels were much higher than gp from gpΔmld, confirming that gpΔmld is more effectively processed than the full-length gp. nevertheless, gp expressions from both full-length gp and gpΔmld were completely inhibited by march proteins but not by the w a mutant ( fig. c and d) . to test whether march destabilizes gp , cells were treated with inhibitors that target different degradation pathways, including mg for proteasomes, nh cl and bafilomycin a (bafa ) for lysosomes, and dbeq for er-associated protein degradation (erad). none of these inhibitors rescued gp expression or increased gp expression, indicating that gp is not subjected to degradation by these two pathways (fig. e) . we also tested how march affects the expression of vesicular stomatitis virus glycoprotein (vsv-g), a class iii fusion protein. the vsv-g expression was strongly reduced by march but not by its w a mutant, and the reduction was blocked by nh cl but not mg (fig. f ), confirming that march triggers vsv-g degradation via the lysosome pathway as reported previously ( ) . human march has amino acids (aa) that create a ring-ch finger in the n-terminal cytoplasmic tail (ct), two tm domains, and a c-terminal ct (fig. a ). the critical w residue is in the ring region and is required for recruitment of ubiquitinconjugating e enzyme ( ) . to uncover other important domains, we created three march c-terminal ct deletion mutants by expressing its -to- -aa ( - ), -to- -aa ( - ), and -to- -aa ( - ) regions. when these mutants were expressed with the full-length gp or gpΔmld, wt and mutants - and - blocked the gp processing, whereas the - mutant did not (fig. a ). these mutants were then fused with a green fluorescent protein (gfp) tag, and their subcellular localizations were tracked and compared by confocal microscopy. both wt and the - mutant displayed a scattered punctate localization in the cytoplasm, whereas the - mutant protein with a ring domain and two tm domains is presented. three march c-terminal ct deletion mutants, - , - , and - , were created and expressed with the full-length gp or gpΔmld. march and gp expression was detected by wb using anti-ha or anti-flag. (b) march and its three deletion mutants that have a c-terminal gfp tag were expressed in hela cells. after staining with dapi, their subcellular localizations were detected by confocal microscopy. (c) a schematic diagram of human furin functional domains is presented. flag-tagged cd, p domain, and cd subdomain-deletion mutants were created and were expressed with gpΔmld. proteins were immunoprecipitated by anti-flag and detected by wb. furin was detected by anti-flag, and ebov gp was detected by anti-ebov gp antibodies. (d) the full-length gp was expressed with increasing amounts of furin in t cells. the gp and furin expression was determined by wb. ® exhibited diffuse localization in the cytoplasm and nucleus (fig. b) . the - mutant diffused partially in the cytoplasm. these results demonstrate that the - region determines march intracellular compartmentalization that is critical for its activity. identification of a novel furin cleavage site on ebov gp. human furin is a -aa type i tm protein with a large lumenal region that has a signal peptide (sp), prodomain (pro), subtilisin-like catalytic domain (cd), p domain, cysteine-rich region (crr), tm domain, and a -aa ct ( ) (fig. c) . the cd and p domain are functionally critical for furin convertase activities. we created five furin dominant negative mutants, including Δcd, Δp, Δcd , Δcd , and Δcd , by deleting cd ( to aa), p domain ( to aa), and the three cd subdomains cd ( to aa), cd ( to aa), and cd ( to aa). when these five mutants were expressed with gp, they all blocked the gp cleavage from gp to gp (fig. c , lanes to ), confirming that ebov gp is processed by furin ( ) . consistently, when their interactions were determined by immunoprecipitation, gp was pulled down by the Δp mutant, but not by the Δcd, Δcd , Δcd , and Δcd mutants (fig. c) , indicating that gp interacts with the furin catalytic domain. to detect furin-cleaved ebov gp products, full-length gp was expressed with increasing amounts of furin. gp and gp were detected even in the absence of ectopic furin, which is produced by endogenous furin (fig. d , lane ). in the presence of ectopic furin, although the levels of gp were not changed, those of gp and gp were decreased or increased in a dose-dependent manner by furin (fig. d , lanes to ). in addition, there appeared another gp at ϳ kda that was also increased by furin. accordingly, a novel furin cleavage site, rkir , was found in gp in front of mld, and this novel -kda product was named gp * (see fig. b and also fig. s in the supplemental material). we conclude that gp * is predominantly processed from gp by furin. analysis of the gp glycosylation becomes complex due to proteolytic processing. to detect gp glycosylation, we blocked gp processing by removal of the furin cleavage site between gp and gp . two furin cleavage site-deficient (Δfr) mutants were created from the full-length gp and gpΔmld, and gp glycosylation was analyzed by treatments with endoglycosidase h (endo h) and peptide-n-glycosidase f (pngase f) that trim off nh-glycans or all n-glycans, respectively. gpΔfr was detected at and kda, and gpΔmldΔfr was detected at and kda (fig. a , lanes and ). all these proteins were sensitive to pngase f, confirming that they are modified by n-glycans (fig. a, lanes and ) . a ϳ -kda, pngase f-resistant protein was detected from gpΔfr after treatment with pngase, confirming that gp but not gpΔmld is modified by o-glycans (fig. a, lane ) . the -kda gpΔfr and -kda gpΔmldΔfr were resistant to endo h (fig. a, lanes and ) , indicating that they are modified by nc-glycans. the -kda gpΔfr and -kda gpΔmldΔfr were sensitive to endo h (fig. a, lanes and ) , indicating that they are modified by nh-glycans. when march was expressed, only the endo h-sensitive -kda gpΔfr and -kda gpΔmldΔfr were detected (fig. a, lanes to and lanes to ) . additional experiments were conducted to verify these results by using the fulllength gp and gpΔmld that have an active furin cleavage site. much more o-glycosylated gp proteins were detected from the full-length gp, which should come from gp (fig. a, lane ) . all gp proteins were sensitive to, whereas all gp proteins were resistant to, endo h (fig. a, lanes and ) , confirming that gp and gp are modified by nh-or nc-glycans, respectively. again, march selectively inhibited the endo-h-resistant and pngase f-resistant gp expressions (fig. a, lanes to and lanes to ) . collectively, these results demonstrate that march blocks gp modifications by nc-and o-glycans, but not by nh-glycans. modification of ebov gp by nc-and o-glycans is required for its proteolytic cleavage by furin and incorporation into virions. because gp is packaged into ebov virions ( ), we addressed how glycosylation affects its incorporation into ebov vlps. gpΔfr and gpΔmldΔfr were expressed with ebov vp and march , and their expressions in vlps were compared. we confirm again that march selectively inhibited the expression of gp with nc-and o-glycans (fig. b, lanes , , , and ) . notably, only gp with these two types of glycans was detected from vlps (fig. b) , indicating that only gp proteins with nc-and o-glycans are selectively incorporated into virions. when march was replaced with furin in this experiment, the expression of gp with nc-and o-glycans was also inhibited, but gp * showed up that was also incorporated into virions (fig. b, lanes and ) . gp * was produced much more from gpΔmldΔfr than gpΔfr, and it was also detected from gpΔmldΔfr in the absence of ectopic furin expression that was also enriched in virions (fig. b, lane ) . collectively, these results demonstrate that furin selectively cleaves gp with nc-and o-glycans. in addition, because gp * is incorporated into vlps (fig. b, lanes , , and ), we confirm that only fully glycosylated gps are incorporated into virions. because gp is modified by nc-and o-glycans, it further confirms that furin selectively processes fully glycosylated gp. march blocks ebov gp shedding and secretion. after maturation, gp /gp trimers on the plasma membrane are further cleaved by the tumor necrosis factor alpha (tnf-␣) converting enzyme (tace) at the gp membrane-proximal external region to release a soluble shed gp ( ) . to verify march 's inhibitory effect on ebov gp march inhibits viral fusion protein maturation ® maturation, we tested how march affects gp shedding. after expression of gp and gpΔmld with march or furin, gp proteins were immunoprecipitated from supernatants and analyzed by wb. march strongly blocked gp shedding, and in contrast, furin strongly increased it (fig. a ). gp * was also detected as soluble proteins. in addition to gp, ebov produces secreted gp (sgp) and small secreted gp (ssgp) via rna editing, all of which share the n-terminal aa (fig. b) . to address how march affects the gp secretion, we expressed sgp with march proteins and measured its secretion. unlike its w a mutant, march retained sgp in cells and effectively reduced its secretion (fig. c, lanes to ) . next, we expressed only gp or gp Δmld that should also be secreted as sgp and tested how march affects their secretion. march proteins from different species strongly increased gp and gp Δmld expression in cells but completely blocked their secretions (fig. c, lanes to ) . march blocks hiv- env and h n ha maturation. unlike hiv- env, most iav has are processed by trypsin-like serine proteases. however, a highly pathogenic avian strain such as h n has gained an additional furin cleavage site to expand its tropism ( ) . thus, we used h n ha as well as hiv- env to confirm the march antiviral activity. when hiv- env and h n ha were expressed with furin and march , furin pulled down gp or ha with march (fig. a) , confirming that march interacts with env/furin or ha/furin complex. in addition, when march was expressed with these fusion proteins, hiv- gp /gp and h n ha /ha expression levels were all decreased (fig. b) , confirming that march also blocks the env and ha cleavage by furin. when the env and ha glycosylations were analyzed after treatments with glycosidases, they were all sensitive to pngase f (fig. c, lanes and ) , confirming that they are modified by n-glycans. hiv- gp was completely sensitive to endo h, indicating that it is modified by nh-glycans (fig. c, lane ) . hiv- gp was only partially sensitive to endo h (fig. c, lane ) , indicating that it is modified by both nh-and nc-glycans. h n ha and ha were completely sensitive or resistant to endo h (fig. c, lane ) , indicating that ha and ha were modified by nh-or nc-glycans, respectively. when march was expressed, the gp expression was reduced likely due to inhibition of the furin activity (fig. c, lane ) . in addition, endo h-sensitive ha expression was increased (fig. c, lane ) . collectively, these results demonstrate that march does not block the env and ha modification by nh-glycans in the er, but rather they suggest that it should target the other glycosylation steps. class i fusion proteins project from the virion surface as highly glycosylated heterotrimeric spikes and initiate infection by binding to receptors on the surface of target cells. although it is clear that glycosylation and proteolytic cleavage are critically important, it remains incompletely understood how these steps are coopted to generate mature spikes that are incorporated from the plasma membrane into virions. we march inhibits viral fusion protein maturation ® now show that march specifically inhibits these processes, which provides new understanding for this poorly defined mechanism. our results highlight the fundamental role of n-glycosylation in the spike formation. first, n-glycosylation is absolutely required for ebov gp maturation. although march does not affect the gp modification by nh-glycans in the er, it inhibits the gp modification by nc-and o-glycans as well as its proteolytic processing in the tgn. these results suggest that march inhibits the conversion of nh-glycans to nc-glycans, likely by blocking the mannose trimming step in the cgn and/or the following glycosylation steps in the medial-golgi and the tgn. in addition, it is possible that o-glycosylation and furin cleavage are both dependent on the mannose trimming. in fact, when gpΔfr and gpΔmldΔfr were expressed with furin, only gp proteins with nc-and o-glycans were processed into gp * (fig. b, lanes and ) . consistently, when gp was expressed with furin, only gp but not gp was effectively processed into gp * (fig. d) . these results strongly suggest that the gp processing by furin is dependent on the completion of the glycosylation processes. second, n-glycosylation is required for ebov gp transport from the tgn to the plasma membrane. march not only blocked gp shedding and secretion (fig. ) , but also downregulated gp expression on the cell surface ( fig. e and f) . these inhibitory effects were likely caused by an inhibition of the gp glycosylation in the golgi by march . in fact, we found that only gp proteins modified with o-and/or nc-glycans were selectively incorporated into virions (fig. b) . thus, glycosylation is required for gp externalization and incorporation into virions. because we detected the gp interaction with march as well as the gp retention in the tgn by march , it is possible that march directly or indirectly retains viral glycoproteins at an early location so they never physically reach the location where they would be glycosylated. in that case all the glycosylation machinery would be intact but the gp would never reach it. in addition to the three class i fusion proteins, march also targets a class iii fusion protein, vsv-g, via a different mechanism. instead of inhibiting maturation, march triggers vsv-g degradation via the lysosomal pathway. although march does not trigger class i fusion protein degradation, its activity still depends on the ring domain, because the w a mutant that has a dead ring domain is inactive. these results demonstrate that the march activity depends on the ubiquitination pathway. we speculate that march might ubiquitinate and degrade a critical cellular transport facilitator, which blocks gp from access to the glycosylation and furin cleavage machinery. however, the degradation of vsv-g by march follows a similar mechanism by which march proteins downregulate immune receptors from the cell surface ( ) . march proteins usually ubiquitinate a lysine residue in the cytoplasmic tail of these immune receptors, which redirects them from the endosomes to the late endosomes and the lysosomes for degradation when they are endocytosed from the cell surface ( ) . thus, march proteins use two different mechanisms to target different proteins. for class i fusion proteins, march blocks their anterograde transport from the tgn to the plasma membrane; for vsv-g and immune receptors, march proteins block their recycling back to the tgn after retrograde transport from the plasma membrane to the endosomes. in addition to these four viral proteins, march proteins can target an amazingly large number of cellular proteins ( ) . most of these targets are immune receptors on the cell surface that play an important role in innate and adaptive immunity, including mhc i, mhc ii, cd , cd , cd , cd , cd , cd , intracellular adhesion molecule (icam)- , nkg d ligand mult , transferrin receptor (tfr), trail receptor , and interleukin- receptor (il rap). thus, the expression of march proteins is tightly regulated, which causes their poor expression in immortalized cell lines and a major difficulty in studying their endogenous function ( ) . thus, we were unable to further confirm the march antiviral activity by silencing its endogenous gene expression. in addition, due to lack of structural data on march proteins, it is difficult to understand how they have so many different targets. the role of tm domains of march proteins in substrate recognition has been suggested ( ) . a gxxxg motif is conserved in march tm domains, and it is also present in the mhc ii tm domain, which may mediate their interactions. in addition, the recognition could be mediated by a common adaptor protein. in addition to march , the maturation of viral fusion proteins is inhibited by guanylate-binding proteins (gbp) and ( ) , and interferon-induced transmembrane (ifitm) protein ( ) . gbp / directly target the cytoplasmic part of furin and inhibit its enzymatic activities, which exert broad antiviral activity by inhibiting furin-mediated cleavage of viral fusion proteins. ifitm interrupts viral glycoprotein processing of murine leukemia virus (mlv) env, hiv- env, and vsv-g, but not ebov gp, suggesting that it does not affect furin function. in addition, it interferes with env trafficking and promotes its degradation in lysosomes. thus, mammalian cells have evolved diverse mechanisms to intercept the production of mature viral fusion proteins for assembly of infectious virus particles. march is naturally expressed in terminally differentiated myeloid cells such as macrophages and dendritic cells (dcs). these cells are the primary targets for hiv- and ebov infection, transmission, and dissemination, and establishment of persistent tissue virus reservoirs in the case of hiv- . these cells can also be infected by h n , although the infection may not necessarily contribute to the significant part of pathogenesis. thus, march should play a role in defending these viruses during natural infection. in fact, when the march expression was silenced in macrophages, hiv- replication was strongly promoted ( ) . although macrophages and dcs express all hiv- receptors, they are much less permissive to hiv- infection than cd ϩ t lymphocytes. the hiv- resistance in these myeloid cells is currently attributed to their high expression levels of the host restriction factor samhd . however, the role of march should not be ignored. in addition, it should be addressed how viruses counteract march to establish productive infection. cell lines, rna oligonucleotides, and transfection reagents. hek t, hela, hep g , huh , vero e , and tzm-bi cells were cultured in dulbecco's modified eagle medium (dmem). thp- cells were cultured in roswell park memorial institute (rpmi) medium. media were supplemented with % fetal bovine serum and % penicillin-streptomycin (gibco), and cells were cultured at °c in a humidified atmosphere in a % co incubator. march sense/antisense rna oligonucleotides =-ccu ucucucgcacuucuautt- =/ =-auagaagugcgagagaaggtt- = and negative-control sense/antisense rna oligonucleotides =-uucuccgaacgugucacgutt- =/ =-acgugacacguucggagaatt- = were ordered from genepharma. t cells were transfected with polyethylenimine (pei) from sigma, huh cells were transfected with lipofectamine from thermo fisher, and hep g cells were transfected by amaxa nucleofector using program t- from lonza. plasmids. the human march expression plasmid pcaggs- xha-hmarch and its w mutant pcaggs- xha-hmarch -w a were provided by kenzo tokunaga ( ) . bovine and murine march cdnas were amplified from bovine peripheral blood mononuclear cells or mouse raw . cells by rt-pcr and cloned into pcaggs with a c-terminal ha tag after ecori/xhoi digestion. the human march c-terminal deletion mutant - , - , and - expression vectors were created by pcr in pcaggs- xha-hmarch vector. ebov gp and ebov vp expression vectors were described previously ( ) . the n-terminal flag-tagged ebov gpΔmld expression vector was provided by shan-lv liu. pcdna . -flag-sgp, pcdna . -flag-gp , and pcdna . -flag-gp Δmld were created by pcr from the ebov-gp and ebov gpΔmld vector and cloned into pcdna . (ϩ) vector by bamhi/ecori digestion. pcmv -furin-flag/myc was purchased from origene (catalogue number rc ). the furin Δsd, Δsd , Δsd , Δsd , and Δp mutants were created in the same furin expression vector by overlapping pcr. pcdna . -hmarch -gfp and pcdna . -gpΔmld-gfp were constructed by inserting march and ebov gpΔmld cdnas into pegfp-n vector (genbank accession no. u ) via ecori/agei digestion. to construct bifc expression vectors, the furin, march , and gp-Δmld cdnas were inserted into pcdna . -vn, pcdna . -vn-ha, or pcdna . -flag-vc (c-terminal vn/vc) vectors by ecori/agei digestion, which generated pcdna . -furin-vn, pcdna . -furin-vn-ha, pcdna . -march -vn-ha, or pcdna . -gpΔmld-flag-vc, respectively. pcdna . -serinc -flag-vc was reported previously ( ) . mcherry-tgnp-n- and mcherry-calnexin-n- expression vectors were obtained from michael davidson via addgene. h n ha expression vector was obtained from gary nabel. confocal microscopy assay. a total of ϫ hela cells were seeded on a glass slide h before transfection. cells were transfected with lipofectamine reagent (thermo fisher). after h, cells were washed and fixed with % paraformaldehyde and permeabilized with . % triton x- . after washing, cells were blocked with % bovine serum albumin. for immunofluorescence assay, cells were incubated with anti-ha primary antibody for h at room temperature (rt). after washing times with phosphate-buffered saline (pbs), cells were stained with alexa fluor- conjugated secondary antibody h at rt. following washing times with pbs, cells were stained with =, -diamidino- -phenylindole (dapi) for min, washed for h at rt, and observed by a confocal microscopy assay (zeiss lsm ). at least random cells per slide were analyzed, and the most representative images from each slide were selected for presentation. flow cytometry analysis. to detect ebov gp expression on the cell surface, a total of ϫ t were seeded in each well of a -well plate h before transfection. cells were transfected with pcdna . -flag-gpΔmld and increasing amounts of pcaggs- xha-hmarch expression vectors. after h, cells were centrifuged at , rpm for min and fixed with % paraformaldehyde for min at rt. after brief centrifugation, cells were blocked with % bovine serum albumin and incubated with anti-flag primary antibody for h at rt. after centrifugation and washing three times with pbs, alexa fluor -conjugated secondary antibody was added for h at rt. after washing times, cells were collected and analyzed by flow cytometry. to detect bifc, t cells in -well plates were transfected with the pcdna . -march -vn-ha/pcdna . -gpΔmld-flag-vc, pcdna . -furin-vn-ha/pcdna . -gpΔmld-flag-vc, or pcdna . -march -vn-ha/pcdna . -serinc -flag-vc pairs similarly and directly analyzed by flow cytometry. western blotting. forty-eight hours posttransfection, cells were lysed with ripa buffer (sigma) and cytosolic fractions were collected. after incubation with loading buffer, samples were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds-page). some samples were subjected to endo h or pngase f treatment before applying them to the sds-page gel. for endo h (catalogue no. p l; neb) treatment, a total of g of cell lysate was first denatured by heating at °c for min in denaturing buffer. denatured proteins were added to a -l reaction system containing l of glycobuffer and , units of endo h and incubated at °c for h. for pngase f (catalogue no. p l; neb) treatment, the same amount of cell lysate was added to l of pngase f buffer ( ϫ) to make a -l total reaction volume and incubated at °c for min. after cooling down, l of rapid pngase f was added and samples were incubated h at °c. after transferring to polyvinylidene difluoride (pvdf) membrane, % nonfat milk was used to block the membrane for h at rt. the membrane was incubated with primary antibody and horseradish peroxidase (hrp)-conjugated secondary antibody. alternatively, the membrane was incubated with hrp-conjugated primary antibody. membranes were exposed after incubation with enhanced chemiluminescence (ecl) substrate (thermo fisher). mouse anti-march antibody was purchased from proteintech; mouse anti-actin, -ha, and -flag monoclonal antibodies were purchased from sigma; rabbit anti-ebov gp and rabbit anti-iav-ha polyclonal antibodies were purchased from sino biology (china). the mouse anti-hiv gp , anti-hiv gp , anti-hiv gp , and anti-hiv p /p monoclonal antibodies were obtained from the nih aids reagent program. hrp-conjugated anti-human, anti-rabbit, or anti-mouse immunoglobulin g secondary antibodies were purchased from pierce. immunoprecipitation. a total of ϫ t cells were seeded in each well of a -well plate h before transfection. forty-eight hours posttransfection, cells were lysed by ripa buffer. cytosolic fractions were collected and incubated with anti-flag antibody-conjugated magnetic beads (sigma) overnight at °c. the beads were isolated by magnetic shelf, washed several times, and used for wb assay. viral infectivity assay. a total of ϫ t cells were seeded in each well of a -well plate h before transfection. cells were transfected with pnl-luc-Δenv, pcdna . -flag-gpΔmld, and march expression vectors. forty-eight hours posttransfection, virion-containing supernatants were collected and subjected to ultracentrifugation at , ϫ g for h. pellets were collected and analyzed by wb. in addition, -l supernatants were collected for quantification by p gag enzyme-linked immunosorbent assay (elisa). supernatants with equal amounts of p gag were used to infect vero e cells that were seeded in a -well plate. after another h, infected cells were washed times with pbs and lysed with l ripa buffer. twenty-five microliters was collected and mixed with an equal amount of substrate from the bright-glo luciferase assay system (promega), and luminescence activity was determined by a luminometer. supplemental material is available online only. fig s , pdf file, . mb. unconventional protein secretion in animal cells er to golgi-dependent protein secretion: the conventional pathway glycosylation in health and disease arms race between enveloped viruses and the host erad machinery common features of enveloped viruses and implications for immunogen design for next-generation vaccines influenza virus n-linked glycosylation and innate immunity gp on hiv- virions lacks o-linked carbohydrate the secret life of viral entry glycoproteins: moonlighting in immune evasion structures and mechanisms of viral membrane fusion proteins: multiple variations on a common theme the membrane associated ring-ch proteins: a family of e ligases with diverse roles through the cell downregulation of cell surface receptors by the k family of viral and cellular ubiquitin e ligases downregulation of major histocompatibility complex class i by human ubiquitin ligases related to viral immune evasion proteins c-mir, a human e ubiquitin ligase, is a functional homolog of herpesvirus proteins mir and mir and has similar activity overview of the membrane-associated ring-ch (march) e ligase family march inhibits hiv- infection by reducing virion incorporation of envelope glycoproteins membrane-associated ring-ch (march) and are march family members that inhibit hiv- infection covalent modifications of the ebola virus glycoprotein mechanistic understanding of n-glycosylation in ebola virus glycoprotein maturation and function ebola virus glycoprotein with increased infectivity dominated the - epidemic human adaptation of ebola virus during the west african outbreak characterization of ebola virus entry by using pseudotyped viruses: identification of receptor-deficient cell lines bimolecular fluorescence complementation (bifc) analysis as a probe of protein interactions in living cells solution structure of the kaposi's sarcomaassociated herpesvirus k n-terminal domain reveals a novel e -binding c hc -type ring domain furin at the cutting edge: from protein traffic to embryogenesis and disease processing of the ebola virus glycoprotein by the proprotein convertase furin endoproteolytic processing of the ebola virus envelope glycoprotein: cleavage is not required for function ectodomain shedding of the glycoprotein gp of ebola virus the proteolytic regulation of virus cell entry by furin and other proprotein convertases . march inhibits viral infection by two different mechanisms guanylate-binding proteins and exert broad antiviral activity by inhibiting furin-mediated processing of viral envelope proteins ifitm reduces retroviral envelope abundance and function and is counteracted by glycogag murine leukemia virus glycosylated gag reduces murine serinc protein expression at steady-state levels via the endosome/lysosome pathway to counteract serinc antiretroviral activity we thank bin wang and wen-bo tan for reagents or technical assistance. we thank ian a. york for reading and very insightful comments on the manuscript. we thank kenzo tokunaga, shan-lv liu, michael davidson, and gary nabel, as well as the nih aids reagent program, for providing various reagents.changqing yu and sunan li are supported by national natural science foundation of china grants and , respectively. key: cord- -p b b t authors: wolf, yuri i.; kazlauskas, darius; iranzo, jaime; lucía-sanz, adriana; kuhn, jens h.; krupovic, mart; dolja, valerian v.; koonin, eugene v. title: origins and evolution of the global rna virome date: - - journal: mbio doi: . /mbio. - sha: doc_id: cord_uid: p b b t viruses with rna genomes dominate the eukaryotic virome, reaching enormous diversity in animals and plants. the recent advances of metaviromics prompted us to perform a detailed phylogenomic reconstruction of the evolution of the dramatically expanded global rna virome. the only universal gene among rna viruses is the gene encoding the rna-dependent rna polymerase (rdrp). we developed an iterative computational procedure that alternates the rdrp phylogenetic tree construction with refinement of the underlying multiple-sequence alignments. the resulting tree encompasses , rna virus rdrps and consists of major branches; of the branches include positive-sense rna viruses, is a mix of positive-sense (+) rna and double-stranded rna (dsrna) viruses, and consist of dsrna and negative-sense (−) rna viruses, respectively. this tree topology implies that dsrna viruses evolved from +rna viruses on at least two independent occasions, whereas −rna viruses evolved from dsrna viruses. reconstruction of rna virus evolution using the rdrp tree as the scaffold suggests that the last common ancestors of the major branches of +rna viruses encoded only the rdrp and a single jelly-roll capsid protein. subsequent evolution involved independent capture of additional genes, in particular, those encoding distinct rna helicases, enabling replication of larger rna genomes and facilitating virus genome expression and virus-host interactions. phylogenomic analysis reveals extensive gene module exchange among diverse viruses and horizontal virus transfer between distantly related hosts. although the network of evolutionary relationships within the rna virome is bound to further expand, the present results call for a thorough reevaluation of the rna virus taxonomy. required for polymerase activity ( ) ( ) ( ) . rdrps belong to the expansive class of polymerases containing so-called palm catalytic domains along with the accessory fingers and thumb domains ( , ) . in addition to viral rdrps, palm domain polymerases include reverse transcriptases (rts) of retroelements and reverse-transcribing viruses and the dna polymerases that are responsible for genome replication in cellular organisms and diverse dna viruses. within the palm domain class of proteins, rt and the ϩrna virus rdrps are significantly similar in sequence and structure and appear to comprise a monophyletic group ( , ( ) ( ) ( ) . more specifically, the highest similarity is observed between the ϩrna virus rdrps and the rts of group ii introns. these introns are widespread retrotransposons in prokaryotes that are thought to be ancestral to the rts of all other retrotransposons as well as retroviruses and pararetroviruses (recently jointly classified as the order ortervirales) of eukaryotes ( ) ( ) ( ) ( ) . a phylogenetic analysis of the ϩrna virus rdrps revealed only a distant relationship between the leviviruses and the bulk of the eukaryotic ϩrna viruses, leaving the ancestral relationships uncertain ( ) . the origin of eukaryotic ϩrna viruses from their prokaryotic counterparts is an obvious possibility. however, given the dramatically greater prevalence of ϩrna viruses among eukaryotes compared to the narrow spread of leviviruses and "levi-like viruses" in bacteria, an alternative scenario has been proposed. in this scenario, rdrps of the prokaryotic and eukaryotic ϩrna viruses independently descended from distinct rts ( , ) . among eukaryotic ϩrna viruses, phylogenetic analysis of the rdrps strongly supports the existence of picornavirus and alphavirus "supergroups," which are further validated by additional signature genes ( , ) . however, both the exact compositions of these supergroups and the evolutionary relationships among many additional groups of viruses remain uncertain. some rdrp phylogenies suggest a third supergroup combining animal "flavi-like viruses" and plant tombusviruses, but this unification is not supported by additional shared genes and thus remains tenuous ( , , ) . the similarity of rdrps among dsrna viruses is limited, but these rdrps are similar to various degrees to ϩrna virus rdrps. therefore, different groups of dsrna viruses might have evolved from different ϩrna viruses independently, on multiple occasions ( , ) . although it is not entirely clear how prokaryotic dsrna viruses fit into this concept, evidence of an evolutionary affinity between cystoviruses and reoviruses has been presented ( , ) . for a long time, the evolutionary provenance of Ϫrna virus rdrps remained uncertain due to their low sequence similarity to other rdrps and rts ( ) . however, recent protein structure comparisons point to a striking similarity between the rdrps of Ϫrna orthomyxoviruses and those of ϩrna flaviviruses and dsrna cystoviruses ( ) . all these findings notwithstanding, the overall evolutionary relationships among the rdrps of ϩrna, Ϫrna, and dsrna viruses and rts remain unresolved. in particular, whether the rdrps of ϩrna and Ϫrna viruses are mono-or polyphyletic is unclear. many deep evolutionary connections between rna virus groups that were originally thought to be unrelated have been delineated using the results of pre-metagenomicera evolutionary studies. these discoveries culminated in the establishment of rna virus supergroups ( , , ) . however, the evolutionary provenance of many other rna virus groups remained unclear, as did the relationships between the rna viruses of the baltimore classes and retroelements and their ultimate origins. the prospects of substantial progress appeared dim because of the extreme sequence divergence among rna viruses, which could amount to irrevocable loss of evolutionary information. recent revolutionary developments in virus metagenomics (metaviromics) dramatically expanded knowledge of the diversity of rna viruses and provided an unprecedented amount of sequence data for informed investigation into rna virus evolution ( , , ) . the foremost development was the massive expansion of the known invertebrate virome, which was achieved primarily through meta-transcriptome sequencing of various holobionts. the subsequent phylogenetic analysis revealed previously unknown lineages of ϩrna and Ϫrna viruses and prompted reconsideration of high-rank virus unifications, such as ϩrna virus supergroups ( , ( ) ( ) ( ) ( ) . the rna viromes of fungi and prokaryotes also underwent notable expansion, albeit it was not as extensive as that of invertebrates ( , , ) . here we reexamine the evolutionary relationships among and within the baltimore classes of rna viruses through a comprehensive analysis of the available genomic and metagenomic sequences. in particular, to build a phylogenetic tree of thousands of viral rdrps, we designed an iterative computational procedure that alternates phylogeny construction with refinement of the underlying multiple alignments. although rna viruses have relatively short genomes (ϳ to kb), the combined gene repertoire (pangenome) of these viruses includes numerous genes that are shared, to various degrees, by related subsets of rna viruses. to obtain further insight into virus evolution, we therefore attempted to reconstruct the history of gain and loss of conserved proteins and domains in different virus lineages. we also investigated evolution of the single jelly-roll capsid protein (sjr-cp), the dominant type of capsid protein among ϩrna viruses. our analysis revealed patterns that are generally congruent with the rdrp phylogeny and provide further insights into the evolution of different branches of rna viruses. finally, we analyzed a bipartite network in which rna virus genomes are connected via nodes representing virus genes ( , ) to identify distinct modules in the rna virosphere. the results shed light on the evolution of rna viruses, revealing, in particular, the monophyly of Ϫrna viruses and their apparent origin from dsrna viruses, which seem to have evolved from distinct branches of ϩrna viruses on at least two independent occasions. comprehensive phylogeny of rna virus rna-dependent rna polymerases: overall structure of the tree and the major branches. amino acid sequences of rdrps and rts were collected from the nonredundant national center for biotechnology information (ncbi) database and analyzed using an iterative clustering-alignmentphylogeny procedure (see fig. s in the supplemental material) (see materials and methods for details). this procedure ultimately yielded a single multiple alignment of the complete set of , virus rdrp sequences (see data set s in the supplemental material) and , rt sequences organized in ϩ clusters ( clusters of rdrps and clusters of rts; see materials and methods for details). this sequence set did not include rdrps of members of the families birnaviridae and permutatetraviridae, distinct groups of rna viruses that encompass a circular permutation within the rdrp palm domain ( ) and therefore could not be confidently included in the alignment over their entire lengths. the phylogenetic tree of rdrps and rts ( fig. ; see also data set s ) was assembled from a set of trees that represent three hierarchical levels of relationships. at the lowest level, full complements of sequences from each cluster were used to construct clusterspecific trees. at the intermediate level, up to representatives from each cluster were selected to elucidate supergroup-level phylogeny. at the highest level, up to representatives from each cluster were taken to resolve global relationships (data set s a and s ). the final tree (data set s ) was assembled by replacing the cluster representatives with the trees from the previous steps. the large number and immense diversity of the viruses included in our analysis create serious challenges for a systematic, phylogeny-based nomenclature of the identified evolutionary lineages of rna viruses. many such lineages consist of viruses newly discovered by metaviromics and are not yet formally classified by the international committee on taxonomy of viruses (ictv) and therefore cannot be assigned formal names. for the purpose of the present work, we adopted a semiarbitrary naming scheme using the following approach. (i) we use taxon names that had been fully accepted by the ictv as of march ( ) whenever possible. these names are recognizable through their capitalization and italicization and rank-specific suffixes (e.g., -virales for orders and -viridae for families). as is the common practice in virus taxonomy, the officially classified members of each ictv-approved taxon are referred to via vernacular designations (recognizable through their lack of capitalization and italicization). for instance, the members of the ictv-approved order bunyavirales are called bunyaviruses, whereas those of the family tombusviridae are called tombusviruses. however, in this work, both taxon and vernacular terms are to be understood sensu lato: if our analysis indicates certain viruses to be members of or very closely related to an ictv-established taxon, we consider them members of that taxon despite the lack of current ictv recognition. as a result, the order bunyavirales has more members in our analysis than in the official taxonomy. (ii) we use vernacular names in quotation marks for viruses/lineages that are clearly distinct from those covered by the official ictv framework. whenever possible, we use names that circulate in the literature (e.g., "hepeliviruses," "statoviruses"). in the absence of such unofficial names, we name the lineage reminiscent of the next most closely related lineage (e.g., "levi-like viruses" are a clearly distinct sister group to leviviridae/leviviruses). (iii) monophyletic clusters that transcend the currently highest ictv-accepted rank (i.e., order) are labeled according to terms circulating in the literature (i.e., "alphavirus supergroup," "flavivirus supergroup," and "picornavirus supergroup"). (iv) lineages represented by a single virus are labeled with the respective virus name. rooting the phylogenetic tree, generated using phyml ( ) , between the rts and rdrps resulted in a well-resolved topology of rna viruses in which the tree splits into major branches, each including a substantial diversity of viruses (fig. ) . branch consists of leviviruses and their eukaryotic relatives, namely, "mitoviruses," "narnaviruses," and "ourmiaviruses" (the latter three terms are placed in quotation marks as our analysis contradicts the current ictv framework, which classifies mitoviruses and narnaviruses as members of one family, narnaviridae, and ourmiaviruses as members of a free-floating genus, ourmiavirus). branch ("picornavirus supergroup") consists of a large assemblage of ϩrna viruses of eukaryotes, in particular, those of the orders picornavirales and nidovirales; the families caliciviridae, potyviridae, astroviridae, and solemoviridae, a lineage of dsrna viruses that includes partitiviruses and picobirnaviruses; and several other, smaller groups of ϩrna and dsrna viruses. branch consists of a distinct subset of ϩrna viruses, including the "alphavirus supergroup" along with the "flavivirus supergroup," nodaviruses, and tombusviruses; the "statovirus," "wèivirus," "yànvirus," and "zhàovirus" groups; and several additional, smaller groups. branch consists of dsrna viruses, including cystoviruses, reoviruses, and totiviruses and several additional families. branch consists of Ϫrna viruses. each of these major branches of the tree is strongly supported by bootstrap replications (fig. ). assuming the rt rooting of the tree, branch , which consists of leviviruses and their relatives infecting eukaryotes, is a sister group to the rest of rna viruses; this position is highly robust with respect to the choice of the phylogenetic method and parameters. this tree topology is compatible with the monophyly of the rdrps and, by inference, of rna viruses and the origin of eukaryotic rna viruses from a prokaryotic rna virus ancestor shared with leviviruses. the deeper history remains murky. we have no information on the nature of the common ancestor of retroelements and rna viruses, let alone on whether the ancestor was an rna virus or a retroelement. however, parsimony considerations suggest that a retroelement ancestor is more likely given that capsids first appeared in the virus part of the tree rather than having been lost in retroelements. the next split in the tree occurs between branch and the short stem that formally joins branches , , and . however, the unification of branch with branches and is weakly supported and might not reflect actual common ancestry. arguably, the most striking feature of the rna virus tree topology is the paraphyly of ϩrna viruses relative to dsrna and Ϫrna viruses. indeed, according to this phylogeny, Ϫrna viruses evolved from within dsrna viruses, whereas dsrna viruses are polyphyletic (fig. ). one major group of dsrna viruses that includes partitiviruses and picobirnaviruses is firmly embedded within ϩrna virus branch , whereas another, the members of a larger dsrna virus group that includes cystoviruses, reoviruses, totiviruses, and viruses from several other families comprise a distinct branch, branch , which might be related to ϩrna virus branch (fig. ). this placement of the two branches of dsrna viruses is conceptually compatible with the previous evolutionary scenarios of independent origins from ϩrna viruses. however, the presence of a strongly supported branch combining lineages of dsrna viruses that infect both prokaryotes and eukaryotes suggests a lesser extent of polyphyly in the evolution of dsrna viruses than originally proposed ( , ) . an alternative phylogenetic analysis of the same rdrp alignment using raxml yielded the same main branches, albeit some with weak support (data set s b). furthermore, although the dsrna viruses are split the same way using raxml as in the phyml tree, the nested tree topology, in which branch (the bulk of dsrna viruses) is lodged deep within ϩrna viruses and branch (Ϫrna viruses) is located inside branch , is not reproduced (data set s b). instead, branches and are separate and positioned deep in the tree, immediately above the split between branch and the rest of the rdrps. given the poor resolution of the raxml tree and a strong biological argument, namely, the absence of identified Ϫrna viruses in prokaryotes or protists (with the exception of the "leishbuviruses" infecting kinetoplastids [ , ] ; see also discussion below), we believe that the tree topology presented in fig. carries more credence than that shown in data set s b. nevertheless, these discrepancies emphasize that utmost caution is due when biological interpretation of deep branching in trees of highly divergent proteins is attempted. the rdrp is the only universal protein of rna viruses. accordingly, other viral genes must have been gained and/or lost at different stages of evolution. thus, after performing the phylogenetic analysis of rdrps, we assigned the proteins and domains shared by diverse viruses to the branches of the rdrp tree. multiple alignments and hidden markov model (hmm) profiles were constructed for , proteins and domains encoded by rna viruses, and a computational pipeline was developed to map these domains on the viral genomes (data set s ). the resulting patterns of domain presence/absence in the branches of the rdrp tree were used to reconstruct the history of the gains and losses of rna virus genes (or proteins and domains, for simplicity), both formally, using the ml-based gloome method ( ) , and informally, from parsimony considerations. these reconstructions reveal a high level of branch specificity in the evolution of the gene repertoire of rna viruses. the only protein that is likely to have been gained at the base of the eukaryotic virus subtree (branches to ) (which also includes the bacterial cystoviruses) is the single jelly-roll capsid protein (sjr-cp) (fig. s a ). retroelements lack capsid proteins, and therefore, there is no indication that sjr-cp was present in the hypothetical element that encoded the common ancestor of rdrps and rts. furthermore, reconstruction of the evolution of branch (leviviruses and their relatives) argues against the ancestral status of sjr-cp in this branch. (ii) branch : leviviruses and their descendants. the current rdrp tree topology combined with gene gain-loss reconstruction suggests the following evolutionary scenario for branch ( fig. a) : a levivirus-like ancestor that, like the extant members of the leviviridae, possessed a capsid protein unrelated to sjr-cp ( , ) gave rise to naked eukaryotic rna replicons known as "mitoviruses" and "narnaviruses." these replicons consist of a single rdrp gene (fig. b ) and replicate in mitochondria and in the cytosol of the host cells of fungal and invertebrate hosts, respectively (the latter hosts were identified in metaviromic holobiont analyses) ( , ) . recently, the existence of plant "mitoviruses" has been reported although it is not known whether these viruses reproduce in the mitochondria ( ) . the "narnavirus" rdrp is also the ancestor of the rdrp of the expanding group of "ourmiaviruses" (fig. a) . "ourmiaviruses" were originally identified in a narrow range of plants, and genomic analysis revealed the chimeric nature of these viruses, with a "narnavirus"-like rdrp but sjr-cps and movement proteins (mps) which were apparently acquired from "picorna-like" and "tombuslike" viruses, respectively ( ) . use of metaviromics has led to the identification of numerous related viruses associated with invertebrates, many of which encode distinct sjr-cp variants and some of which acquired an rna helicase ( fig. b and s ) ( ) . thus, the evolution of this branch apparently involved the loss of the structural module of leviviruses, which yielded naked rna replicons that reproduced in the mitochondria of early eukaryotes. a group of these replicons subsequently escaped to the cytosol, which was followed by the reacquisition of unrelated structural modules from distinct lineages of eukaryotic viruses inhabiting the same environment (fig. b ). this complex evolutionary scenario emphasizes the key role of modular gene exchange in the evolution of rna viruses. (iii) branch : picornavirus supergroup. expansive branch generally corresponds to the previously described "picornavirus supergroup" (fig. and ) ( , ) . some of the virus groups that were previously considered peripheral members of this supergroup, such as totiviruses and nodaviruses, were relocated to different branches in the present tree (branches and , respectively), whereas the viruses of the order nidovirales were moved inside branch from an uncertain position in the tree. nevertheless, the core of the supergroup remains coherent, suggestive of common ancestry. within branch , major clades are strongly supported (fig. a) ; however, many of the internal branches are less reliable, so that the relative positions of partitivirus-picobirnavirus, potyvirusastrovirus, and nidovirus clades within branch remain uncertain. the largest and most coherent of the branch clades includes the cornerstone of the picornavirus supergroup, the ϳ viruses-strong order picornavirales ( ), expanded with caliciviruses, solinviviruses, and a multitude of unclassified viruses infecting invertebrates, vertebrates, fungi, protists, and undefined hosts (for viruses discovered by metaviromics) (fig. ) ( , , , ( ) ( ) ( ) . the second largest, deep-branching clade consists of two lineages that include, respectively, ϩrna and dsrna viruses (fig. ). the ϩrna virus lineage combines astroviruses and potyviruses, the evolutionary affinity of which is well recognized ( , ) . the dsrna lineage includes the members of the families amalgaviridae, hypoviridae, partitiviridae, and picobirnaviridae, with each of these families greatly expanded by unclassified affiliates. finally, the "middle" clade is smaller and less diverse; it encompasses nidoviruses, including the longest of all ϩrna virus genomes ( ) , and solemoviruses with much shorter genomes (fig. ) . notably, some members of the family luteoviridae and heterocapsa circularisquama rna virus, the only known alvernavirus ( ) , are nested within the solemovirus clade. given the lack of support beyond the phylogenetic affinity of the rdrps and the dramatic differences in the genomic architectures of nidoviruses and solemoviruses, the possibility that this unification was caused by a tree construction artifact is difficult to rule out (the branch support notwithstanding). hypoviruses, representing a group of fungal capsidless rna replicons, have been traditionally viewed as dsrna viruses. however, comparisons of genome architectures and phylogenetic analyses suggested that hypoviruses are derivatives of potyviruses (continued on next page) global rna virome ® that have lost the capsid protein ( , ) . in the current rdrp tree, hypoviruses cluster with the dsrna viruses of the partitivirus-picobirnavirus clade rather than with potyviruses (fig. ). whether this position is an artifact of tree construction or whether hypoviruses actually share the rdrps with dsrna viruses is unclear. the partitivirus-picobirnavirus clade within branch represents a transition to the bona fide dsrna baltimore class (fig. ) . typical partitiviruses and picobirnaviruses have minimalist genomes that consist of two dsrna segments encapsidated separately into distinct -subunit tϭ capsids ( ) ( ) ( ) ( ) . these genome segments encode, respectively, rdrps and cps that are clearly homologous between the two families. the cps of the partitivirus-picobirnavirus clade have been suggested to be distantly related to those of other dsrna viruses that belong to branch ( , ) . notably, this clade also includes some naked rna replicons that reproduce in algal mitochondria or chloroplasts, use a mitochondrial genetic code, and, in terms of lifestyle, resemble "mitoviruses" ( , , ) . by analogy, the origin of the partitivirus-picobirnavirus group in an as-yet-undiscovered lineage of prokaryotic rna viruses seems likely. more specifically, this group of dsrna viruses could have evolved through reassortment of genomic segments encoding, respectively, a ϩrna virus rdrp of branch (possibly a naked rna replicon) and a dsrna virus capsid protein related to those of branch viruses. the most recently evolved branch of partitiviruses is characterized by larger, -to -partite genomes, in contrast to the mono-or bipartite genomes in the deeper branches ( ) . this observation emphasizes a major tendency in virus evolution: increases in genome complexity via gradual acquisition of accessory genes ( ) . apart from the sjr-cp, an apparently ancestral protein that is likely to represent a shared derived character (synapomorphy) of branch is a serine protease that is present in members of the order picornavirales (with the diagnostic substitution of cysteine for the catalytic serine), members of the potyvirus-astrovirus clade, solemoviruses, alvernavirus, and nidoviruses ( fig. b ; see also fig. s b ). as demonstrated previously, this viral protease derives from a distinct bacterial protease, probably of mitochondrial origin, which is compatible with an early origin of branch in eukaryotic evolution ( ) . the reconstruction of protein gain and loss, together with the comparison of genome architectures in this branch, revealed extensive rearrangements as well as gene and module displacement ( fig. b ; see also fig. s b). branch includes viruses with relatively long genomes and complex gene repertoires (nidoviruses, potyviruses, and many members of picornavirales) along with viruses with much shorter genomes and minimal sets of genes (astroviruses and solemoviruses). clearly, evolution of branch viruses involved multiple gene gains. of special note is the gain of distinct helicases in clades within this branch: superfamily helicases (s h) in members of picornavirales, superfamily helicases (s h) in potyviruses, and superfamily helicases (s h) in nidoviruses ( fig. b ; see also fig. s b ). this independent, convergent gain of distinct helicases reflects the trend noticed early in the study of rna virus evolution, namely, that most viruses with genomes longer than ϳ kb encode helicases, whereas smaller ones do not. this difference conceivably exists because helicase activity is required for the replication of longer rna genomes ( ) . another notable feature is the change of virion morphologies among potyviruses (replacement of ancestral sjr-cp by an unrelated cp forming filamentous virions) and nidoviruses (displacement by a distinct nucleocapsid protein). the dramatic change in virion morphology and mode of ge- where a conserved domain comprises only a part of the larger protein, the rest of this protein is shown in light gray. the locations of such domains are approximated (indicated by fuzzy boundaries). c pro , c chymotrypsin-like protease; cp, capsid protein; e, envelope protein; en, nidoviral uridylate-specific endoribonuclease (nendou); exo, =-to- = exoribonuclease domain; fcp, capsid protein forming filamentous virions; m, membrane protein; md, macrodomain; mp, movement protein; mt, ribose- -o-methyltransferase domain; n, nucleocapsid protein; n , guanine-n -methyltransferase; ppro, papain-like protease; sjr and sjr , single jelly-roll capsid proteins of type and type ; spike, spike protein; s h, superfamily helicase; s h, superfamily helicase; s h, superfamily helicase; vp , virion protein ; z, zn-finger domain; spro, serine protease; p , protein . distinct hues of same color (e.g., green for mps) are used to indicate cases where proteins that share analogous function are not homologous. wolf et al. ) that most likely were acquired independently of the capping enzymes of other rna viruses. nidoviruses also gained ribonucleases and other accessory proteins that are involved in genome replication, virulence, and other aspects of the infection cycle of these largest known rna viruses ( , ( ) ( ) ( ) . (iv) branch : "alphavirus supergroup" and "flavivirus supergroup" and the extensive diversity of "tombus-like viruses." branch is the part of the ϩrna virus rdrp tree that underwent the most dramatic rearrangements compared to previous versions. this branch consists of two strongly supported clades of ϩrna viruses: (i) the assemblage that originally was defined as the "alphavirus supergroup" ( , ) joined by several additional groups of viruses and (ii) flaviviruses and related viruses ("flavivirus supergroup"; fig. and ). in the former clade, the alphavirus supergroup encompasses an enormous diversity of plant, fungal, and animal ϩrna viruses and consists of well-supported lineages, namely, tymoviruses, virgaviruses/alphaviruses/endornaviruses, and hepeviruses/benyviruses, each accompanied by related viruses that often form as-yet-unclassified lineages (fig. a ). within the "alphavirus supergroup" alone, the genome lengths range from ϳ to ϳ kb. despite this length variation, all supergroup members harbor a conserved rna replication gene module encoding a cape, s h, and rdrp; the conservation of this module attests to the monophyly of the supergroup (fig. b) . in contrast, virion architectures differ dramatically even within each of the three lineages of the "alphavirus supergroup." the major structural themes include variants of icosahedral capsids formed by sjr-cp (e.g., bromoviruses and tymoviruses); unrelated icosahedral capsids enveloped in a lipoprotein bilayer (togaviruses); flexuous filamentous capsids formed by a distinct type of cp (alphaflexiviruses, betaflexiviruses, gammaflexiviruses, and closteroviruses); and rigid rod-shaped capsids assembled from another distinct cp (benyiviruses and virgaviruses). it was traditionally thought that the latter capsid type is specific to viruses of flowering plants ( ) . however, the recent discovery of a virgavirus-like cp in invertebrate viruses (e.g., bě ihǎi charybdis crab virus [ fig. b ]) ( ) suggests that the emergence of this unique cp fold antedates land colonization by plants at ϳ million years ago (mya). yet another "structural" theme is offered by endornaviruses, naked rna replicons which, similarly to the hypoviruses in branch (see above), originally were classified as dsrna viruses. however, endornaviruses possess all the hallmarks of the alphavirus supergroup and are clearly derived from ϩrna viruses of this group. they seem to have been mislabeled dsrna viruses due to the accumulation of dsrna replication intermediates in infected cells ( , ) . a parallel loss of the cp genes apparently occurred in the deltaflexiviruses, which, in rdrp phylogenies, form a sister group to the flexible filamentous gammaflexiviruses ( ) , and in the umbraviruses that are included in the family tombusviridae based on the rdrp phylogeny. notably, unlike most other capsidless viruses that are vertically inherited, umbraviruses can hijack capsids of coinfecting luteoviruses for aphid transmission ( ) . within branch , the phylogenetically compact alphavirus supergroup is embedded within the radiation of diverse virus groups, including the well-known tombusviruses and nodaviruses, along with several newcomers discovered via metaviromics, such as the "statovirus," "wèivirus," "yànvirus," and "zhàovirus" groups ( , , ) (fig. a ). our rdrp analysis revealed remarkable phylogenetic heterogeneity within and among these groups and split the "tombus-like viruses" into lineages with distinct evolutionary affinities (groups "uncl. [unclassified] inv. [invertebrates]" and subsets of "tombus-like viruses" and "nodaviruses" in fig. a ). this subdivision is also supported by the analysis of the cps of these viruses (see section on sjr-cp evolution) (fig. ) . therefore, in contrast to the alphavirus supergroup, nodaviruses, and the flavivirus supergroup, the term "tombus-like" loses its evolutionary and taxonomic coherence. accordingly, we use the term "tombusviruses" (without quotation marks) only for one lineage that includes the members of the current family tombusviridae along with a broad variety of related plant and invertebrate holobiont viruses ( ) . the previously suggested, tenuous flaviviridae-tombusviridae affinity is gone in the present tree, although members of both families belong to the same major branch, branch . plant tombusviruses (and members of closely related plant virus genera), representing the only group of "tombus-like viruses" that was available at the time of previous analyses ( , ) , now form but a small twig deep within the large assemblage that we refer to as tombusviruses. tombusviruses are affiliated with "statoviruses" ( ) and with a subset of unclassified viruses from invertebrate holobionts rather than with flaviviruses (fig. a) . flaviviruses now form a separate clade within branch , the flavivirus supergroup that includes members of four recognized flaviviral genera (pegivirus, hepacivirus, flavivirus, and pestivirus), the newly discovered "jı ngménviruses" with segmented genomes ( ) , and a variety of unclassified, extremely divergent "flavi-like viruses" of animals and plants. this clade is split into two well-supported lineages; one includes pegiviruses and hepaciviruses, and the other consists of the rest of flaviviruses (fig. a ). flaviviral virions are enveloped, with the envelope proteins forming an external icosahedral shell, whereas the core nucleocapsid is apparently disordered; the evolutionary provenance of the core protein, with its distinct fold, is unclear ( , , ) . notably, flaviviral envelope proteins are class ii fusion proteins that are closely related to alphavirus envelope e proteins ( ) . the theme of gene swapping between these distantly related virus groups of branch is further emphasized by the homology between the alphavirus cps that form icosahedral capsids under the lipid envelopes and the flavivirus nonstructural ns proteases that share a chymotrypsin-like fold ( ) . because the rdrp tree topology implies that the alphavirus ancestor is more recent than the ancestor of flaviviruses (fig. ) , such adoption of the ns protease for a structural role is suggestive of emerging alphaviruses borrowing their structural module from preexisting flaviviruses ( ) . the hallmark of branch is the capping enzyme (cape), which is present in the entire "alphavirus supergroup" and in flaviviruses ( fig. ; see also fig. s b). a highly divergent version of cape has been identified in nodaviruses ( ) and, in our present analysis, in the additional subset of viruses that grouped with nodaviruses, as well as in a few viruses scattered throughout the clade. formally, cape is inferred to be ancestral in the entire branch . however, capes of "alphavirus supergroup" members, nodaviruses, and flaviviruses are related only distantly to one another, and at least the latter have closer eukaryotic homologs, namely, the ftsj family methyltransferases ( , ) . furthermore, tombusviruses, statoviruses, yànviruses, zhàoviruses, wèiviruses, and members of the pegivirus-hepacivirus lineage of flaviviruses lack cape, putting into question its presence in the ancestor of this branch ( to an even greater extent than in branch , the apparent routes of virus evolution in branch involve lineage-specific gene capture that resulted in evolution of complex cp-spro, capsid protein-serine protease; e, envelope protein; fcp, divergent copies of the capsid protein forming filamentous virions; hsp h, hsp homolog; mp, movement protein; ns, nonstructural protein; nsp to nsp , nonstructural proteins; ppro, papain-like protease; prm, precursor of membrane protein; rcp, capsid protein forming rod-shaped virions; ris, rna interference suppressor; s h, superfamily helicase; s h, superfamily helicase; sjr , single jelly-roll capsid proteins of type ; spro, serine protease; votu, virus otu-like protease; ns, nonstructural protein. distinct hues of same color (e.g., green for mps) are used to indicate the cases when proteins that share analogous function are not homologous. global rna virome ® genome architectures (fig. b) . the most notable cases are the closteroviruses and divergent flaviviruses that have genomes of up to to kb, rivalling coronaviruses in terms of genome length and the complexity of the gene repertoire ( , ( ) ( ) ( ) . the lack of genes assigned to the common ancestor of branch (with the obvious exception of the rdrp) prevents development of a coherent evolutionary scenario for the entire branch. in the case of the clade encompassing the "alphavirus supergroup" and related viruses, a potential common ancestor could be a simple virus that encoded only an rdrp and a sjr-cp, a cp fold most broadly represented in this clade, including diverse tombusviruses, nodaviruses and members of bromoviridae, and tymoviridae within the "alphavirus supergroup." proposal of such an ancestor for the flavivirus clade is challenged by the lack of viruses with short and simple genomes among flaviviruses. indeed, the lengths of the genomes in this clade range from ϳ kb to ϳ kb, with even the shortest ones encoding at least three of the flavivirus signature genes (serine protease [spro], s h, and rdrp). one potential clue, however, is provided by "jı ngménviruses," with tetrapartite genomes in which the protease-helicase modules and the rdrps are encoded by separate genome segments; two other segments apparently encode structural proteins of unclear provenance ( ) . this genome architecture could hint at an ancestral flavivirus genome that was assembled from genes borrowed from preexisting viruses, one of which possessed a divergent "tombus-like virus" rdrp. although the origins of branch are murky, major trends in its subsequent evolution clearly included lineage-specific gene capture, starting with helicases and capes in the ancestors of the major lineages and followed by diverse genes in smaller groups (fig. b) . (v) branch : dsrna viruses. branch , which joins branch with weak support, includes the bulk of the dsrna viruses ( fig. and ). all dsrna viruses in this branch share a unique virion organization and encode homologous cps. in particular, the specialized icosahedral capsids of these viruses, involved in transcription and replication of the dsrna genome inside cells, are constructed from homo-or heterodimers of cp subunits organized on an unusual tϭ (also known as pseudo-tϭ ) lattice ( , ) . the only exceptions are the chrysoviruses, which encode large cps corresponding to the cp dimers of other dsrna viruses and form genuine tϭ capsids ( ) . the icosahedral capsids of partitiviruses and picobirnaviruses, which encode rdrps belonging to branch , are also constructed from homodimers ( , , ) and have been suggested to be evolutionarily related to those of the dsrna viruses from rdrp branch ( ) despite little structural similarity between the corresponding cps. totiviruses, many of which have "minimal" genomes encoding only rdrps and cps, comprise one of the two major clades in branch , whereas cystoviruses, the only known prokaryotic dsrna viruses, together with members of the vast family reoviridae, which consists of multisegmented dsrna viruses infecting diverse eukaryotes, comprise the second clade (fig. ) . the concept of the closer phylogenetic affinity between cystoviruses and reoviruses appears to be corroborated by the fact that the inner tϭ icosahedral capsid is uniquely encased by the outer icosahedral shell constructed on a tϭ lattice in both families ( ) . both cystoviruses and reoviruses appear to have gained many cladespecific genes, in particular, reca-like packaging atpases of the former ( ) and the capes of the latter that are only distantly related to capes of other rna viruses and likely were acquired independently ( , ) ( (vi) branch : ؊rna viruses. branch , the % supported lineage combining all Ϫrna viruses, is lodged within branch as the sister group of reoviruses, and this position is upheld by two strongly supported internal branches in the rdrp tree ( fig. and ). the Ϫrna branch splits into strongly supported clades. the first clade encompasses the viruses-strong membership of the order mononegavirales ( ), along with the members of the distantly related family aspiviridae ( ), groups of Ϫrna viruses discovered through metaviromics ("chǔ viruses," "qínviruses," and "yuèviruses") ( , ) , and a group of unclassified fungal viruses (fig. a) ( , ) . in contrast to the members of the mononegavirales, most of which possess unsegmented genomes, the remainder of this clade is characterized by bi-, tri-, or even tetraseg-mented genomes (fig. b) . the second clade combines the family orthomyxoviridae, the genus tilapinevirus ( ), and the large order bunyavirales ( viruses) ( ) . the latter order consists of two branches, one of which is the sister group to the orthomyxovirus/tilapinevirus clade (albeit with weak support). the numbers of negativesense or ambisense genome segments in this clade range from to in most of bunyaviruses to in viruses of the genus emaravirus and the orthomyxovirus/tilapinevirus group ( , , , ) . a notable acquisition in the first clade is a cape, whereas members of the second clade share "cap-snatching" endonucleases (en) ( ) . patterns of the single jelly-roll capsid protein evolution. the sjr-cp is the dominant type of cp among ϩrna viruses and is also found in members of one family of dsrna viruses (birnaviridae). structural comparisons indicate that sjr-cps of rna viruses form a monophyletic group and likely were recruited from cellular sjr proteins on a single occasion during the evolution of rna viruses ( ) . the short length and high divergence of sjr-cps preclude adequate resolution in phylogenetic analysis; thus, we performed profile-profile sequence comparisons and clustering of all viral sjr-cp sequences in our data set (see materials and methods for details). analysis of the resulting network reveals patterns that are generally congruent with the rdrp phylogeny and provides further insights into the evolution of different branches of rna viruses. at conservative p value thresholds (p Ͻ e Ϫ ), the majority of sjr-cps segregated into two large clusters, both of which contained representatives from rdrp branch . cluster included the members of picornavirales and caliciviridae and diverse "picornalike viruses" of invertebrates, whereas cluster consisted of the members of the families astroviridae, luteoviridae, and solemoviridae and "sobemo-like viruses" (fig. s ). in addition, cluster contained members of several families from rdrp branch , namely, tombusviridae (and diverse "tombus-like viruses"), hepeviridae, a subgroup of nodaviridae, and "statoviruses." at less-restrictive p value thresholds (p Ͻ e Ϫ ), all sjr-cps were interconnected, largely through making contacts to the core of cluster . only "ourmiaviruses" had stronger affinity to picornaviruses in cluster (fig. ) . this pattern of connectivity is consistent with the radiation of sjr-cps from a common ancestor, likely resembling sequences from cluster of branch . this analysis also reveals high cp sequence divergence among members of some families (e.g., bromoviridae) and numerous cases of apparent cp gene replacement. for instance, the cps of nodaviruses fall into two groups; one is related to the turreted cps of tetraviruses, and the other is similar to cps of tombusviruses, mirroring the rdrp phylogeny (fig. ) . at a greater phylogenetic distance, cps of astroviruses and hepeviruses are closely related despite their affiliation with branches and , respectively, suggesting cp gene replacement in the ancestor of one of the two families. given that the cps of hepeviruses connect to sjr-cps of other viruses through astroviruses, cp gene replacement most likely occurred in the ancestor of hepeviruses (fig. s ) . notably, the cps of "zhàoviruses," "wèiviruses," and "tombus-like" and "sobemo-like" viruses (representing a diverse virus assemblage within the solemovirus branch, to the exclusion of bona fide solemoviridae) did not form discrete clusters but rather were affiliated with diverse virus groups, suggesting extensive recombination in these viruses, with multiple cp gene exchanges (fig. ) . in the case of unclassified "narnaviruses" and "ourmiaviruses," the cp genes apparently were acquired on more than independent occasions from different groups of viruses, emphasizing the impact of recombination and gene shuffling in the evolution of rna viruses. previously, a similar extent of chimerism was also observed among singlestranded dna (ssdna) viruses ( , ) , highlighting the evolutionary and functional plasticity of short viral genomes. the modular gene-sharing network of rna viruses: gene transfer and module shuffling. the pronounced structural and functional modularity of virus proteomes and the pervasive shuffling of the genomic regions encoding distinct protein modules are key features of virus evolution ( , , ) . therefore, a productive approach to the study of the virosphere that complements phylogenetics is the construction and analysis of networks of gene sharing. bipartite networks, in which one type of node corresponds to genes and another to genomes, have been employed to investigate the double-stranded dna (dsdna) domain of the virosphere ( ) . this analysis revealed hierarchical modular organization of the network, with several modules that included global rna virome ® nonobvious connections between disparate groups of viruses ( , ) . although this type of analysis is less informative for rna viruses due to the small number of proteins encoded in each viral genome, the "pan-proteome" of rna viruses is large (data set s ), prompting us to experiment with bipartite gene sharing networks for rna viruses. the initial search for statistically significant modularity identified distinct modules, most of which included a single virus family (fig. a and b) . remarkably, the family reoviridae has been split into modules, highlighting the vast diversity of this family, comparable to that seen in order-level taxa. among the exceptions, the most expansive module included the viruses of the order picornavirales (module ), together with those of the family caliciviridae (module ), which are linked through the conserved suite of genes that includes those encoding sjr-cp, chymotrypsin-like protease, s h, and the rdrp (fig. a and c) . viruses of the order bunyavirales were also recovered in a single module characterized by the presence of a conserved nucleocapsid (with the exception of the families nairoviridae and arenaviridae) and the cap-snatching endonuclease (module ; fig. a and c) . the next stage of the network analysis aimed at detecting the supermodules that are formed from the primary modules via connecting genes. the results of the analyses of the supermodules of rna viruses failed to attain statistical significance due to the small number of shared genes; nevertheless, some notable connections were revealed by this analysis. specifically, overlapping supermodules were identified (fig. c) . the largest and, arguably, most remarkable is a supermodule that combines ϩrna, dsrna, and Ϫrna viruses that share the capping enzymes, s h (with the exception of reoviridae and mononegavirales), and additional connector genes (e.g., the otu family protease) that link some of the constituent modules. the second largest supermodule combines large subsets of viruses from rdrp branches and that are connected through the sjr-cp and the chymotrypsin-like protease. another supermodule encompasses enveloped ϩrna viruses of the families flaviviridae and togaviridae and Ϫrna viruses of the order bunyavirales (except for arenaviridae) that share homologous envelope glycoproteins (except for flaviviruses) and class ii fusion proteins. information from gene sharing is inherently limited for rna viruses due to the small number of genes in each genome. nevertheless, the bipartite network analysis revealed prominent "horizontal" connections that are underlain either by actual gene exchange or by parallel acquisition of homologous genes by distinct rna viruses. host ranges of rna viruses: evolutionary implications and horizontal virus transfer (hvt). rna viruses have been identified in representatives of all major divisions of eukaryotes, whereas in prokaryotes, members of two families of rna viruses are known to infect only a limited range of hosts ( , , , ) . for branch in our phylogenetic tree of rdrps, the route of evolution from leviviruses infecting prokaryotes to eukaryotic "ourmiaviruses" of plants and invertebrates is readily traceable and involves a merger between a levivirus-derived naked rna replicon that eukaryotes most likely inherited from the mitochondrial endosymbiont with the sjr-cp of a eukaryotic "picorna-like virus." notably, such a merger seems to have occurred on at least three other independent occasions in branch because several groups of invertebrate holobiont "narnaviruses" and some "ourmiaviruses" encode distantly related sjr-cps that apparently were acquired from different groups of plant and animal viruses (fig. ) . the case of cystoviruses is less clear given that this clade is sandwiched between eukaryotic viruses in branch and therefore does not seem to be a good candidate for classification as the ancestor of this branch. it appears more likely that the ancestor was a toti-like virus, whereas cystoviruses were derived forms, which would imply virus transfer from eukaryotes to prokaryotes. however, an alternative scenario might be considered. no known prokaryotic viruses are classified in branch , but it has been proposed that the picobirnaviruses, for which no hosts have been reliably identified, actually are prokaryotic viruses. this proposal is based on the conspicuous conservation of functional, bacterium-type, ribosome-binding sites (shine-dalgarno sequences) in picobirnavirus genomes ( , ) . should that be the case, viruses of prokaryotes might be lurking among totiviruses as well. then, branch would stem from a prokaryotic ancestor, obviating the need to invoke virus transfer from eukaryotes to prokaryotes to explain the origin of the cystoviruses. we made an attempt to quantify the potential horizontal virus transfer (hvt) events in the rna viruses that represent the major branches of the rdrp tree. the leaves of the tree were labeled with the known hosts, the entropy of the host ranges for each subtree was calculated, and the resulting values were plotted against the distance from the root (fig. ). by design, for all branches, entropy (host diversity) drops from the maximum values at the root to zero at the leaves. all branches show substantial host range diversity such that, for example, at the half-distance from the root to the leaves, all branches, except for branch , retain at least half of the diversity (fig. ). furthermore, the differences between the branches are substantial, with the highest entropy observed in branch (dsrna viruses) and branch (Ϫrna viruses). with all the caveats due to potential errors and ambiguities in host assignment, this analysis strongly suggests that hvt played an important role in the evolution of all major groups of rna viruses. rna virus evolution coming into focus. this work was prompted by the advances of metaviromics, which have dramatically increased the known diversity of rna viruses ( , , , , ) . we reasoned that this expansion of the rna virosphere could provide an improved understanding of virus evolution. although further progress of metaviromics and enhanced phylogenomic methods will undoubtedly change current ideas, we believe that some key aspects of rna virus evolution are indeed coming into focus. the expanded diversity of rna viruses, combined with the iterative procedure for phylogenetic analysis, allowed us to obtain a tree of all rdrps and the most closely related rts in which the main branches are strongly supported and thus appear to be reliable (fig. ) . to our knowledge, the picture of rna virus evolution emerging from the tree has not been presented previously. the tree seems to clarify the relationships between the baltimore classes of rna viruses by revealing the nested tree structure in which dsrna viruses evolved, on at least two occasions, from ϩrna viruses, whereas Ϫrna viruses evolved from a distinct group of dsrna viruses. the derivation of Ϫrna viruses from dsrna viruses is, arguably, the most unexpected outcome of the present analysis, considering the lack of genes (other than the rdrp gene) shared by these virus classes. clearly, given that the primary evidence behind the derivation of Ϫrna viruses from within dsrna viruses comes from deep phylogeny, extreme caution is due in the interpretation of this observation. however, the pronounced similarity between the three-dimensional ( d) structures of the rdrps of Ϫrna influenza virus a and bacteriophage dsrna cystovirus ( ) is compatible with our findings. further, because virtually no Ϫrna viruses are known in prokaryotes or unicellular eukaryotes (with the single exception of the "leischbuviruses" in parasitic trypanosomatids [ ] , which were likely acquired from the animal hosts of these protists), their later origin from a preexisting group of ϩrna or dsrna viruses appears most likely. the ϩrna to dsrna to Ϫrna scenario of rna virus genome evolution also makes sense in terms of the molecular logic of genome replication-expression strategies. indeed, ϩrna viruses use the simplest genomic strategy and, in all likelihood, represent the primary pool of rna viruses. the dsrna viruses conceivably evolved when a ϩrna virus switched to encapsidating a replicative intermediate (dsrna) together with the rdrp. naked replicons similar to those of "mitoviruses," hypoviruses, and endornaviruses might have been evolutionary intermediates in this process. this switch does not seem to be as "easy" and common as previously suspected ( , ) but, nevertheless, appears to have occurred at least twice during the evolution of rna viruses. the global rna virome ® origin of Ϫrna viruses is the next step during which the plus strand is discarded from the virions, perhaps simplifying the processes of transcription and replication. conceivably, the evolution of dsrna and Ϫrna viruses, in which transcription and replication of the viral genomes were confined to the interior of virions or nucleocapsid transcription/replication complexes and no dsrna accumulated in the infected cells, was driven by the advantage of escaping some of the host defense mechanisms, in particular, rna interference (rnai) ( , ) . the membrane-associated replication complexes of ϩrna viruses could represent an initial step in this direction ( ) . obviously, evolution of the rdrp does not equal evolution of viruses; other genes, in particular, those encoding capsid and other structural proteins, are crucial for virus reproduction, and these genes often have different histories. the reconstruction of gene gain and loss sheds some light on these aspects of rna virus evolution. the ancestors of each of the major branches of rna viruses (except for branch ) appear to have been simply organized ϩrna viruses resembling tombusviruses (fig. ) . thus, these types of viruses encoding rdrps and sjr-cps might have been ancestral to the bulk of eukaryotic rna viruses (apart from those in branch that derive directly from prokaryotic leviviruses). the subsequent parallel capture of different helicases enabled evolution of increasingly complex genomes via accumulation of additional genes (fig. ) . notably, similar levels of complexity, with a complete coding capacity of to kb, were reached independently in branches of the rdrp tree, namely, branch (coronaviruses), branch (closteroviruses and flaviviruses), branch (reoviruses), and branch (filoviruses and paramyxoviruses) (fig. and ) . gene exchange and shuffling of gene modules are important factors of rna virus evolution. for example, it appears that all dsrna viruses in the two major clades within fig. legend. only the genes corresponding to rdrp, cp, and helicases (s h, s h, and s h for the helicases of superfamilies , , and , respectively) are shown systematically. the helicases appear to have been captured independently and in parallel in branches of ϩrna viruses, facilitating the evolution of larger, more complex genomes. additional genes, namely, the endo and exo genes (for endonuclease and exonuclease, respectively) and the hsp h gene (heat shock protein homolog), are shown selectively, to emphasize the increased genome complexity, respectively, in nidovirales and in closteroviridae. the virion architecture is shown schematically for each included group of viruses. icosahedral capsids composed of unelated cps are indicated by different colors (see the text for details). the question mark at the hypothetical ancestral eukaryotic rna virus indicates the uncertainty with regard to the nature of the host (prokaryotic or eukaryotic) of this ancestral form. the block arrow at the bottom indicates the time flow and the complexification trend in rna virus evolution. wolf et al. branches and share homologous structural modules that combine with distinct rdrps. at least in the case of partiti-picobirnaviruses in branch , the dsrna virus ("toti-like virus") cp apparently displaced the ancestral sjr-cp. however, this particular protein structure does not seem to be essential to encapsidate a dsrna genome; birnaviruses, whose provenance is uncertain due to the permutation in their rdrps, retain sjr-cp, which is most closely related to sjr-cp of nodaviruses and tetraviruses ( ) . an even more striking example of module shuffling is presented by amalgaviruses, dsrna viruses that group with partitiviruses in the rdrp tree but encode a distant homolog of the nucleocapsid protein of Ϫrna bunyaviruses ( ) ( ) ( ) ( ) . more generally, structural and replication modules have been repeatedly shuffled during the evolution of ϩrna viruses. examples include displacement of the ancestral sjr-cp by a filamentous cp in potyviruses and by a helical nucleocapsid protein in nidoviruses and multiple cases of displacement with rod-shaped-like cp and unique nucleocapsid proteins in branch . thus, exchange of genes and gene modules among rna viruses is pervasive and can cross the boundaries of baltimore classes. another recurring trend in rna virus evolution is the loss of the structural module, resulting in the emergence of naked rna replicons such as "narnaviruses" and "mitoviruses" in branch , hypoviruses in branch , and endornaviruses, umbraviruses, and deltaflexiviruses in branch ( ) . on some occasions, broad horizontal spread of a gene led to a major shift in the lifestyle of viruses, such as adaptation of viruses to new types of hosts. the primary cases in point are the movement proteins (mps) of plant viruses that are represented in all branches of the rdrp tree and, outside the rna part of the virosphere, in plant caulimoviruses and badnaviruses and in ssdna viruses ( ) . the prevalence of hvt and the overall course of rna virus evolution. arguably, the most striking realization brought about by metaviromics analyses concerns the diverse host range of numerous groups of viruses, even tight ones that occupy positions near the tips of the rdrp tree. extending early observations on the highly unexpected similarities between viruses of animals and plants, recent metaviromic analyses revealed numerous clusters of indisputably related viruses infecting animals and plants or plants and fungi or, in some cases, animals or plants and protists. the results of the analyses of evolutionary relationships between viruses with distinct host ranges are supported not only by the phylogeny of the rdrp but also by the fact that these viruses share additional conserved domains, such as, for example, sjr , s h, and c-pro in the case of picornavirales members infecting protists, plants, fungi, invertebrates, and vertebrates (fig. ) . invertebrates are particularly promiscuous hosts for viruses, often sharing the same virus group with distantly related organisms. certainly, much caution is due in the interpretation of host range assignments from metaviromics, especially those resulting from holobiont studies. viruses identified in holobiont samples of (for example) invertebrates could actually infect protists associated with these animals ( ) or could represent contamination from fungal or plant or even prokaryotic sources. these uncertainties notwithstanding, the extensive diversity of hosts even within small branches of the rdrp tree is undeniable. a coevolution scenario in which the ancestors of all these viruses originated from the common ancestor of the respective groups of eukaryotes and coevolved with the hosts implies an enormous diversity of rna viruses in early eukaryotes. this scenario appears to be highly unlikely given the apparent paucity of rna viruses in the extant protists (although new metaviromic studies might substantially expand the range of protist viruses). the pervasive hvt alternative seems much more plausible, especially given that arthropods, nematodes, and other invertebrates are well known as virus vectors and thus fit the role of rna virus reservoirs ( ) . in addition to invertebrates, which appear to be the dominant hvt agents, fungi could also play an important role in hvt within the global rna virome. indeed, fungi that are tightly associated with plants and insects often share closely related viruses with these organisms ( ) ( ) ( ) . furthermore, an indisputable case of cross-kingdom transfer of an insect iflavirus to an entomopathogenic fungus has been recently described ( ) . these findings appear to be most extensively compatible with a grand evolutionary scenario (fig. ) in which the ancestor of the eukaryotic rna virome was a levi/ narnavirus-like naked rna replicon that originally reproduced in mitochondria and then combined with a host carbohydrate-binding sjr protein ( ) or a preexisting sjr-cp from a dna virus to form a simple ancestral virus. given that viruses of branches , , and are present in modern protists, it appears likely that these branches emerged in early eukaryotes. however, because of the apparent dominance of the viruses of rdrp branch (members of the "picornavirus supergroup" in general and of the "aquatic picorna-like viruses" clade in particular) in protists, this branch likely diversified first, whereas the diversification of branches and occurred later, after ancestral protist viruses were transferred to marine invertebrates during the cambrian explosion. results of the recent analysis of the viromes of ctenophores, sponges, and cnidarians suggest that substantial diversification of rna viruses had already occurred in these deeply branching metazoa ( ) . invertebrates brought their already highly diverse rna virome to land at the time of terrestrialization and subsequently inoculated land plants. in land plants, rna viruses, particularly those of branch , dramatically expanded, perhaps in part because of the exclusion of competing large dna viruses. finally, it seems plausible that, given the high prevalence of Ϫrna viruses in metazoa and their virtual absence in protists (with the exception of the recently discovered "leishbuviruses" that likely invaded their parasitic protist hosts via hvt from an animal host [ , ] ), the viruses that comprise rdrp branch evolved in animals via mixing and matching genes from reovirus-like and flavivirus-like ancestors. the impending overhaul of rna virus taxonomy. the expansion of the global rna virome thanks to the advances of metaviromics, combined with the phylogenomics results, seems to call for an overhaul of the current virus taxonomy on multiple levels. most importantly, creation of a coherent, hierarchical system with multiple taxonomic ranks seems to be imminent. this process has already started with the proposal of a phylum rank for Ϫrna viruses, for which monophyly is unequivocally supported by the present analysis ( fig. and ). this phylum could consist of two subphyla with multiple classes and orders. at least additional phyla of rna viruses can be confidently predicted to emerge, including the dsrna viruses of branch and the ϩrna viruses of branches , , and . each of these phyla will undoubtedly have a rich internal structure. in addition, some of the current families do not seem to be compatible with the expansive rdrp trees presented here and in a previous analysis ( ) . for instance, the families coronaviridae, togaviridae, and rhabdoviridae are likely to be split into two families each. while the present study was in preparation, a major attempt at a comprehensive virus classification was published ( ) . this work analyzed a dendrogram that was produced from matrices of distances between viruses derived from sequence similarity scores combined with measures of gene composition similarity. unlike our present analysis, this approach presupposes monophyly of each of the baltimore classes. furthermore, given that their analysis was based on measures of similarity rather than on phylogenetic analysis proper, this approach is best regarded as producing a phenetic classification of viruses rather that an evolutionary reconstruction as such. some of the groups delineated by this method, particularly those among ϩrna viruses, are closely similar to those reported here. others, however, are extensively different; for instance, the order mononegavirales is not classified as monophyletic in their dendrograms. we did not attempt a complete comparison; such an exercise could be useful in the future for better understanding the routes of rna virus evolution. concluding remarks. through metaviromics, many aspects of the global rna virome evolution can be clarified. certainly, reconstruction of the deepest events in this evolutionary history is bound to remain tentative, especially because the rdrp gene is the only universal gene of the rna viruses and is hence the only one that can serve as the template for evolutionary reconstructions. at the depth of divergence characteristic of rdrps, the relationship between the major branches in the tree cannot be established with confidence. nevertheless, monophyly of several expansive groups, in particular, the main branches in the rdrp tree, is strongly supported. because of the stability of these branches, biologically plausible scenarios of evolution emerge under which dsrna viruses evolved from different groups of ϩrna viruses whereas Ϫrna viruses evolved from dsrna viruses. evolutionary reconstructions suggest that the last common ancestors of each major lineage of eukaryotic ϩrna viruses were simple viruses that encoded only the rdrp and a cp, most likely of the sjr fold. subsequent evolution involved independent capture of distinct helicases which apparently facilitate replication of larger, more complex ϩrna genomes. the helicase-assisted replication of ϩrna genomes created the opportunities for parallel acquisition of additional genes encoding proteins involved in polyprotein processing and virus genome expression, such as proteases and capping enzymes, respectively, and proteins involved in virus-host interactions, such as mps or rnai suppressors of plant viruses. in addition to these processes of vertical evolution of rna viruses, the results from phylogenomic analyses reveal multiple cases of gene module exchange among diverse viruses and pervasive hvt, often between distantly related hosts, such as animals and plants. together, these processes have shaped a complex network of evolutionary relationships among rna viruses. the much-anticipated comprehensive exploration of the rna viromes of prokaryotes and unicellular eukaryotes, such as free-living excavates, chromalveolates, rhizaria, amoebozoa, and choanoflagellates, as well as deeply rooted metazoa, will undoubtedly help in developing better-supported evolutionary scenarios for each of the major branches of the rna virus tree. nevertheless, it is already clear that the current taxonomy of rna viruses is due for a complete overhaul. phylogeny of rna-dependent rna polymerases. protein sequences belonging to rna viruses (excluding retroviruses) and to unclassified viruses were downloaded from the ncbi genbank database in april ( ) . initial screening for rdrp domains was performed using psi-blast ( ) (e value of . , effective database size of ϫ sequences) with position-specific scoring matrices (pssms) produced from the available rdrp alignments. the sources included group-specific alignments for ϩrna viruses and dsrna viruses ( , ) and the pfam alignments for the Ϫrna viruses (pfam , pfam , pfam , and pfam ) from the ncbi conserved domain database (cdd) ( ) . additionally, a set of rts from group ii introns and non-long-terminal-repeat (non-ltr) retrotransposons was extracted from genbank as an outgroup. extracted rdrp footprints were filtered for the fraction of unresolved amino acids (at most %) and clustered using uclust ( ) with a similarity threshold of . . one representative from each cluster was selected for further analysis. the resulting set contained , virus rdrps. this set went through several rounds of semimanual curation whereby sequences were clustered using uclust, aligned using muscle ( ) , and cross-searched against each other and their parent sequences (often representing complete viral polyproteins) using psi-blast and hhsearch ( ) . upon obtaining the results of these searches, the boundaries of the rdrp domain were expanded or trimmed to improve their compatibility with each other. the rdrp and rt sequences were subjected to an iterative clustering and aligning procedure, organized as follows. initially, sequences were clustered using uclust with a similarity threshold of . . the clustered sequences were aligned using muscle, and singletons were converted to pseudoalignments consisting of just one sequence. sites containing more than % gaps were temporarily removed from alignments, and pairwise similarity scores were obtained for clusters by the use of hhsearch. scores for a pair of clusters were converted to distances as follows: the d a,b ϭ Ϫlog[s a,b /min(s a,a ,s b,b ) formula, in which d a,b is the distance between cluster a and cluster b and s a,b is the hhsearch score for the comparison of these clusters, was used to convert scores s to distances d. the matrix of pairwise distances was used to calculate the mean by the unweighted pair group method using average linkages (upgma) ( ) . shallow tips of the tree were used as the guide tree for a progressive pairwise alignment of the clusters at the tree leaves using hhalign ( ) , resulting in larger clusters. this procedure was reiterative, ultimately resulting in the single alignment of the whole set of , virus rdrp sequences and , rt sequences. during the clustering procedure, virus rdrp clusters, consisting of to sequences, were defined. these clusters represented either well-established groups of related viruses (roughly comparable to the ictv family rank) or, in case of poorly characterized and unclassified viruses, groups of well-aligned rdrps that were clearly distinct from others. in all cases, uncertainties were treated conservatively, i.e., favoring placing sequences with questionable relatedness into separate clusters. additionally, rt sequences were placed into two clusters consisting of group ii intron rts and non-ltr retrotransposon rts. for each cluster consisting of three or more sequences, an approximate maximum likelihood (ml) phylogenetic tree was constructed from the cluster-specific alignment using the fasttree program ( ) (whelan and goldman [wag] evolutionary model with gamma-distributed site rates) with sites that contained more than % gaps removed from the alignment. trees were rooted using a variant of the midpoint rooting procedure such that the differences between the (weighted) average root-to-tip distances in the subtrees on the opposite sides of the root were minimized. to resolve the structure of the global relationships, up to five representatives from each cluster were selected using the within-cluster trees to ensure the diversity of the selected sequences. this procedure resulted in a set of virus rdrps and rts. the alignments of the selected sequences were extracted from the master alignment and filtered for sites containing more than % gaps. a ml phylogenetic tree was reconstructed for the resulting alignment using phyml ( ) (le gascuel [lg] evolutionary model with gamma-distributed site rates and empirical amino acid frequencies; support values were calculated using abayes method implemented in phyml). another form of branch support, i.e., bootstrap support provided by the transfer (booster) phylogenetic bootstrap method ( ) , was also used to assess the reliability of the major tree divisions. alternatively, the same rdrp alignment was used as the input for ml phylogenetic analysis using raxml (lg evolutionary model with gamma-distributed site rates and empirical amino acid frequencies). rdrps in the global tree were divided into major branches (supergroups). up to representatives from each cluster were selected to form supergroup-level alignments. the respective trees were reconstructed from these alignments using the same procedure (phyml tree with lg evolutionary model with gamma-distributed site rates and empirical amino acid frequencies). the overall tree was assembled manually by first replacing the supergroup representatives in the global tree with the supergroup trees and then replacing the cluster representatives with the cluster trees. the lower-level trees were rooted according to the arrangement of the representatives in the upper-level tree. identification of protein domains. protein domains were identified using a representative set of rna virus genomes, including representative members of ictv-approved virus families and unclassified virus groups. this set was annotated manually using sensitive profile-profile comparisons with the hhsuite package ( ) , and hmm profiles for annotated proteins or their domains were generated by running one iteration of hhblits against the latest (october ) uniclust database ( ) . each annotated profile was assigned to a functional category (e.g., Љcapsid protein_jelly-roll,Љ Љchymotrypsinlike proteaseЉ). these profiles were used to annotate the genomes of all of the viruses that were included in the rdrp phylogenetic analysis and for which complete (or near-complete) genome sequences were available. profiles for the latter proteins were generated by running one iteration of jackhmmer ( ) against the uniref database. protein regions that did not have significant hits were extracted and clustered with cluster analysis of sequences (clans) ( ) , and groups containing at least three members were identified, annotated (if possible), and added to the manually annotated profile database. the last step of the domain identification procedure was then repeated using the updated rna virus profile database. highly similar viruses (with identical domain organizations and Ͼ % identical rdrps) were removed from the data set. in addition, only one representative genome was retained for some members of overrepresented species (e.g., hepacivirus c). many genomes (e.g., alphaviruses, tombusviruses, and nidoviruses) encoded readthrough proteins, resulting in domains from the shorter protein contained within the longer readthrough protein. such redundancies were removed by filtering out the shorter of the two proteins sharing Ͼ % identity. the final set of annotated genomes included , viruses. reconstruction of the history of gene gain and loss. the protein domains identified in the , virus genomes were mapped onto the composite tree of virus rdrps. a set of representatives was chosen among the (mostly complete) genomes. the domain complement data and the respective tree were analyzed using the gloome program ( ) . domain gains and losses were inferred at each tree branch using the difference of posterior probabilities for the domain occurrence in the nodes at the proximal and the distal ends of the branch (differences with values above . imply gain; differences with values below Ϫ . imply loss). clustering analysis. the sjr cp network was generated by performing all-against-all comparisons of the sjr cp profiles. to generate hmm profiles, sjr cp sequences were extracted from the total proteome of rna viruses and two iterations of hhblits were performed against the uniprot _ _ database. the resulting profiles were compared to each other with hhsearch ( ) . their similarity p values were extracted from the result files and used as an input for clans program ( ) . clusters were identified using a network-based algorithm implemented in clans. resulting clusters were manually inspected and refined. analysis of bipartite gene-genome networks. a bipartite network was built to study the patterns of gene sharing among viral genomes ( , ) . after removal of genomes with fewer than domains and of domains that appeared in less than genomes, the network consisted of , nodes, of which , corresponded to genomes and to domains. genome and domain nodes were connected by links wherever a domain was present in a genome. a consensus community detection approach was used to identify the modules of the network ( , ) . first, we ran replicas with infomap ( ) (bipartite setting, using domains as factors) and built a similarity matrix by assigning to each pair of nodes a similarity value equal to the fraction of replicas in which both nodes were placed in the same module. then, hierarchical clustering was performed on the similarity matrix, setting the number of clusters equal to the median number of modules obtained in the replicas. order statistics local optimization method (oslom) software ( ) was subsequently used to filter significant modules with a p value threshold equal to . . to detect higher-order (super)modules, we first identified connector domains as those present in at least modules with prevalence greater than . . the second-order network, composed of modules and connector domains, was searched for second-order modules with infomap ( replicas, bipartite setting with connector domains as factors). due to the small size of the second-order network, consensus community detection did not qualitatively improve the results of the search, and therefore we took the replica with the best infomap score and skipped hierarchical clustering for this and subsequent steps of the higher-order module search. after assessing statistical significance with oslom, second-order modules were recovered, encompassing of the original modules associated with closely related virus families. a third-order network was built by pooling the second-order modules with the modules that remained unmerged. the third-order network was analyzed in the same manner to obtain the supermodules. none of these supermodules was assessed by oslom as significant. quantification of virus host range diversity. known hosts or, in case of viruses isolated from holobionts, virus sources were identified for , viruses. the host taxonomy (analyzed to the phylum level) was mapped to the leaves of the combined tree. the tree was ultrametrized, and the weights were computed for all leaves as described in reference . each internal node of the tree therefore defines a set of leaves with assigned hosts; the distribution of (weighted) relative frequencies of hosts can be characterized by its shannon entropy. each leaf has one host defined such that the entropy of the host range distribution is whereas the host 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across multicellular eukaryotes the genomic underpinnings of eukaryotic virus taxonomy: creating a sequence-based framework for family-level virus classification gapped blast and psi-blast: a new generation of protein database search programs cdd: ncbi's conserved domain database search and clustering orders of magnitude faster than blast muscle: a multiple sequence alignment method with reduced time and space complexity protein homology detection by hmm-hmm comparison a statistical method for evaluating systematic relationships fasttree -approximately maximum-likelihood trees for large alignments renewing felsenstein's phylogenetic bootstrap in the era of big data uniclust databases of clustered and deeply annotated protein sequences and alignments accelerated profile hmm searches clans: a java application for visualizing protein families based on pairwise similarity consensus clustering in complex networks community detection in networks: a user guide maps of random walks on complex networks reveal community structure finding statistically significant communities in networks extreme deviations from expected evolutionary rates in archaeal protein families key: cord- -fjd rsrf authors: imperiale, michael j.; casadevall, arturo title: vagueness and costs of the pause on gain-of-function (gof) experiments on pathogens with pandemic potential, including influenza virus date: - - journal: mbio doi: . /mbio. - sha: doc_id: cord_uid: fjd rsrf nan ried about gof experiments ( ), seem to have precipitated the government action. pauses and moratoriums are blunt instruments in science and carry the potential for unintended consequences. we recognize that the pause is a response from well-meaning government officials who are tasked with trying to find ways to minimize potential dangers from gof experiments. we note, however, that depending on which interruptions of work are counted, this is at least the third pause/moratorium in this field, with the first being voluntary, the second requested by the usg ( , ) , and the third being the current pause. we have numerous concerns with this third stoppage, including the timing of the announcement relative to the ongoing debate, the vagueness in the wording of the statement, and the potential effects on the fields of influenza virus and coronavirus research. each concern will be discussed separately. the timing of this pause is perplexing given that one might have expected this action to follow a concerted effort to explore the issues rather than to precede detailed discussions. many have drawn the analogy between the current situation and that surrounding the advent of recombinant dna technologies. however, there are significant differences: the discussions at asilomar preceded a self-imposed moratorium by molecular biologists working on recombinant dna technology ( ) . it seems that this should have been the case now: the nsabb could have been deliberating on this topic in the years that have passed since the gof debate began with the publication of two manuscripts describing mammalian transmission of h n influenza virus ( , ) . instead, the nsabb did not even meet, and this created a vacuum of discussion that may have contributed to the current crisis. in contrast, the government has responded to the heightened controversy by reactivating the nsabb while simultaneously calling for a pause of gof work before a meaningful discussion. although this course of action seems to emphasize safety and caution, it carries significant risks that we discuss below. it is also unclear to us why the pause is necessary, given that the government is already presumably providing an extra layer of review of gof experiments that followed the prior moratoriums (http://www.phe.gov/s / dualuse/documents/us-policy-durc- .pdf) and has asked institutions to do the same (http://www.phe.gov/s /dualuse/ documents/durc-policy.pdf). we are concerned that the wording of the pause is vague and could have unintended consequences. first, the pause has no end date. will the nsabb and the academies be nimble enough to make concrete recommendations that are broadly acceptable within months? given the pace at which these committees generally function, we worry that this will not be the case. having the pause drag on for too long will affect not only research progress but also the careers of the scientists engaged in that research. second, we worry about the meaning of "reasonably anticipated." obviously this phrase is very subjective, and similar wording in the definition of dual use research of concern (durc) has already made assessments of what constitutes durc very problematic for journals and authors ( ) . at one extreme, cautious researchers could over-interpret the vague wording and stop experiments that were not intended for inclusion in the pause order. for example, albeit somewhat extreme, any time one grows an rna virus in the laboratory, even in cell culture, the error-prone nature of the viral rna polymerase results in each progeny genome containing more or less one mutation. any scientist versed in rna virus biology could "reasonably anticipate" that some of these mutations would impose a gain of function on the virus. however, if one does not select for that function, it is extremely unlikely that that mutant will overtake the population. we therefore suggest that the intent of the experiment must be considered before making a determination of whether it should be paused. the pause will almost certainly have a disruptive effect on several laboratories at a time when information derived from gof experiments is beginning to bear fruit in pandemic preparedness. the accompanying articles from stacey schultz-cherry et al. and nancy cox et al. describe how mutational information from gof is producing actionable information on surveillance studies and selection of strains for vaccines ( , ) . the pause means that some information from gof experiments will cease to become available, with potential negative consequences on preparedness. ongoing experiments will stop, and the vagueness of the wording raises the possibility that other work will not be done due to an abundance of caution. for example, there is tremendous need for rodent models of coronaviruses with pandemic potential, including the agents responsible for mers and sars. such models could greatly facilitate the discovery of new drugs and vaccines. however, developing such models requires changing the host tropism of the virus, and as such they fall under experiments of concern despite the fact that human viruses often lose virulence as they adapt to other species. the current pause affects two dozen studies that include experiments to develop rodent models of coronavirus research ( ) . in this regard, the reader may want to listen to a story on national public radio in which researchers discuss how the pause is affecting coronavirus research ( ) . the inclusion of this work in the stoppage is an example of how pauses and moratoriums can be blunt instruments with major unintended consequences. finally, we worry that work being carried out by graduate students and postdoctoral fellows will be put on hiatus, causing disruption to their plans for completing their training. although some will argue that this is a small price to pay for ensuring safety, we worry that this could have a tremendous effect downstream, as investigators may be discouraged from resuming such studies in the future. furthermore, bright young scientists who have a choice of what research to pursue may avoid this area of investigation because of its controversy, unpredictability, and increased restrictions. research output is not like a factory line that can be shut down and restarted depending on supply and demand. instead, research output is dependent on the presence of ongoing projects by dedicated scientists who carried them out in good faith, hoping to generate useful information. when students and postdoctoral fellows stop such projects, they inevitably move to other projects and it may be difficult to jump-start gof experiments once laboratories cease doing that type of work. as such, we are more concerned about pausing ongoing projects than delaying the start of new lines of investigation. given that a healthy research enterprise is humanity's best defense against future threats from these respiratory pathogens, the pause could hurt future progress by discouraging the best and the brightest from joining this field. hence, this pause, which is presumably intended to safeguard society from laboratory accidents and unintentional releases, could have the paradoxical effect of leaving humanity more vulnerable to future pandemics by virtue of the information that was not obtained. as we have written previously, understanding the pathogenicity of these viruses is necessary if we want to develop new therapies and vaccines and ensure useful surveillance ( , ) . clearly, the research must be performed under biocontainment conditions that minimize the risk of accidental release. the discussion that the white house is asking for must occur because the status quo is not acceptable. we call on the government to provide clarity regarding what truly should be paused and for how long. we call on the nsabb and the national academies to move rapidly on this issue, to consider whether the current biosafety practices put in place after the earlier moratoriums are sufficient, and if found to be so, to state so without a need for new layers of mandates for what is already a highly supervised field. to repeat ourselves ( ), we must get this right. risks and benefits of gain-of-function experiments with pathogens of pandemic potential, such as influenza virus: a call for a science-based discussion an epistemological perspective on the value of gain-of-function experiments involving pathogens with pandemic potential doing diligence to assess the risks and benefits of life sciences gainof-function research the irrationality of gof avian influenza virus research h n . flu controversy spurs research moratorium avian influenza. h n researchers ready as moratorium nears end summary statement of the asilomar conference on recombinant dna molecules airborne transmission of influenza editorial a/h n virus between ferrets experimental adaptation of an influenza h ha confers respiratory droplet transmission to a reassortant h ha/h n virus in ferrets on the need for a national board to assess dual use research of concern how a tilt toward safety stopped a scientist's research influenza gain-of-function experiments: their role in vaccine virus recommendation and pandemic preparedness use of highly pathogenic avian influenza a(h n ) gain-of-function studies for molecularbased surveillance and pandemic preparedness the views expressed in this article do not necessarily reflect the views of the journal or of asm key: cord- -sht yx a authors: tal, michal caspi; torrez dulgeroff, laughing bear; myers, lara; cham, lamin b.; mayer-barber, katrin d.; bohrer, andrea c.; castro, ehydel; yiu, ying ying; lopez angel, cesar; pham, ed; carmody, aaron b.; messer, ronald j.; gars, eric; kortmann, jens; markovic, maxim; hasenkrug, michaela; peterson, karin e.; winkler, clayton w.; woods, tyson a.; hansen, paige; galloway, sarah; wagh, dhananjay; fram, benjamin j.; nguyen, thai; corey, daniel; kalluru, raja sab; banaei, niaz; rajadas, jayakumar; monack, denise m.; ahmed, aijaz; sahoo, debashis; davis, mark m.; glenn, jeffrey s.; adomati, tom; lang, karl s.; weissman, irving l.; hasenkrug, kim j. title: upregulation of cd is a host checkpoint response to pathogen recognition date: - - journal: mbio doi: . /mbio. - sha: doc_id: cord_uid: sht yx a it is well understood that the adaptive immune response to infectious agents includes a modulating suppressive component as well as an activating component. we now show that the very early innate response also has an immunosuppressive component. infected cells upregulate the cd “don’t eat me” signal, which slows the phagocytic uptake of dying and viable cells as well as downstream antigen-presenting cell (apc) functions. a cd mimic that acts as an essential virulence factor is encoded by all poxviruses, but cd expression on infected cells was found to be upregulated even by pathogens, including severe acute respiratory syndrome coronavirus (sars-cov- ), that encode no mimic. cd upregulation was revealed to be a host response induced by the stimulation of both endosomal and cytosolic pathogen recognition receptors (prrs). furthermore, proinflammatory cytokines, including those found in the plasma of hepatitis c patients, upregulated cd on uninfected dendritic cells, thereby linking innate modulation with downstream adaptive immune responses. indeed, results from antibody-mediated cd blockade experiments as well as cd knockout mice revealed an immunosuppressive role for cd during infections with lymphocytic choriomeningitis virus and mycobacterium tuberculosis. since cd blockade operates at the level of pattern recognition receptors rather than at a pathogen or antigen-specific level, these findings identify cd as a novel potential immunotherapeutic target for the enhancement of immune responses to a broad range of infectious agents. adaptive immune responses. indeed, results from antibody-mediated cd blockade experiments as well as cd knockout mice revealed an immunosuppressive role for cd during infections with lymphocytic choriomeningitis virus and mycobacterium tuberculosis. since cd blockade operates at the level of pattern recognition receptors rather than at a pathogen or antigen-specific level, these findings identify cd as a novel potential immunotherapeutic target for the enhancement of immune responses to a broad range of infectious agents. importance immune responses to infectious agents are initiated when a pathogen or its components bind to pattern recognition receptors (prrs). prr binding sets off a cascade of events that activates immune responses. we now show that, in addition to activating immune responses, prr signaling also initiates an immunosuppressive response, probably to limit inflammation. the importance of the current findings is that blockade of immunomodulatory signaling, which is mediated by the upregulation of the cd molecule, can lead to enhanced immune responses to any pathogen that triggers prr signaling. since most or all pathogens trigger prrs, cd blockade could be used to speed up and strengthen both innate and adaptive immune responses when medically indicated. such immunotherapy could be done without a requirement for knowing the hla type of the individual, the specific antigens of the pathogen, or, in the case of bacterial infections, the antimicrobial resistance profile. keywords cd , host response, immune checkpoint, innate immunity, pathogen recognition receptors t he earliest immune responses to invasion by pathogenic microorganisms begin with the sensing of pathogen-associated molecular patterns (pamps) by pattern recognition receptors (prrs) such as toll-like receptors (tlrs). ligation of prrs initiates signal transduction pathways that ultimately lead to the activation of broad innate and highly specific adaptive immune responses. discoveries in recent years have demonstrated that the induction of adaptive immune responses involves not only activation mechanisms but also inhibitory mechanisms or "checkpoints," which regulate immune function at a cellular level to prevent immunopathological damage by overactivated effector responses ( ) . antibody (ab)-mediated blockade of checkpoint molecules such as ctla and pd- is being used therapeutically to enhance anticancer immune responses ( , ) , and blockade of cd is now in clinical trials to activate macrophagemediated phagocytosis of cancer cells ( ) ( ) ( ) ( ) , which upregulate cd expression as an immune evasion mechanism ( ) ( ) ( ) ( ) . cd is an abundantly expressed transmembrane cell surface glycoprotein that can act as a receptor for thrombospondins, form complexes with integrins, and bind to the inhibitory receptor signal-regulatory protein alpha (sirp␣) ( ) ( ) ( ) . cd binding to sirp␣ has emerged as an important innate immune checkpoint by regulating immune cell clearance and inflammatory signaling ( ) . cd engagement of sirp␣ results in the phosphorylation of cytoplasmic itim motifs by inhibitory protein tyrosine phosphatases, shp- and shp- ( ) . given the well-established role of cd in cancer cell immune evasion, we investigated whether cd expression is modified in other disease contexts, specifically infectious diseases. the cd -sirp␣ axis has immunomodulatory functions that impact phagocytosis, chemokine and cytokine responses, innate and adaptive immune cell homeostasis and activation, t cell killing, and b cell antibody production ( ) ( ) ( ) ( ) . viruses have evolved mechanisms to evade host defenses ( ) and take advantage of inhibitory signaling pathways for selective advantage. of interest, poxviruses, which devote many genes toward immune suppression and evasion, encode a cd mimic ( ) . the cd mimic of myxomavirus, m l, can be deleted with no effect on in vitro replication, but the deletion mutant loses pathogenicity in vivo. this loss of pathogenicity was associated with increased monocyte/macrophage activation ( ) . the present study examines cd expression in the context of infectious agents that encode no cd mimic. both mouse and human cells showed a significant upregulation of cd upon infection with various pathogens. the results indicated that stimulation of either cytosolic or endosomal pprs resulted in cd upregulation. in addition, inflammatory cytokines present in the serum of hepatitis c virus (hcv)-infected patients could also induce cd upregulation, even in the context of no virus. in addition to viruses, clinically relevant bacteria such as mycobacterium tuberculosis induce the upregulation of cd that limits host resistance. our results indicate that cd upregulation is a very early innate checkpoint response and that immunological inhibitory mechanisms are activated not only at the effector phase of immune responses but also already at the induction phase of prr sensing. thus, cd is a promising target for checkpoint therapies against a wide range of infectious diseases. to examine the role of cd expression during the innate response to infection, we investigated whether hematopoietic cells upregulated cd expression in several unrelated infection models during the first days after infection. we began by analyzing cd expression on cells from mice inoculated with friend virus (fv), a naturally occurring retroviral infection in mice ( ) . fv primarily infects erythroid progenitor cells in the spleen but can also infect immune cells ( ) . cd was significantly upregulated on several hematopoietic cell lineages from mouse spleens at days postinfection (dpi) compared to cells from naive mice (fig. a) . cd expression was also analyzed at dpi in mice infected with lymphocytic choriomeningitis virus (lcmv). compared to naive controls, all of the spleen cell types analyzed showed significantly increased cell surface expression of cd (fig. b) . a significant upregulation of cd expression was also observed in response to lcmv at dpi in a previous report ( ) . infections with la crosse arbovirus were also analyzed at dpi, and we also observed significantly upregulated cd expression in hematopoietic spleen cells compared to naive controls (fig. c) . cd expression is upregulated on human cells in response to in vitro infection. examination of a publicly available gene expression data set (gene expression omnibus [geo] accession number gse ) from severe acute respiratory syndrome coronavirus (sars-cov- ) infection of a human lung cells also showed a significant upregulation of cd compared to mock-infected controls (fig. d ). to determine whether bacterial infection would also upregulate cd expression, human peripheral blood mononuclear cells (pbmcs) were examined and h after infection with borrelia burgdorferi in vitro. multiple pbmc subsets showed significantly upregulated cd expression in response to borrelia burgdorferi infection compared to naive cells (fig. e) . we also investigated cd expression on human pbmcs infected in vitro with mcherry-expressing strains of salmonella enterica serovar typhi. wild-type salmonella typhi (ty wt) and salmonella typhi Δflic (ty Δflic), a mutant strain that lacks flagella, were examined. infections were done by centrifugation to compensate for the lack of motility of the flagellum mutant. compared to naive cells, mcherry-positive b cells significantly upregulated cd expression when infected with wild-type salmonella typhi for h (fig. f ). in contrast, cd expression was not significantly upregulated in b cells infected with the mutant strain lacking flagella, salmonella typhi Δflic (fig. f) . the reduced upregulation of cd expression by salmonella typhi Δflic compared to wild-type salmonella typhi suggested that the sensing of pathogen-associated molecular patterns (pamps) by pattern recognition receptors (prrs) might play a role in cd upregulation, as flagellin is a potent pamp recognized by extracellular tlr ( ) and the nlr family of apoptosis-inhibitory proteins (naips) ( ) . overall, the combined in vivo and in vitro results from multiple pathogen infections in both human and mouse cells indicated that the upregulation of cd was a conserved host response possibly related to host sensing mechanisms. cd is upregulated in response to host recognition of pathogens. to determine whether cd upregulation was initiated by prr stimulation, we specifically stimulated prrs using small-molecule agonists rather than infectious agents. cd upregulation on human dendritic cells (dcs) and monocytes was tested in vitro using pbmcs stimulated with either muramyl dipeptide (mdp) to activate the bacterial peptidoglycan prr, nucleotide-binding oligomerization domain-containing protein (nod ), or cl to activate the single-stranded rna (ssrna) endosomal prr, tlr . flow cytometry was used to identify cell subsets and measure cd expression at h poststimulation. both human dcs and monocytes responded to either type of prr stimulation with a significant upregulation of cd surface expression ( fig. a) . we also tested tlr stimulation using a dual tlr / agonist, r . since this dual agonist, which also has in vivo activity ( ) , produced dose-dependent upregulation of cd on human pbmcs (fig. b) , we proceeded to test prr stimulation in an in vivo mouse model. daily intraperitoneal injections of r into mice led to strongly upregulated cd expression on both macrophages and dcs isolated from spleens on day (fig. c) . together, these data demonstrated that cd was upregulated on both human and mouse immune cells via pathogen-sensing mechanisms. furthermore, tlr stimulation via endosomal uptake indicated that cd could be upregulated not only tal et al. ® by infected cells, as would be reflected by cytosolic nod stimulation, but also by surveilling immune cells. to examine cd expression in human viral infection, we first compared transcriptional levels of cd from publicly available microarray data (geo accession number gse ) from healthy and hepatitis c virus (hcv) patient liver biopsy specimens. the analysis revealed significantly higher expression levels of cd in the liver biopsy specimens from acutely infected hcv patients than for healthy controls (fig. a) . we then used cytometry by time of flight (cytof) to examine cd expression on pbmcs during hcv infection in the context of sofosbuvir (sof) therapy ( ) , comparing healthy controls to hcvinfected patients prior to treatment, midway through treatment, and at months posttreatment. compared to healthy controls, monocytes and dcs from hcv patients demonstrated a sustained upregulation of cd at all treatment time points, including the -month posttreatment time point ( fig. b (b) cd mfi on human total pbmcs from separate donors stimulated with titrated concentrations of r from . g/ml to g/ml, as indicated, for h. statistics were done by a paired two-way t test with bonferroni correction. (c) mice (n ϭ /group) were injected intraperitoneally with mg/kg r for days, and on day , splenocytes were isolated and macrophages and dcs were analyzed for cd mfi. statistics were done by an unpaired two-way t test. (ns, p Ͼ . ; *, p Ͻ . ; **, p Ͻ . ; ***, p Ͻ . ). error bars represent sem. upregulation of cd is a host checkpoint response ® differences between pre-, mid-, and posttreatment cd levels in either monocytes or dcs, and all of these time points were significantly different from those for healthy controls ( fig. b and c). conventional dendritic cells (cdcs) are classified into cdc s and cdc s, which can be distinguished in part through the specific expression of the cd receptor, sirp␣, on cdc s ( ) . when we compared cd expression levels within sirp␣ lo and sirp␣ hi dcs, we observed that cd expression was highest on sirp␣ hi dcs (fig. d ). there was no correlation between viral titers and cd expression on either monocytes or dc subsets in this patient cohort (see fig. s a in the supplemental material). between healthy controls and pretreatment hcv patients, there was significant cd upregulation in sirp␣ lo dcs but not in sirp␣ hi dcs (fig. d ). however, compared to healthy controls, the proportion of sirp␣ hi dcs was significantly increased at both pretreatment and midtreatment, which could also contribute to higher cd expression levels in dcs (fig. s b) . thus, hcv infections were associated with the increased expression of both cd and its receptor, sirp␣, on antigen-presenting cells (apcs). hcv patient plasma-induced cd expression ex vivo. to determine whether inflammatory factors present in hcv patient plasma could affect cd upregulation, we derived dcs from healthy donor monocytes with plasma added from either patients in the hcv patient cohort or healthy controls. we found that monocyte-derived dcs (mdcs) cultured in the presence of hcv patient plasma significantly upregulated cd compared to plasma from healthy donors (fig. a ). luminex analysis of patient plasma was used to characterize the inflammatory milieu over the course of hcv infection and treatment. plasma isolated from patients at the pretreatment time point contained both virus and inflammatory cytokines, indicating that cd upregulation could have been due to infection of the dcs. however, plasma isolated from patients at the midtreatment and posttreatment time points contained no detectable virus (data not shown) but still had increased levels of inflammatory cytokines, including tumor necrosis factor alpha (tnf-␣), cxcl , and interferon alpha (ifn-␣), compared to healthy controls (fig. b to d). cd expression was increased under all hcv patient plasma conditions compared to healthy controls despite undetectable virus at the midtreatment and posttreatment time points. the differences between the pre-, mid-, and posttreatment time points within hcv patients were not statistically significant. these results suggested that cytokines in the inflammatory milieu of hcv patient plasma could upregulate cd expression. to confirm the ability of cytokines to upregulate cd surface expression, we performed in vitro stimulations of human pbmcs isolated from healthy donors using tnf-␣, cxcl , and ifn-␣, as single treatments and in combinations. at h poststimulation, the only single cytokine that induced significant cd upregulation was ifn-␣ (fig. e) . combinations of these inflammatory cytokines enhanced the upregulation of cd surface expression, with the triple combination of tnf-␣, cxcl , and ifn-␣ having the strongest effect (fig. e) . these experiments demonstrated that in addition to pathogen recognition, host immune cells could also upregulate cd surface expression in response to the inflammatory milieu induced by the pathogen. to determine the effects of cd blockade during a live viral infection, c bl/ mice were injected with anti-cd (blocking) antibody daily beginning days prior to infection with lcmv. to ascertain whether both anti-cd -treated and mock-treated animals were equivalently infected, lcmv titers in the spleens and liver were determined at dpi. the analyses showed no significant differences in viral titers at this time point regardless of treatment (fig. a) . anti-cd injections were continued through dpi, and plasma virus levels were then measured at and dpi. significantly reduced viremia levels were observed in anti-cd -treated mice compared to mock-treated mice at both time points (fig. b ). since it is known that host clearance of lcmv is highly dependent on cd ϩ t cell responses ( , ) , it was of interest to determine if cd blockade affected those responses. total cd ϩ t cell levels were determined tal et al. ® from blood samples of lcmv-infected mice at , , and dpi in the presence or absence of cd blockade. at both and dpi, there were significantly increased cd ϩ t cell numbers responding to lcmv infection in the treated mice compared to the isotype-matched antibody control-treated mice (fig. c) . genetic inactivation of cd expression prolongs survival from mycobacterium tuberculosis infection in vivo. we next investigated the role of cd during mycobacterium tuberculosis infection in vitro and in vivo. first, human macrophages derived from healthy pbmc samples were used for in vitro infection with m. tuberculosis to examine cd expression levels. cd expression levels are shown for four donors, and compare naive macrophages from cultures that were not infected to macrophages from cultures that were infected with phrodo-labeled m. tuberculosis. in comparison to naive macrophages, cd was not upregulated on m. tuberculosis-infected (phrodopositive [phrodo ϩ ]) macrophages (fig. d) . interestingly, m. tuberculosis-uninfected (phrodo-negative [phrodo Ϫ ]) macrophages from the m. tuberculosis-infected cultures downregulated cd expression in comparison to naive macrophages, an observation for which we do not yet have an explanation (fig. d ). the lack of cd upregulation by m. tuberculosis-infected macrophages was supported by metasignature analysis upregulation of cd is a host checkpoint response ® comparing microarrays for gene expression signatures specific to hcv and tuberculosis (tb) (fig. s ) . the disease-specific gene expression signature for hcv consistently showed an upregulation of cd , whereas the disease-specific gene expression signature for tb showed downregulation. for an in vivo assessment of a possible functional role for cd in host resistance to m. tuberculosis infection, cd knockout (ko) mice were infected via the aerosol route with a low dose ( to cfu) of m. tuberculosis strain h rv. cd -deficient mice displayed significantly increased host resistance to m. tuberculosis infection, with significantly longer survival (humane endpoints), compared to wild-type c bl/ mice (fig. e) . furthermore, spleens and lungs from the cd -deficient mice at humane endpoints yielded significantly lower mycobacterial cfu (fig. f ). these results indicated that cd expression can exert significant suppressive effects on immune responses to infectious pathogens in vivo. previous results demonstrating that malignant cells upregulate cd expression to evade cellular clearance ( , ) led us to hypothesize that pathogens might also induce cd surface expression as an immune evasion mechanism. in fact, poxviruses encode a cd mimic that acts as a potent virulence factor ( ) . curiously, we repeatedly observed an upregulation of cd surface expression across diverse infections with both viruses and bacteria, none of which are known to encode a cd mimic or have any cd sequence homology. flagella are potent inducers of tlrs, so the finding that salmonella typhi Δflic mutants lacking flagella were poor at inducing cd suggested a possible connection to host sensing via prr signaling (fig. f) . indeed, direct stimulation of prrs with synthetic ligands such as mdp, cl , and r induced cd upregulation in vitro and in vivo (fig. ) . since sirp␣-mediated recognition of cd on infected cells by macrophages and dcs transduces an inhibitory signal that attenuates phagocytosis and downstream antigen presentation functions, it was counterintuitive that cd upregulation would be due to a host response. why would the host dampen its immune response to infection? we have shown that in vivo, cd blockade improved the control of lcmv infection (fig. a) , increased cd ϩ t cell responses (fig. c) , and, in a previous report, increased the expression of costimulatory molecules on apcs as well ( ), thus confirming that cd induces immunosuppressive signals. similarly, mice with genetic inactivation of cd had reduced bacterial loads and longer survival times when infected with m. tuberculosis ( fig. e and f) . we conclude that, like coinhibitory molecules such as pd- that dampen adaptive immune responses, cd upregulation acts as an intrinsic governor of the innate immune response to prevent overactivation that can lead to immunopathology. thus, the initial immune response to infections is attenuated until proinflammatory signals overcome anti-inflammatory signals. cd was previously identified by microarray analysis as an interferon-stimulated gene (isg), upregulated as part of a coordinated program of host defense mechanisms upon ifn-␣ stimulation ( ) . in addition, tnf-␣ was demonstrated to induce the upregulation of cd on vascular smooth muscle cells in vitro ( ) . in breast cancer, tnf-␣-mediated cd upregulation is transcriptionally controlled by nf-b through an nf-b motif within an enhancer of the cd gene ( ) . these reports are consistent with our findings showing that the inflammatory milieu in patient blood during hcv infection is sufficient to upregulate cd surface expression (fig. e) . while our study focused on tnf-␣, cxcl , and ifn-␣, redundant mechanisms of cd upregulation by additional inflammatory mediators are possible and should be examined in depth in future experiments. in the experimental models utilized, it is difficult to distinguish the relative contributions of cytokine-induced cd upregulation from those of direct pathogen-induced cd upregulation where both are present and either is sufficient. the results clearly indicate that cd surface expression can be upregulated either in a cell-intrinsic manner via stimulation of prrs or by surveilling immune cells in response to extrinsic signaling by inflammatory cytokines. it may also be possible that signaling via cis interactions within a cell could occur in cells such as macrophages, which express both cd and sirp␣. of the infectious agents that we analyzed, m. tuberculosis infection was unique in failing to induce cd expression upon infection. of interest, uninfected cells from m. tuberculosis-infected cultures downregulated cd expression. this may not be too surprising because the life cycle of m. tuberculosis is dependent on phagocytosis by alveolar macrophages, and m. tuberculosis is known to induce strong inflammatory responses via the induction of cytokines and chemokines. further research will be required to identify the mechanisms involved in these unique responses to m. tuberculosis, but despite its failure to upregulate cd , improved survival from m. tuberculosis infection was observed in mice genetically deficient in cd compared to wildtype mice (fig. e ). better recoveries from malaria parasites ( , ) and escherichia coli infections ( ) have also been shown in cd ko mice. in addition, cd ko mice have improved influenza virus vaccine responses ( ) . however, from an evolutionary standpoint, the upregulation of cd expression in response to pathogens must result in a competitive advantage for the host. as an example, cd ko mice show poorly controlled inflammatory responses to and increased morbidity and mortality from candida albicans infection ( ) . the contrasting results from various infections in cd ko mice illustrate how tightly balanced the immune system has evolved to be and the care that must be taken when immune interventions are undertaken. that said, the results from cd ko mice, in which the entire immune system has developed in the absence of a critical immunomodulatory molecule, might produce different results than the same infection in an immune-replete mouse treated with anti-cd antibodies during the infectious process. indeed, while we found that treating wild-type mice with anti-cd before lcmv strain we (lcmv-we) infection improved recovery, it has been reported that cd ko mice have decreased resistance to lcmv clone- infections ( ) . additional experiments will be required to determine which experimental factors may account for the differences in these outcomes. the accelerated cd ϩ t cell responses and clearance of lcmv infections following prophylactic blockade of cd were most likely due to enhanced apc function evidenced by the increased expression of costimulatory cd on dendritic cells observed at dpi ( ). however, it is also possible that there were direct effects on cd ϩ t cells since it was previously shown that activated cd ϩ t cells express sirp␣, the receptor for cd ( ) . it was shown that only activated effector cells and not naive cd ϩ t cells express sirp␣, and any direct effects on cd ϩ t cells would occur only after expansion and development of effector functions. furthermore, in contrast to the negative signal delivered to cells of the monocytic lineage via sirp␣ ligation to cd , evidence suggested that cd -sirp␣ signaling in cd ϩ t cells delivered a positive signal associated with improved cytolytic killing of infected target cells in vivo. the cd ϩ t cell responses measured in this prophylactic anti-cd study were total cd ϩ t cell responses rather than tetramer-stained cells known to be virus specific. thus, the expansion of bystander cd ϩ t cells by anti-cd was not excluded. however, when anti-cd was used in a therapeutic setting against lcmv infections, the predominant cd ϩ t cell expansions were virus specific, and the mechanism of protection was dependent on dcs and cd ϩ t cells ( ) . the current results demonstrate that cd plays a prominent role in modulating inflammatory responses to infections. while these findings open new possibilities for therapeutic intervention against pathogenic agents, it is important to note that the context of the host response to specific types of infections will determine whether cd blockade would be protective or detrimental. there may be circumstances where host responses need boosting, and cd represents a novel target for host-directed therapies in such cases. possibilities include viruses such as sars-cov- , human immunodeficiency virus, human papillomavirus, cytomegalovirus, epstein-barr virus, varicella-zoster virus, and ebola virus, etc. there is also a potential application for treating infections with bacteria, including m. tuberculosis and multidrug-resistant bacterial strains that might otherwise be untreatable. although not addressed in this study, other infectious agents, such as fungi or parasites, that elicit prr responses might also be tractable to anti-cd therapy. a key factor is that infected cells also express damage-associated molecular patterns (damps), which act as "eat me" signals that are being masked by the cd "don't eat me" signal ( , ) . therefore, releasing the inhibition of phagocytosis of these cells would need to be weighed cautiously with the extent of infection and the replaceability of the infected cell types. . virus stocks were passaged no more than times in vero cells. for analysis of cd expression in mice during lacv infection, -day-old c bl/ (jackson laboratories) male or female mice were inoculated intraperitoneally with a -pfu dose of virus diluted into a -l volume of sterile pbs. mice of the same strain, age, and sex inoculated with an equivalent volume of a vero cell culture supernatant in pbs were used as controls. at dpi, whole blood and spleen were isolated from mice and processed for flow cytometry as described above. lcmv viral titers were detected by plaque-forming assays on mc fibroblasts (obtained from by the ontario cancer institute, canada). organs were dissociated, and plasma was diluted in dulbecco's modified eagle medium (dmem) containing % fetal calf serum (fcs), titrated : over steps, and incubated on mc cells. after h of incubation at °c, a methylcellulose overlay was added, and the cells were incubated for h, followed by staining of lcmv plaques using an anti-lcmv-np antibody (clone vl ). c bl/ mice were infected with ϫ pfu of lcmv strain we and treated with anti-cd via daily intraperitoneal injections of g of anti-cd (clone , catalog number be ; bioxcell) or an isotype control (rat igg a isotype) (bioxcell) from day Ϫ to day postinfection. to analyze infected spleen cells, splenocytes were isolated by tissue homogenization through a -m filter, and red blood cells were removed using ack lysis buffer ( . m nh cl, mm khco , . m edta). the gating strategy for spleen cell subset analyses was the same as the one described previously ( ) . all antibodies were from bd biosciences, biolegend, or ebioscience/thermo fisher scientific, including brilliant violet -anti-cd b (clone m / ), phycoerythrin (pe)-cf -anti-cd (clone id ), pe-cy -anti-cd c (clone hl ), pe-cy -anti-ter (clone ter- ), alexa fluor -anti-cd (clone miap ), and fluorescein isothiocyanate (fitc)-anti-major histocompatibility complex class ii (mhcii) (i-a/i-e) (catalog number fab f); lymphocyte populations were initially gated on single live cells on the basis of forward scatter (fsc) versus side scatter (ssc). mouse dcs were defined as cd c ϩ cd b Ϫ , and macrophages were defined as cd c Ϫ cd b ϩ . human dcs were also gated on the basis of high mhcii expression levels. the cd c ϩ subset contained a minor population of cd bintermediate cells that could have been inflammatory macrophages. the multiparameter data were collected with an lsrii instrument (bd biosciences) and analyzed using flowjo software. bacterial strains. bacterial strains included a b borrelia burgdorferi clone (gcb ) with the cp plasmid replaced by a cp -based ptm construct containing green fluorescent protein (gfp) ( ) . salmonella enterica serovar typhi ty mcherry mutant strains were generated via red recombination and included the salmonella enterica serovar typhi ty mcherry Δfla (flic::kan) mutant ( ) . m. tuberculosis h rv Δlysa and Δpancd, used for the in vitro studies, were provided by william r. jacobs, jr. ( ) . m. tuberculosis strain h rv was used for the in vivo studies. affymetrix array profiles of liver biopsy specimens. affymetrix arrays were obtained as cel files, mas normalized using the "affy" package in bioconductor, mapped to ncbi entrez gene identifiers using a custom chip definition file (brainarray version [http://brainarray.mbni.med.umich.edu/ brainarray/]), and converted to hugo gene symbols ( ) . a publicly available gene expression data set from sars-cov- infection of a cells (accession number gse ) (n ϭ ) was downloaded from the gene expression omnibus (geo). independent biological replicates of transformed lung alveolar (a ) cells were mock infected (uninfected [un]) (n ϭ ) or infected (inf) (n ϭ ) with sars-cov- (usa-wa / ). cdna libraries were sequenced from each sample using the illumina nextseq platform. raw sequencing reads were aligned to the human genome (hg ) using the rna-seq alignment app (v . . ) on basespace (illumina, ca). gene expression values were summarized using counts per million (cpm) and converted to a log scale using the formulas log (cpm) if the cpm were Ͼ and cpm Ϫ if the cpm were Ͻ . a standard t test was performed using the python scipy.stats.ttest_ind package (version . . ) with welch's two-sample t test (unpaired, unequal variance [equal_varϭfalse], and unequal sample size) parameters. the results were independently validated with r statistical software (r version . . , july ). cd was significantly upregulated (p ϭ . ) in sars-cov- -infected samples. ( ) . hcv sofosbuvir cohort. pbmcs, plasma, and serum were studied in hcv-infected patients previous to direct-acting antiviral therapy (sofosbuvir [sof] and simeprevir [sim] ; sof and ribavirin [rbv]; and sof, rbv, and pegylated interferon [peg]) before treatment, during treatment, and after treatment. ten patients underwent at least one previous treatment with interferon, and the other four were treatment naive. thirteen patients experienced a sustained virologic response (svr) after weeks of therapy. pbmcs, plasma, and serum were collected from noninfected patients as a control (table ) . one patient relapsed. patients provided written informed consent for research testing under protocols by the stanford university institutional review board. phospho-cytof sample processing and staining. cryopreserved pbmcs stored at Ϫ °c were thawed in warm rpmi medium supplemented with % fbs, benzonase, and a penicillin-streptomycin mixture (complete rpmi medium). cells were transferred into serum-free rpmi medium containing mm edta and benzonase, incubated with cisplatin for min, and immediately quenched with volumes of complete rpmi medium. next, million cells per sample were transferred into complete rpmi medium and rested for min at °c. following this rest period, cells were fixed in pbs with % paraformaldehyde (pfa) at room temperature for min. cells were then washed twice with cyfacs buffer and barcoded as previously described ( ) . following barcoding, samples were combined for surface marker staining, performed at room temperature for h. subsequently, cells were washed and permeabilized in methanol (meoh) at Ϫ °c overnight. the next day, cells were washed and incubated with the intracellular cytokine cocktail at room temperature for h. dna staining was performed for min with iridium ( / ) in pbs with % pfa at room temperature. finally, cells were washed twice with cyfacs buffer and then twice with milliq water before data acquisition on the cytof instrument. data were debarcoded and manually analyzed on cytobank (www.cytobank.org/). monocyte-derived dc and macrophage cultures. healthy donor leukocyte reduction system cones were provided by the stanford blood center. pbmcs were isolated by a . -g/ml ficoll gradient using sep-mate tubes. monocytes were selected for by plastic adherence after min of incubation at °c with % co in rpmi medium plus % human serum (gemini). selected monocytes were then cultured for h in rpmi medium supplemented with % serum, ng/ml interleukin- (il- ), and iu/ml granulocyte-macrophage colony-stimulating factor (gm-csf); the concentration of gm-csf was increased to , iu/ml for the final h. immature dcs were matured by replacing culture medium with rpmi medium supplemented with % healthy donor or hcv patient plasma, ng/ml il- , iu/ml gm-csf, mg/ml lipopolysaccharide (lps), and iu/ml ifn-␥. for macrophage derivation, monocytes were cultured in rpmi medium supplemented with % human serum for days. human and murine luminex assays. the human samples were analyzed at the human immune monitoring center at stanford university. human -plex or mouse -plex kits were purchased from ebioscience/affymetrix and used according to the manufacturer's recommendations, with modifications as described below. briefly, beads were added to a -well plate and washed in a biotek elx washer. samples were added to the plate containing mixed antibody-linked beads and incubated at room temperature for h, followed by incubation overnight at °c with shaking. cold and room-temperature incubation steps were performed on an orbital shaker at to rpm. following incubation overnight, plates were again washed in a biotek elx washer, and a biotinylated detection antibody was then added for min at room temperature, with shaking. the plate was washed as described above, and streptavidin-pe was added. after incubation for min at room temperature, another wash was performed as described above, and reading buffer was added to the wells. each sample was measured in duplicate. plates were read using a luminex instrument with a lower bound of beads per sample per cytokine. custom assay control beads by radix biosolutions were added to all wells. in vitro stimulations and infections of human pbmcs and macrophages. healthy donor leukocyte reduction system cones were provided by the stanford blood center. human pbmcs were isolated by a . -g/ml ficoll gradient using sep-mate tubes. isolated pbmcs were cultured in rpmi medium supplemented with % fbs and u/ml penicillin-streptomycin at a concentration of ϫ cells/ml. to activate prrs, cells were stimulated with either g/ml cl -rhodamine (invivogen), g/ml muramyl dipeptide (invivogen), or r at concentrations of . g/ml, g/ml, and g/ml or left unstimulated. cells were collected at h poststimulation prior to flow cytometry. pbmcs were stimulated with single treatments or combination treatments of ng/ml tnf-␣, ng/ml cxcl , and ng/ml ifn-␣. cells were then analyzed for cd expression h after cytokine stimulation. in vitro bacterial infections of pbmcs were performed at a multiplicity of infection (moi) of for salmonella enterica serovar typhi strains and at an moi of for borrelia burgdorferi, for and h, respectively. salmonella enterica serovar typhi strains were spun onto the cells to compensate for the motility differences. indeed, the strain of salmonella enterica serovar typhi infected a lower percentage of cells, but infected versus uninfected cells were differentiated for the analyses. for m. tuberculosis in vitro infection of macrophages, m. tuberculosis was stained for h in pbs with a : , dilution of phrodo (essen biosciences) at °c to fluorescently label infected macrophages. macrophages were plated into -well u-bottom plates. fluorescently labeled m. tuberculosis bacteria were used to infect macrophages at an moi of : for h. for antibodies for flow cytometry, anti-cd c (clone . ), anti-hla-dr (clone l ), anti-cd b (clone m / ), anti-cd (clone m e ), anti-cd (clone g ), and anti-sirp (clone se a ) were purchased from biolegend, except for allophycocyanin (apc)-anti-cd (clone b h ; ebioscience), which was purchased from invitrogen. cells were analyzed with =, -diamidino- phenylindole (dapi) for dead-cell exclusion and then gated on single cells using fsc-a (forward scatter all human in vitro experiments were repeated in at least two independent experiments with a minimum of biological replicates. in vivo and in vitro stimulation of mouse cells. female c bl/ rrid:imsr_jax: (wt) mice were bred at the stanford university stem cell institute barrier facility (stanford, ca) and used at between and weeks of age at the beginning of the experiments. for r in vivo analysis, naive mice were injected intraperitoneally with either mg/kg of body weight of r or pbs, all at a volume of . ml, for three consecutive days. on day after the first treatment, splenocytes were isolated. spleens were dissociated by collagenase treatment in the presence of dnase i and mechanical dissociation to obtain a single-cell suspension of splenocytes. red blood cells were removed using ack lysis buffer (gibco), and the remaining splenocytes were seeded at a density of ϫ splenocytes/well in a -well u-bottom low-adherence plate. cells were then stained for the macrophage and dc markers cd b, mhcii, and cd c, as well as sirp␣ and cd , with dapi for live/dead exclusion. for in vitro stimulation, splenocytes were isolated from naive mice as described above and then stimulated with either g/ml cl -rhodamine (invivogen) overnight or g/ml poly(i·c)-rhodamine (invivogen) complexed with lipofectamine for h or left unstimulated. cells were collected at h poststimulation for analysis by flow cytometry. antibodies for flow cytometry were purchased from biolegend or bd biosciences. m. tuberculosis infections. m. tuberculosis infections were done in c bl/ mice or cd ko rrid:imsr_jax: (cd ko) mice that were bred at niaid facilities. for infections with m. tuberculosis h rv ( to cfu), -to -week-old male and female mice were placed in a whole-body inhalation system (glas-col, terre haute, in) and exposed to aerosolized m. tuberculosis. delivery doses were set by measuring lung cfu at to h postexposure from three to five control mice through mechanical homogenization using precellys evolution (precellys, atkinson, nh). lung homogenates were then serially diluted in pbs-tween and cultured on middlebrook h agar plates supplemented with oleic acid-albumin-dextrose-catalase (difco, detroit, mi), and cfu were counted days later. cell isolation from m. tuberculosis-infected lung tissue and flow cytometry. lungs were digested and dissociated using gentle magnetically activated cell sorting (macs) and lung cell isolation buffer (miltenyi biotec). the digested lung was passed through a -mm cell strainer, and an aliquot was removed for the determination of cfu. cells were washed and purified with % percoll. cells for sorting were washed, counted, and subsequently surface stained in a biosafety level (bsl ) containment area under sterile conditions. the following cell populations were sorted to to % purity and plated for cfu counts: cd . ϩ (wt) or cd . Ϫ (il r / ) cd b ϩ , cd b ϩ gr high (neutrophils), and cd b ϩ gr low (myeloid). abs against i-ab (clone m / . . ), ly g (clone a ), cd c (clones hl and n ), cd . (clone a ), cd . (clone ), t cell receptor ␤ (tcr␤) (clone h - ), nk . (clone pk ), cd b (clone m / ), cd (clone -f ), and gr- (clones rb and c ) and live/dead fixable cell stains were obtained from ebioscience/thermo fisher scientific, biolegend, or bd pharmingen. samples were acquired on a symphony flow cytometer or sorted on a facsaria instrument (bd biosciences, san jose, ca) and analyzed using flowjo software. supplemental material is available online only. was supported by grant f dk and the stanford medical scientist training program. the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. k.j.h. and i.l.w. are listed as inventors on u.s. patent / a cd , targeted therapies for the treatment of infectious disease. i.l.w. is a cofounder, director, and stockholder in forty seven inc., a public company that was involved in cd -based immunotherapy of cancer during this study but was acquired by gilead. at the time of this submission, i.l.w. has no formal relationship with gilead. restoring function in exhausted cd t cells during chronic viral infection immune checkpoints and their inhibition in cancer and infectious diseases combination anti-ctla- plus anti-pd- checkpoint blockade utilizes cellular mechanisms partially distinct from monotherapies cd blockade by hu f -g and rituximab in non-hodgkin's lymphoma first-in-human, first-in-class phase i trial of the anti-cd antibody hu f -g in patients with advanced cancers the macrophage 'do not eat me' signal, cd , is a clinically validated cancer immunotherapy target therapeutic targeting of the macrophage immune checkpoint cd in myeloid malignancies the cd -signal regulatory protein alpha (sirpa) interaction is a therapeutic target for human solid tumors cd is upregulated on circulating hematopoietic stem cells and leukemia cells to avoid phagocytosis dysregulated gene expression networks in human acute myelogenous leukemia stem cells calreticulin is the dominant pro-phagocytic signal on multiple human cancers and is counterbalanced by cd integrin-associated protein is a receptor for the c-terminal domain of thrombospondin integrin-associated protein is a ligand for the p neural adhesion molecule integrin-associated protein (cd ) and its ligands the interaction between signal regulatory protein alpha (sirp␣) and cd : structure, function, and therapeutic target cd plays a role as a negative regulator in inducing protective immune responses to vaccination against influenza virus a functional subset of cd ϩ t cells during chronic exhaustion is defined by sirp␣ expression functional cd /signal regulatory protein alpha (sirp(alpha)) interaction is required for optimal human t-and natural killer-(nk) cell homeostasis in vivo recent advances in understanding viral evasion of type i interferon myxoma virus m l is expressed as a cell surface cd -like virulence factor that contributes to the downregulation of macrophage activation in vivo friend retrovirus studies reveal complex interactions between intrinsic, innate and adaptive immunity suppression of acute anti-friend virus cd ϩ t-cell responses by coinfection with lactate dehydrogenase-elevating virus immunotherapeutic blockade of cd inhibitory signaling enhances innate and adaptive immune responses to viral infection toll-like receptors stimulate human neutrophil function cutting edge: inflammasome activation in primary human macrophages is dependent on flagellin human tlr or tlr independently confer responsiveness to the antiviral compound r- sofosbuvir: a novel treatment option for chronic hepatitis c infection dendritic cells, monocytes and macrophages: a unified nomenclature based on ontogeny immune response against lymphocytic choriomeningitis virus infection in mice without cd expression lymphocytic choriomeningitis virus cd is an adverse prognostic factor and therapeutic antibody target on human acute myeloid leukemia stem cells functional classification of interferon-stimulated genes identified using microarrays cd -blocking antibodies restore phagocytosis and prevent atherosclerosis a cd -associated super-enhancer links pro-inflammatory signalling to cd upregulation in breast cancer cd regulates the phagocytic clearance and replication of the plasmodium yoelii malaria parasite cd -sirp␣ interactions regulate macrophage uptake of plasmodium falciparum-infected erythrocytes and clearance of malaria in vivo cd deficiency protects mice from lipopolysaccharide-induced acute lung injury and escherichia coli pneumonia cd promotes protective innate and adaptive immunity in a mouse model of disseminated candidiasis cd expression in natural killer cells regulates homeostasis and modulates immune response to lymphocytic choriomeningitis virus macrophages eat cancer cells using their own calreticulin as a guide: roles of tlr and btk enhancement of spleen focus formation and virus replication in friend virus-infected mice real-time high resolution d imaging of the lyme disease spirochete adhering to and escaping from the vasculature of a living host long-term protection against tuberculosis following vaccination with a severely attenuated double lysine and pantothenate auxotroph of mycobacterium tuberculosis robust enumeration of cell subsets from tissue expression profiles sars-cov- launches a unique transcriptional signature from in vitro, ex vivo, and in vivo systems platinum-conjugated antibodies for application in mass cytometry we thank members of the weissman, hasenkrug, davis, and glenn laboratories, especially da yoon no and nathaniel fernhoff, for helpful advice, discussions, and reagents. we specifically acknowledge aaron newman for assistance and guidance in the analysis of microarray data. we thank susan matlock brewer for assistance and guidance with salmonella experiments. we thank yael rosenberg-hasson and the staff of the stanford human immune monitoring center for their technical expertise and for running the mouse and human luminex assays. we also acknowledge the laboratory of benjamin r. tenoever and daniel blanco-melo for the rapid sharing of critical sars-cov- data at the onset of this pandemic (geo accession number gse ).research key: cord- -lnrgom authors: björnsdóttir, sigríður; harris, simon r.; svansson, vilhjálmur; gunnarsson, eggert; sigurðardóttir, Ólöf g.; gammeljord, kristina; steward, karen f.; newton, j. richard; robinson, carl; charbonneau, amelia r. l.; parkhill, julian; holden, matthew t. g.; waller, andrew s. title: genomic dissection of an icelandic epidemic of respiratory disease in horses and associated zoonotic cases date: - - journal: mbio doi: . /mbio. - sha: doc_id: cord_uid: lnrgom iceland is free of the major infectious diseases of horses. however, in an epidemic of respiratory disease of unknown cause spread through the country’s native horse population of , . microbiological investigations ruled out known viral agents but identified the opportunistic pathogen streptococcus equi subsp. zooepidemicus (s. zooepidemicus) in diseased animals. we sequenced the genomes of isolates of s. zooepidemicus to differentiate epidemic from endemic strains. we found that although multiple endemic clones of s. zooepidemicus were present, one particular clone, sequence type (st ), was likely to have been responsible for the epidemic. concurrent with the epidemic, st was also recovered from a human case of septicemia, highlighting the pathogenic potential of this strain. epidemiological investigation revealed that the incursion of this strain into one training yard during february provided a nidus for the infection of multiple horses that then transmitted the strain to farms throughout iceland. this study represents the first time that whole-genome sequencing has been used to investigate an epidemic on a national scale to identify the likely causative agent and the link to an associated zoonotic infection. our data highlight the importance of national biosecurity to protect vulnerable populations of animals and also demonstrate the potential impact of s. zooepidemicus transmission to other animals, including humans. t he resident horse population of iceland is geographically isolated; it arose from animals introduced by settlers in the th and th centuries, with virtually no import of horses for the last , years. this isolation of the population has meant that icelandic horses have remained free from most common contagious diseases of equidae, including equine influenza, equine rhinopneumonitis, equine viral arteritis, rhodococcus equi infection, and strangles (streptococcus equi subsp. equi infection) ( ) ( ) ( ) . as such, the population is extremely vulnerable to these and other infectious agents, and strict biosecurity measures are in place to maintain a high health status. between april and november of , an epidemic of respiratory disease in horses that was typified by a persistent dry cough and mucopurulent nasal discharge spread across iceland, affecting almost the entire population of an estimated , horses. in addition to the stoppage of all equine activities, it resulted in a self-imposed ban on the export of horses, with important economic consequences for the icelandic horse industry. here, we describe how the opportunistic bacterial pathogen streptococcus equi subsp. zooepidemicus (s. zooepidemicus) was identified as the causative agent of the respiratory disease and how, by analyzing the genome sequences of s. zooepidemicus isolates, a distinct rapidly expanding clone was identified. furthermore, we demonstrate that this clone was responsible for zoonotic infections during the course of the epidemic and that this strain has become endemic within the icelandic horse population. epidemiological network analysis identifies an infection source. the veterinary authority of iceland received notifications from horse owners and practicing veterinarians indicating cases of the disease all over the country during the spring and summer of . questionnaires were sent electronically to equine premises throughout the country. data from ( %) of these premises that responded confirmed that the disease was already widespread throughout iceland by the first week of april , with affected premises in the south, west, and north of iceland. these data provided evidence that all horses residing at these premises appeared to be susceptible to the infectious agent, with a morbidity of % but low mortality. dry cough was usually the first clinical sign noted, most often coexisting with symptoms of mucopurulent nasal discharge and mild conjunctivitis, although rectal temperature remained normal in most horses. laryngoscopy revealed laryngitis and inflammation of the upper trachea. the incubation period was between and weeks and the duration of clinical signs varied from to weeks. the first cases of the epidemic were reported to the veterinary authority of iceland on april by an equine center in the north of iceland (farm ). at that time, out of the resident horses showed clinical signs of respiratory tract infection, one of which had been coughing for days. three other horses at farm had a history of similar clinical signs to weeks earlier. network analysis of affected farms identified a single common training yard (yard a) as a primary center of transmission (fig. ) . two horses leaving yard a on february transmitted the disease both to farm and to a stable in the reykjavik area. all horses (n ϭ ) that left yard a in march transmitted the disease to new premises (n ϭ ). these new premises each had up to horses stabled and became secondary centers of transmission. however, two horses from yard a which had returned to farm on february were not incubating the disease. therefore, it is likely that the transmission of the infectious agent to horses at yard a began between and february . identification of s. zooepidemicus as a potential causative agent. the initial assumption due to the rate of spread was that a virus was responsible. nasal swabs were tested by pcr for viruses known to cause respiratory disease in horses and some common respiratory viruses of humans and animals. paired blood samples were used to determine if horses developed an antibody response to equine respiratory viruses during the course of their disease. established and primary cell lines were also used toward the isolation of a potential causal virus. all of these tests proved negative, with the exception of equine gammaherpesviruses, which were isolated from small numbers of both healthy and clinically affected horses (see table s in the supplemental material). therefore, these viruses were not regarded as being related to the epidemic. in the absence of a viral pathogen, it was noted that the gram-positive bacterium s. zooepidemicus was isolated from almost all of the nasal swabs taken from coughing horses and from the diseased tissues of occasional fatal cases (table s ). initially this was not considered of relevance, because although s. zooepidemicus is an opportunistic pathogen that is associated with a range of equine infections ( , ) and infections of other animals, including humans ( , ) , it is routinely isolated from healthy horses and is widely considered a commensal. the population structure of icelandic s. zooepidemicus isolates is dominated by a few dominant clones. to determine if the epidemic was associated with the introduction and spread of a specific s. zooepidemicus strain, we employed wholegenome sequencing (wgs) to interrogate the relationships of s. zooepidemicus isolates. a total of isolates were recovered during from horses residing on different premises across iceland, and also from two cats, one dog, and three people. ten archived icelandic isolates of s. zooepidemicus from seven horses, two sheep, and a dog were included to provide an insight into the identity of historical isolates of s. zooepidemicus from iceland. thirty-eight isolates that were representative of the wider population diversity of s. zooepidemicus beyond iceland were also included in our analysis (table s ) . phylogenetic analysis of the wgs data provided a high-resolution view of the icelandic s. zooepidemicus population structure. the majority of icelandic s. zooepidemicus isolates recovered during the epidemic ( of isolates [ %]) fell into four distinct clades (fig. ) that corresponded to four separate multilocus sequence typing (mlst) sequence types (sts). clade (st ) contained isolates obtained from eight horses from the institute for experimental pathology, icelandic veterinary institute at keldur, reykjavik, that were sampled over a -day period from september to november . six of these horses had signs of respiratory disease over the sampling period. clade (st ) contained isolates obtained from horses and a dog. the horses were resident at different farms, and the isolates were recovered over a -day period between may and october . twelve of the horses had signs of disease, including two with pyrexia. the remaining two horses were both from farm , had died with signs of interstitial pneumonia identified postmortem, and were infected with st or both st and st (table s ). clade (st ) contained isolates obtained from horses residing at farms over a -day period between may and october and also one human isolate. two of the horses infected with st had died with evidence of pneumonia or interstitial pneumonia at postmortem examination. all of the remaining horses had clinical signs of disease, including with pyrexia at the time of sampling. finally, clade (st ) contained isolates from horses residing at premises across iceland over a -day period between may and october . st was also recovered from a human and a cat. forty-three of the horses from which st isolates were recovered had clinical the neighbor joining phylogenetic tree was built using core snps with snps in regions of recombination removed. included in the phylogeny were s. zooepidemicus isolates from outside iceland that were representative of the genetic diversity of the species. the branches of the tree containing these isolates are shown in gray. the four main clades in the iceland population, clades (st ), (st ), (st ), and (st ), are shown in magenta, blue, red, and green, respectively. for each of the clades, the distribution of the pairwise snp distances calculated for the core genome are displayed (top graph), as is the geographic distribution of the isolates' origins within iceland (bottom image). maps were created using www.spatialepidemiology.net. signs of respiratory disease at the time of sampling (n ϭ ) or had died (n ϭ ). s. zooepidemicus was thought to have contributed to the death of six of these horses, with signs of sepsis, bronchopneumonia, pleuropneumonia, or interstitial pneumonia apparent at postmortem examination. the remaining isolates of s. zooepidemicus were recovered from horses, a human, and a cat. mixed populations of two different isolates of s. zooepidemicus were recovered from horses, and three different isolates were recovered from horses. the genetic variation within each of the four clades, with duplicate isolates from the same horse removed (table s ) , was summarized using their nucleotide diversity ( ) , , calculated using the dendropy python library ( ) . st isolates from icelandic horses on farms differed by a maximum of single nucleotide polymorphisms (snps) (fig. ) , with a value of . eϪ ( % confidence interval, . eϪ to . eϪ ) (fig. ). clade (st ) isolates from horses at farms differed by a maximum of snps, with a of . eϪ ( % confidence interval, . eϪ to . eϪ ). clade (st ) isolates from horses at farms differed by a maximum of snps with a of . eϪ ( % confidence interval, . eϪ to . eϪ ). finally, clade (st ) isolates from horses at the icelandic veterinary institute differed by a maximum of snps, with a of . eϪ ( % confidence interval, . eϪ to . eϪ ). the st isolates of clade had significantly less nucleotide diversity than was identified in the other clades (fig. ), but they were recovered from horses at farms which were located over a wide geographic area (fig. ). our data indicate that this strain had been transmitted throughout iceland over a short period of time, providing evidence that this strain was the most likely cause of the epidemic of respiratory disease. st was transmitted from diseased animals and led to disease in previously healthy horses. three healthy horses (horses , , and ) from the university of iceland were transferred into a barn (farm ) on day . the resident horses at farm had been diagnosed with respiratory disease on day Ϫ , and s. zooepidemicus was recovered from the nasal swabs of horses, which were sampled on day Ϫ . eight of these isolates were subsequently identified as st by genome sequencing. following their introduction, the three healthy horses were monitored for clinical signs of disease. nasal discharge was apparent from day , and the discharge became mucopurulent on day and was accompanied by the first observation of coughing. horse was euthanized on day , while horses and were euthanized on day postintroduction. postmortem examination of these three horses revealed signs of respiratory tract infection which included the presence of mucopurulent material in the nasal cavity, larynx, and trachea and enlarged cervical and mandibular lymph nodes. histopathological analysis identified subacute rhinitis, laryngitis, and tracheitis with transmigration of neutrophils through the mucosal epithelium. seven of eight isolates of s. zooepidemicus that were recovered on day or during postmortem examination of horses and on day were st (table s ) . however, none of the seven isolates recovered from horses , , or prior to day were st , and the st strain was not recovered from the postmortem examination of horse on day postintroduction. the failure to recover st from horse may have been due to the onset of clinical signs in this animal and its being euthanized on day , before shedding of st had accumulated to detectable levels in the presence of other strains of s. zooepidemicus. the sampling regimen was, at the time, designed to detect a viral agent and not s. zooepidemicus. a greater depth of bacterial isolates recovered from clinical samples may have enabled the subsequent detection of st in a mixed population of s. zooepidemicus. the very low levels of diversity between st isolates presented a challenge to confirming that the st strains recovered from horses and had been acquired through transmission from the horses at farm . however, st isolates, in common with most other strains of s. zooepidemicus ( ) ( ) ( ) , possess a clustered regularly interspaced short palindromic repeat (crispr), which provides a snapshot of a strain's exposure to mobile genetic elements ( , ) . in keeping with rapid transmission across the population, the icelandic isolates of st predominantly shared the same complement of spacer sequences (fig. ) . however, st isolates from resident horses , , , and on farm were found to contain a novel spacer sequence. this unique spacer was not present in those isolates recovered from four other resident horses , , , and at farm or from any other affected horses from the other farms throughout iceland. the st isolates that were recovered from horses and from day contained the unique spacer sequence, directly linking their infection with this strain to their arrival and exposure to the resident horses at farm . our data show that st was transmitted from horses with signs typical of the respiratory disease epidemic to healthy horses and provide further evidence that st was the most likely cause of the epidemic of respiratory disease in iceland. estimation of the date of common ancestry for the icelandic st isolates. following the resolution of the epidemic, we hypothesized that the st strain had become endemic within the icelandic horse population and that the genetic variation that had accrued since the epidemic could be exploited to estimate the time of most recent common ancestor (tmrca) shared by the icelandic st population. to determine if the st strain persisted within the icelandic horse population, we sampled an additional healthy horses years after the epidemic. a total of s. zooepidemicus isolates were recovered. of these, isolates were identified by quantitative pc (qpcr) as st and had been obtained from four healthy horses stabled at different farms throughout iceland (table s ) . wgs of the isolates from each of the individual horses indicated that they were very closely related. however, isolates from the different horses clustered separately, with longer branch lengths relative to the isolates recovered from the epidemic in . we analyzed a subset of our data set, for which the date of isolation was known, with the bayesian phylogenetics software beast ( ) . removal of strains with identical sequences that were recovered from the same animal on the same date produced a data set of st isolates with polymorphic core genome positions (table s ). this data set comprised isolates from , isolates that were recovered during the epidemic in , and non-icelandic isolates which shared an identical or closely related st to st . beast includes a number of relaxed molecular clock models that permit modeling of variations in substitution rates on different branches of the tree, allowing correction for the observed rate variation in our data. utilizing these models, we found that a strict clock with a skyline population model was the best fit of our data, and the mean substitution rate per core genome site per year was calculated as . ϫ Ϫ . this substitution rate is similar to core genome rates reported for other streptococci, including streptococcus pyogenes ( . ϫ Ϫ ) ( ) and streptococcus pneumoniae ( . ϫ Ϫ ) ( ), and many other gram-positive bacteria, including staphylococcus aureus ( . ϫ Ϫ ) ( ) . interestingly, this rate is faster than that for the closely related host-restricted pathogen streptococcus equi ( . ϫ Ϫ ) ( ), with which s. zooepidemicus shares Ͼ % dna identity ( ) . this difference most likely reflects the unusual lifestyle of s. equi, which can persist within the guttural pouches of recovered horses ( ) . the analysis provided a median estimate for the tmrca of the icelandic st strains of july ( % highest posterior density, august to may ) (fig. ) . this study represents the first time that wgs has been used to investigate an epidemic on a national scale to identify the causative agent. the analysis of wgs data of s. zooepidemicus isolates collected during the epidemic of equine respiratory the presence of shared spacer sequences, from to , is indicated by colored boxes, with the date of isolation, farm, and horse number shown in the columns on the right side of the chart. the location of isolates recovered from farm is highlighted in the right panel. an isolate from farm that contained an additional three spacers in its genome sequence is highlighted in green in the right panel. genomic dissection of an icelandic epidemic ® disease enabled the resolution of recent events from distant transmission events and the identification of the st strain, which was the most likely cause of the epidemic. our data fully support the epidemiological analysis of this epidemic and point to the incursion of a novel strain of s. zooepidemicus, st , into the icelandic horse population. we revealed the diverse pathogenomic properties of s. zooepidemicus and how a novel strain can spread rapidly through a susceptible population devoid of sufficient cross-protective immunity despite a background of concomitant colonization with endemic strains. the endemic strains of s. zooepidemicus recovered from icelandic horses in this study are also likely to have caused clinical signs of disease. st strains were recovered from two fatal cases of pneumonia at farms and in different areas of iceland, and st strains were recovered from two horses that died from pneumonia at farm (one of which was coinfected with st ). however, st or st isolates were each recovered from only horses on and farms, respectively. in comparison, st isolates were recovered from horses that were sampled on farms across iceland. the genetic diversity of st and st strains was significantly higher than that of st , suggesting that these other strains were endemic within the icelandic horse population and were not likely to be responsible for the widespread occurrence of respiratory signs associated with the epidemic in . st strains were only recovered from horses at the icelandic veterinary institute. even then, the nucleotide diversity of these isolates was significantly higher than that of st isolates. our data suggest that st had persisted within this closed population of horses for several years and that it was highly unlikely to have caused the epidemic of respiratory disease in . s. zooepidemicus can cause severe disease in humans ( , , ( ) ( ) ( ) and other animals ( ) ( ) ( ) , and transmission from animals to humans has been demonstrated ( , ) . the epidemic st strain was recovered from a cat and from a blood sample from an icelandic woman who had suffered a miscarriage (e / and e / , respectively) (fig. ) . a closely related strain of s. zooepidemicus st , hum , was recovered from a psoas abscess in a finnish man in ( ) , suggesting that st strains of s. zooepidemicus may have increased potential to cross host boundaries to cause zoonotic disease. several other cases of human infections in iceland during may have been caused by the st strain, but unfortunately these isolates were not available for sequencing. the other two isolates of s. zooepidemicus, e / and e / , that were recovered from humans during the epidemic period were st and st , respectively. both of these sts are representatives of endemic icelandic strains, highlighting that the prevalence of zoonotic transmission of s. zooepidemicus may be underreported. yard a opened in january and offers rehabilitation for up to horses recovering from injury or poor condition as well as equipment training for competition horses. although the epidemic agent was generally transmitted through the coughing of infected horses, coughing was not observed at yard a before april , most probably due to a relatively long incubation period permitting apparently healthy horses to return to their original premises posttraining. instead, the most likely route of transmission of the epidemic strain at yard a was a water treadmill, which horses used on a daily basis. the water used in the treadmill contained no disinfectant and was changed on a once-or twice-weekly basis, providing ideal conditions for the transmission of s. zooepidemicus between the visiting horses. the addition of chlorine coupled with regular cleaning and disinfection of water treadmills may minimize or eliminate the transmission of s. zooepidemicus or other infectious agents via this route. the tmrca for the icelandic isolates was estimated to be july . however, we note that st isolates recovered from eight horses stabled at the icelandic veterinary institute over a period of days had significantly higher nucleotide diversity than the st isolates, which were recovered from horses throughout iceland over a much longer period of time. our data suggest that the st isolates of s. zooepidemicus likely persisted in the horses at the icelandic veterinary institute, accruing nucleotide diversity over time within this isolated population. analysis of s. equi isolates recovered from persistently infected animals identified that they had an increased rate of substitution and that isolates from the same animal could accrue substitutions, amplifications, and deletions ( ) . therefore, it is possible that a mixed population of st variants, which could have originated from one or more horses, may have been introduced to iceland at a date much closer to the start of the epidemic. however, it is also possible that the proposed transmission of st strains, for example, via the submerged treadmill at yard a, provided conditions that led to a selective sweep of a more diverse population of st , with the successful variants establishing the epidemic. in this manner, the introduction of st into iceland may have predated the calculated tmrca. the icelandic epidemic st strains share greatest genetic similarity to strain , which was isolated from a coughing horse in sweden during . to date, with the exception of iceland, st strains have only been isolated from scandinavia, and it is interesting the close relationships between many training yards in iceland and this region of europe. the import of horses to iceland has been prohibited since for biosecurity reasons. although the import of used riding equipment is also prohibited, it is difficult to control. therefore, contaminated tack represents a possible industryrelated route of introduction of the st strain into iceland. the ability of st strains to cross host boundaries provides an alternative import mechanism whereby the infection of a human may have facilitated onward anthroponotic transmission to horses resident in iceland. while it is not possible to identify the precise route by which the st strain entered iceland, a heightened awareness of the routes of transmission will greatly reduce the risk of future epidemics of disease in this important horse population. a questionnaire was sent to premises, including all professional training yards and breeding farms in the country, in june and september to determine whether and when horses had shown signs of respiratory disease. further information pertaining to the movement of horses into affected premises was collected by interviews, enabling the network of connected farms and training centers to be investigated. contact study. three clinically healthy icelandic horses (horses , , and ) , aged to years, from the institute for experimental pathology at keldur were transferred to farm weeks after the first signs of respiratory disease were identified in the resident horses. the experiment was conducted in accordance with the icelandic animal care guidelines for experimental animals (the icelandic animal welfare act / ), the icelandic regulations on animal experimentation / , european treaty series , .iii. , and after formal approval from the icelandic ethical committee on animal research, license - . the horses were examined daily for the onset of clinical signs. nasal swabs (coban) for bacteriological and virological examination and blood samples, serum and with edta (vacuette), were collected every days. postmortem examinations. for postmortem examinations, samples were taken from all major organs in addition to samples from the nasal mucosa, larynx, and trachea. tissues were fixed in % neutral buffered formalin. the formalin-fixed material was embedded in paraffin, and -m-thick sections were cut, mounted, and stained with hematoxylin and eosin. virus isolation and diagnostics. nasal swabs and enriched peripheral blood leukocyte cells (pblc) in plasma from edta-stabilized blood samples from diseased and healthy animals were used to inoculate retroviral vector lxsn e e -transfected fetal lung and kidney cell lines (l. thorsteinsdóttir serological tests and pcr assays for the detection of equine herpesvirus types , , , and equine infectious anemia virus, equine arteritis virus, equine rhinitis virus a and b, equine influenza virus a type (h n ) and type (h n ), equine salemvirus, human and equine adenovirus, mammalian reovirus serotypes , , and , carnivore parvovirus, human rhinovirus, human enterovirus, canine pneumovirus, influenza virus a and b, parainfluenza virus , and mammalian coronaviruses were used according to the methods in the manual of diagnostic tests and vaccines for terrestrial animals produced by the oie and other published methods ( , ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . study isolate collection. the origins and details of the isolates of s. zooepidemicus that were included in this study are listed in table s . a total of isolates were recovered from the nasal swabs of horses that were actively showing signs of respiratory disease or were stabled on affected premises during the epidemic. multiple isolates recovered from the same clinical sample or the same horse sampled over time optimized the isolation of the epidemic strain in the face of concomitant colonization. six icelandic isolates recovered from cases of s. zooepidemicus disease in companion animals (two cats and one dog) and in three people that occurred during the epidemic, and icelandic isolates from seven horses, one dog, and two sheep that predated the epidemic were included in the analysis. ten archived icelandic isolates of s. zooepidemicus from seven horses, two sheep, and a dog were included to provide an insight into the identity of historical isolates of s. zooepidemicus from iceland. we also included the sequenced genomes of strains of s. zooepidemicus from outside iceland that were representative of the known species diversity as a whole, as defined by mlst ( ) , including the published genome sequences for s. zooepidemicus strains mgcs ( ), h ( ), bhs ( ), and atcc ( ) . dna sequencing. dna was purified from s. zooepidemicus isolates by using a genelute column kit as described previously ( ) . dna libraries for isolates recovered pre- were created using a method adapted from the illumina indexing standard protocol. genomic dna was fragmented by acoustic shearing to enrich for -bp fragments by using a covaris e , end repaired, and a tailed. adapter ligation was followed by overlap extension pcr using the illumina primer set to introduce specific tag sequences between the sequencing and flow cell binding sites of the illumina adapter. dna was quantified by qpcr and sequenced on the illumina gaii and hiseq platforms according to the manufacturer's protocols, generating index tag end sequences. for isolates recovered post- , dna libraries were prepared using the illumina nextera xt dna library prep kit, according to the standard protocol. the dna libraries were pooled and quantified using the kapa library quantification kit for illumina platforms prior to sequencing on illumina miseq according to the manufacturer's protocol. variation detection. illumina reads were mapped onto the relevant reference sequences by using smalt (http://www.sanger.ac.uk/resources/software/smalt/), with h (genbank accession number fm ) for the total population and a de novo assembly of clade . a minimum -fold depth of coverage for more than % of the reference genomes was achieved for both reference sequences (table s ). the default mapping parameters recommended for reads were employed but with the minimum score required for mapping increased to to make the mapping more conservative. candidate snps were identified using samtools mpileup ( ) , with snps filtered to remove those at sites with a mapping depth of less than reads and an snp score below . snps at sites with heterogeneous mappings were filtered out if the snp was present in less than % of reads at that site ( ) . identification of the core genomes was performed as previously described ( , ) . recombination was detected in the genomes by using gubbins (http://sanger-pathogens.github.io/gubbins/) ( ) . figure s summarizes the gubbins output for clade (st ) isolates. phylogenetic trees were constructed separately using raxml v . . ( ) for all sites in the core genomes containing snps, with a general time-reversible (gtr) model with a gamma correction for among-site rate variations ( ) . the genetic variation within each of the four clades, with isolates from duplicate horses removed (table s ) , was summarized using their nucleotide diversity ( ) , , calculated using the dendropy python library ( ) . the variances of the nucleotide diversity estimates were calculated as described in reference and used to compute confidence intervals using a normal distribution approximation. we used the bayesian software package beast (v . . ) ( ) to investigate the temporal, spatial, and demographic evolution of the st population, with replicate samples removed from the analysis. to estimate the substitution rates and times for divergences of internal nodes on the tree, a gtr model with a gamma correction for among-site rate variation was used. all combinations of strict, relaxed log normal, relaxed exponential, and random clock models and constant, exponential, expansion, logistic, and skyline population models were evaluated. for each, three independent chains were run for million generations, sampling every generations. a maximum clade credibility tree was created from the resulting combined trees created using the treeannotator program, also from the beast package. st -specific qpcr. st isolates recovered in were initially identified using a specific qpcr for nrde allele prior to genome sequencing. forward ( =-accaaaaaagaaaatgct- =) and reverse ( =-tcaacactataaggactaaagaga- =) primers were used to amplify nrde allele on a steponeplus machine (applied biosystems) with sybr fast abi prism reagents (kapa biosystems) and cycling conditions of °c for min followed by cycles of °c for s and °c for s. all qpcr experiments were performed in triplicate. accession number(s). the illumina sequences generated and used in this study have been deposited in the european nucleotide archive under the accession number erp . the accession numbers for the sequences of each isolate are listed in table s . supplemental material for this article may be found at https://doi.org/ . /mbio . - . fig s , pdf file, . mb. combining two serological assays optimises sensitivity and specificity for the identification of streptococcus equi subsp. equi exposure validation and evaluation of vapa-specific igg and igg subclass enzyme-linked immunosorbent assays (elisas) to identify foals with rhodococcus equi pneumonia study of equid herpesviruses and in iceland with a type-specific polymerase chain reaction association between respiratory disease and bacterial and viral infections in british racehorses inflammatory airway disease, nasal discharge and respiratory infections in young british racehorses zoonotic transmission of streptococcus equi subsp. zooepidemicus from a dog to a handler epidemic nephritis in nova serrana mathematical model for studying genetic variation in terms of restriction endonucleases dendropy: 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gammaherpesviruses and , equine adenoviruses and , equine arteritis virus and equine rhinitis a virus by polymerase chain reaction sybr green real-time reverse transcriptionpolymerase chain reaction assay for the generic detection of coronaviruses detection of mammalian reovirus rna by using reverse transcription-pcr: sequence diversity within the lambda -encoding l gene diagnostic assay recommended by the world health organization for swine origin influenza a (h n ) virus cross-reacts with h n influenza virus detection of adenoviruses and enteroviruses in polluted waters by nested pcr amplification identification and phylogenetic comparison of salem virus, a novel paramyxovirus of horses pneumovirus in dogs with acute respiratory disease a simple touch-down polymerase chain reaction for the detection of canine parvovirus and feline panleukopenia virus in feces evaluation of three influenza a and b real-time reverse transcription-pcr assays and a new h n assay for detection of influenza viruses immune response against equine gammaherpesvirus in icelandic horses isolation and partial sequencing of equid herpesvirus from a horse in iceland development of an unambiguous and discriminatory multilocus sequence typing scheme for the streptococcus zooepidemicus group identification of three novel superantigen-encoding genes in streptococcus equi subsp. zooepidemicus, szef, szen, and szep complete genome sequence of streptococcus equi subsp. zooepidemicus strain atcc the sequence alignment/map format and samtools a genomic portrait of the emergence, evolution, and global spread of a methicillin-resistant staphylococcus aureus pandemic rapid phylogenetic analysis of large samples of recombinant bacterial whole genome sequences using gubbins raxml-vi-hpc: maximum likelihood-based phylogenetic analyses with thousands of taxa and mixed models molecular evolutionary genetics bayesian phylogenetics with beauti and the beast . we thank louise treiberg berndtsson and coworkers at the national veterinary institute in uppsala, sweden, and christine förster at the institute of virology, justus-liebig-univerität in giessen, germany, for their assistance with virological testing. we also thank the personnel at the department of bacteriology at keldur for their technical assistance. the icelandic human isolates were obtained from the department of clinical microbiology, landspítali university hospital, reykjavík, iceland. the human isolates from the united kingdom were provided by androulla efstratiou at public health england. bovine isolates were provided by adrian whatmore of the animal and plant health agency. the human isolate from finland was a kind gift from sinikka pelkonen and tamara tuuminen of the finnish food safety authority and university of helsinki, respectively. we are grateful to alistair darby of the university of liverpool for providing the genome sequence of s. zooepidemicus strain . we also thank the core sequencing and informatics teams at the sanger institute for their assistance.s.r.h., j.p., and m.t.g.h. were supported by the wellcome trust (grant ). key: cord- - lh iqm authors: klotz, lynn c. title: comments on fouchier’s calculation of risk and elapsed time for escape of a laboratory-acquired infection from his laboratory date: - - journal: mbio doi: . /mbio. - sha: doc_id: cord_uid: lh iqm nan i n a letter to the editor of mbio, professor ron fouchier published a calculation ( ) in which he finds a very low probability, p , for a laboratory-acquired infection (lai) for a single lab for a single year. claiming numerous safety precautions in his biosafety level (bsl ϩ) laboratory, fouchier calculates p ϭ ϫ - per person per year, and since there are workers with access to his laboratory, p ϭ ϫ - per lab per year. compare this to p ϭ ϫ - per lab per year for bsl laboratories calculated from cdc statistics for undetected or unreported lais ( , ), here called "community lais," as it is assumed that an undetected or unreported lai represents an infection that has traveled outside the lab and into the community. recently reported escapes of lais from high-level biocontainment at cdc laboratories ( ) and the long history of lais and other escapes from laboratories ( ) also argue that fouchier's value for p is too low. lipsitch and inglesby ( ) have supplied additional arguments as to why the fouchier value for p is likely much too low. fouchier uses a simplistic formula, y ϭ /p , to calculate the elapsed time in years for an lai to escape from his laboratory, y ϭ /( ϫ - ) ϭ ϫ , that is, the million years stated in his letter. it is not clear what this calculation tells us. does it give us the elapsed time for a % chance that an lai occurs? does it give us elapsed time for a % chance, or an % chance? in this regard, the elapsed time for a % chance is infinite, as we can never be absolutely certain that an lai will occur. i suggest attaching little weight to this elapsed time calculation and instead concentrating on risk ϭ likelihood ϫ consequences, starting with the p probability, specifically: potential pandemic fatalities ϭ (probability of a community lai) ϫ (probability that the community lai leads to a pandemic) ϫ (estimated fatalities in a pandemic). my risk calculation estimates the likelihood of a community lai for both a single laboratory and n laboratories conducting this research over y years. the total number of laboratories involved in this potential pandemic pathogen research is called here the "research enterprise." a single, easily derived equation is used to determine the likelihood of a community lai: where e is the probability of at least one community lai from n laboratories in y years. example results are presented in table for three values of p . in table , the number of laboratories is either n ϭ for a single laboratory, such as fouchier's, or n ϭ , which is twice the laboratories currently subject to the nih funding pause. picking n ϭ is a reasonable guess since there are likely many other labs throughout the world conducting this research that are not funded by nih. y ϭ years is a reasonable time frame for this research to be completed. the rationale for picking the probabilities, p , in table is as follows: p ϭ ϫ - is calculated from the cdc statistics ( , ). p ϭ ϫ - is -fold less and is my "guestimate" for a bsl ϩ lab with rigorous safety practices. p ϭ ϫ - is fouchier's calculated value. there are valuable observations to be gleaned from table . for instance, taking into account the whole research enterprise, not just a single lab, is important. furthermore, even for fouchier's very low value for p , there is an estimated probability of e ϭ . that there will be at least one community lai over a -year period for labs (likely exactly one lai, as two is much less probable). as i will soon show, e ϭ . , or . %, is not nearly small enough to reduce risk to an acceptable level. also, this e value assumes that all laboratories involved in this research enterprise have the rigorous safety practices of fouchier's lab, a highly unlikely assumption. summarizing the literature, lipsitch and inglesby ( ) estimate the probability that a community lai leads to a global spread (pandemic) to be to %. this range is consistent with the to % range found by merler and coworkers ( ) and with the to % range found in a focused risk assessment ( ) for infection spread beginning on crowded public transportation. as an illustration, using an intermediate value of % for pandemic probability, which is within the estimated ranges, the probability that a community lai occurs and leads to a pandemic would be . ϫ . ϭ ϫ - . a pandemic could result in million fatalities (world popu- lation of billion, % infected, % fatality rate). thus, in this example the estimated number of fatalities for the research enterprise could be ϫ - ϫ million ϭ , fatalities, and the estimated "fatality burden" for each lab in the research enterprise could be , / ϭ fatalities over years or fatalities per year. to put this fatality burden number in perspective, no institutional review board tasked with assessing human subject research would approve a proposed research project with potential fatalities per year. if instead p ϭ ϫ - or p ϭ ϫ - is more representative of the real probability, then the fatalities and fatality burden for each lab in the enterprise would be much higher. over the assortment of bsl and bsl ϩ labs that may be participating in the research enterprise, frighteningly high fatality burdens may not be unrealistic. to try to understand the meaning of fouchier's simplistic equation y ϭ /p , i substitute into equation the values calculated by fouchier (p ϭ ϫ - , y ϭ ϫ years, n ϭ , for his single lab) to find e ϭ . . as i suspected, the value is high. while i do not know of anything significant that we might learn from this observation that fouchier's calculation implicitly implies a high value for e, it at least answers the question put forth in the second paragraph of this letter about the meaning of his calculation of elapsed time to an escape. recall that the highest value of the probability e is . , which implies absolute certainty of an escape, which would take infinite elapsed time, y. even if every laboratory in the research enterprise is as safe as fouchier claims his is, potential pandemic fatalities and fatality burden are still too great. until the research enterprise is restricted to only a few special bsl ϩ labs, with extraordinary precautions ( ) to reduce significantly the probability of community infections, the international community should agree to pause this research indefinitely. alternatively, the research should be rede-signed to not require the development of live respiratory aerosoltransmissible potential pandemic pathogens ( ) . studies on influenza virus transmission between ferrets: the public health risks revisited monitoring select agent theft, loss and release reports in the united states- - the consequences of a lab escape of a potential pandemic pathogen. front. public health : cdc reports potential ebola exposure in atlanta lab lab escapes and "self-fulfilling prophecy reply to "studies on influenza virus transmission between ferrets: the public health risks revisited moratorium on research intended to create novel potential pandemic pathogens containing the accidental lab escape of potential pandemic influenza viruses the human fatality and economic burden of a man-made influenza pandemic: a risk assessment ethical alternatives to experiments with novel potential pandemic pathogens key: cord- - ecnbqom authors: anthony, s. j.; gilardi, k.; menachery, v. d.; goldstein, t.; ssebide, b.; mbabazi, r.; navarrete-macias, i.; liang, e.; wells, h.; hicks, a.; petrosov, a.; byarugaba, d. k.; debbink, k.; dinnon, k. h.; scobey, t.; randell, s. h.; yount, b. l.; cranfield, m.; johnson, c. k.; baric, r. s.; lipkin, w. i.; mazet, j. a. k. title: further evidence for bats as the evolutionary source of middle east respiratory syndrome coronavirus date: - - journal: mbio doi: . /mbio. - sha: doc_id: cord_uid: ecnbqom the evolutionary origins of middle east respiratory syndrome (mers) coronavirus (mers-cov) are unknown. current evidence suggests that insectivorous bats are likely to be the original source, as several c covs have been described from various species in the family vespertilionidae. here, we describe a mers-like cov identified from a pipistrellus cf. hesperidus bat sampled in uganda (strain predict/pdf- ), further supporting the hypothesis that bats are the evolutionary source of mers-cov. phylogenetic analysis showed that predict/pdf- is closely related to mers-cov across much of its genome, consistent with a common ancestry; however, the spike protein was highly divergent ( % amino acid identity), suggesting that the two viruses may have different receptor binding properties. indeed, several amino acid substitutions were identified in key binding residues that were predicted to block predict/pdf- from attaching to the mers-cov dpp receptor. to experimentally test this hypothesis, an infectious mers-cov clone expressing the predict/pdf- spike protein was generated. recombinant viruses derived from the clone were replication competent but unable to spread and establish new infections in vero cells or primary human airway epithelial cells. our findings suggest that predict/pdf- is unlikely to pose a zoonotic threat. recombination in the s subunit of the spike gene was identified as the primary mechanism driving variation in the spike phenotype and was likely one of the critical steps in the evolution and emergence of mers-cov in humans. express the functional receptor for mers-cov, we further show that recombination was likely to be the critical step that allowed mers to emerge in humans. keywords bat, mers coronavirus, spike, uganda, zoonoses i n , middle east respiratory syndrome (mers) emerged in saudi arabia. clusters of fatal pneumonia in adults were determined to be caused by a novel lineage c betacoronavirus ( c cov), termed mers-cov ( ) . this was the first c cov known to cause disease in humans and at the time of its discovery was most closely related to two known bat coronaviruses ( ) , raising the possibility that bats were a reservoir and source for the virus. concurrently, epidemiologists identified an association between mers infections in patients and their contact with dromedary camels ( , ) . mers-cov was subsequently detected in camels at a farm linked to two human cases in qatar ( ) and in camels in egypt ( ) , followed by surveys that demonstrated widespread exposure to the virus in the middle east and in north and east africa as early as the s ( ) ( ) ( ) ( ) . it is now clear that camels play an important role in the transmission of mers-cov to people ( ) , with seroprevalence highest among those who have had contact with camels ( ) . while camels are thought to be important for the transmission of mers-cov, bats are widely considered to be the evolutionary source of the virus. several c covs have now been described in bats, including hku from tylonycteris pachypus ( ) , hku from pipistrellus abramus ( ) , and the recently identified neocov from neoromicia capensis ( ) . neocov is the closest relative yet discovered ( % identical to mers) and shares sufficient genetic similarity in the replicase genes to be considered part of the same viral species ( ) ; however, despite being closely related across much of the genome, the s subunit of the spike gene is highly divergent as a result of a prior recombination event. recombination in the spike gene is particularly significant because the derived protein is responsible for host receptor recognition and membrane fusion ( ) and thus is central in determining host specificity. the s subunit contains the receptor binding domain and therefore has a specific role in defining host tropism ( ) . other processes are also important, such as the activation of the spike protein by host proteases ( ) , but the ability of s to bind with a host receptor is a critical step in the emergence pathway-and it can be quickly altered by a single recombination event. the sequence variation in the s region of mers-cov and neocov could therefore indicate differences in host binding preferences. predicting the interactions of virus binding domains with a particular host receptor (for example, the human mers-cov receptor dpp ) is possible through the use of structural modeling and the generation of infectious clones. protein-protein interactions can be modeled using a related homologous complex ( , ) while reverse genetic strategies can test the permissiveness of human or other primate cells for infectious clones expressing the novel receptor binding domains or complete spike glycoprotein ( ) ( ) ( ) ( ) . pseudotyped lentivirus systems have also been used, for example, to show that dpp is the receptor for hku but not for the closely related hku ( , ) . and while pseudotypes are not always accurate predictors of spike glycoprotein function ( ) , these findings indicate that multiple cell-entry strategies could exist for c viruses and that not all mers-like covs pose an equal risk of zoonotic emergence. here, we investigated the receptor binding properties of a new strain of mers-like cov found in a bat from uganda. this virus (predict/pdf- ) shares the same putative s subunit recombination that was observed in neocov, allowing us to also consider whether the spike recombination was critical for the emergence of mers-cov in humans. sampling and site characterization. a bat (identifier [id] otba - ) was trapped on february in the nkuringo area of kisoro district, in southwestern uganda (latitude Ϫ . , longitude . ) (fig. ) . this area is an established settlement of villages comprising approximately , inhabitants adjacent to the southwestern boundary of bwindi impenetrable national park. communities include subsistence farmers growing small crops, with some members working inside the national park or supporting tourism-related businesses. livestock, including cattle, pigs, sheep, goats, and poultry, are present in the village and are raised on a small scale primarily for local consumption. the sampled bat weighed . g and had a forearm length of mm (fig. ). it was identified as pipistrellus cf. hesperidus based on % sequence identity in the cytochrome b (cytb) gene. the cytochrome oxidase subunit (co ) was also sequenced, but no corresponding co sequences for p. hesperidus were available in genbank for comparison. we therefore relied on the cytb sequence for species identification. discovery and sequence characterization. the oral swab, rectal swab, and whole blood of bat otba - were assayed for the presence of coronavirus by consensus pcr (cpcr). two separate assays were used, each targeting a different region of the orf b rna-dependent rna polymerase (rdrp). bands of the expected size were amplified from the rectal swab (pdf- ) by both assays and confirmed to represent viral products by traditional sanger dideoxy sequencing. both fragments showed Ͼ % amino acid sequence identity to mers-cov, prompting further characterization of the virus. the oral swab and blood were negative. the near-full-length genome (identified as predict/pdf- ) was assembled from -nucleotide (nt) illumina single-end reads at an average depth of ϫ. only the = and = noncoding regions were left incomplete. the order of all predicted open reading frames (orfs) was consistent with mers-cov and with the recently described neocov (kc ) identified in a bat from south africa. similarly, the hexanucleotide transcription regulatory sequence (aacgaa) was conserved and found in the same position as both mers and neocov upstream of each predicted orf. across the full genome, the sequence had . % amino acid identity to mers-cov and % to neocov; however, considerable variation was observed in different genes. uganda mers-like virus in bats ® amino acid identity could be as high as % to both mers-cov and neocov in orf b or as low as % to mers-cov in subunit of the spike protein. for the full spike protein, identity was % to neocov and % to mers-cov. percent sequence identity of the spike protein (subunits and ) to other c viruses is shown in tables and , respectively. based on the current criteria for species demarcation established by the international committee for the taxonomy of viruses (Ͼ % amino acid sequence identity in the replicase proteins), predict/pdf- shares sufficient genetic identity to mers-cov to be considered a member of the mers-like coronavirus species. phylogenetic analysis. maximum likelihood phylogenetic reconstructions showed that predict/pdf- is most closely related to neocov (fig. ) . the two viruses were basal or formed sister clades to mers-cov in all genes except subunit of the spike. the full-genome alignment was scanned for recombination using seven different algorithms (rdp, geneconv, bootscan, maxchi, chimaera, siscan, and seq) implemented in rdp v . . a single recombination event was detected within the spike gene by rdp, bootscan, maxchi, chimaera, siscan, and seq (bonferroni-corrected p of ϽϽ . ), suggesting that the incongruent phylogenies observed between spike subunit and the rest of the genome are the result of recombination. attempts to date the divergence of these two viruses to estimate the "minimum" number of years since this recombination were prevented by evidence of strong negative (purifying) selection across the genome (fig. ) . given that purifying selection can confound true phylogenetic depth, we felt that attempts to estimate the number of years to common ancestry were inappropriate and would result in artificially "recent" dates. zoonotic potential of predict/pdf- . the high genetic variability in subunit suggests that human and bat strains of mers have different receptor binding properties. to investigate this, we modeled the specific affinity of the predict/pdf- spike protein for the human mers-cov receptor dpp ( ) . we utilized the crystal structure of the mers-cov spike binding domain in complex with dpp to create a homology model for the comparable region of the predict/pdf- spike (fig. ) . previous work has demonstrated specific amino acid residues in mers-cov that ( ) . of these residues, only one is conserved for predict/pdf- . to determine whether the binding interactions may be conserved between dpp and predict/pdf- regardless of the differences in amino acid residues at these positions, we analyzed the predicted interactions between predict/pdf- and dpp , compared to mers-cov and dpp . overall, we found a global reduction in predicted hydrogen bonding interactions in the dpp -predict/ pdf- binding interface compared with dpp -mers-cov (fig. ) . while the interactions in conserved residue y were maintained, dpp interactions with predict/ pdf- residues , , , , , , and were disrupted. the interaction between dpp y and mers d is abolished in the predict/pdf- prediction, where d is replaced by k . this is a charge change from negative to positive. interestingly, a change from r in mers to d in predict/pdf- facilitates a potential interaction with y to replace the hydrogen bond lost with k . regardless, due to the predicted loss of the majority of the dpp binding interactions, the model predicts that predict/pdf- will not bind to dpp . to confirm these results in vitro, a recombinant mers-cov cdna clone was constructed containing the predict/pdf- spike gene in the context of the full-length mers-cov backbone. the chimeric virus maintains the entire ectodomain of the predict/pdf- spike with the exception of the first amino acids of the = end, which were taken from wild-type mers-cov. similarly, the transmembrane domains (tmds) and cytoplasmic tail of the chimeric virus used the wild-type mers-cov sequence in order to minimize incompatibility in virion formation. following transfection into vero cells, pcr amplification of leader-containing transcripts for all of the expected nested subgenomic (sg) mrnas (including the sg spike mrna) confirmed replication of the recombinant virus (fig. ) . however, subsequent passages by supernatant transfer to uninfected monolayers failed to reproduce the infection, suggesting that the predict/pdf- spike protein is unable to mediate cell entry in vero cells as seen with wild-type mers-cov (fig. ) . (fig. a) . similarly, viral rna expression analysis indicated no evidence of replication following infection with the predict/pdf- chimeric virus (fig. b) . in contrast, wild-type the discovery of predict/pdf- in uganda adds to the growing number of group c betacoronaviruses that have now been identified in bats. these include neocov from south africa ( ), mex_cov- from mexico ( ), batcov/kw e from thailand ( ), p.pipi/vm from the netherlands ( ), h.sav/ - from italy ( ), and betacov/sc , hku , and hku , all from china ( ) . collectively, these examples demonstrate that the mers-related covs are highly associated with bats and are geographically widespread. the group c viruses appear to have a particular, though not exclusive, association with vespertilionid bats, which form a highly diverse and widely distributed family within the microchiroptera. neocov, sc , hku , hku , h.sav/ - , p.pipi/ vm , and predict/pdf- were all found in species belonging to this family. if the full diversity of c viruses reflects the number of vespertilionid species described (n ϭ species), there is potential for a substantial diversity of mers-related viruses to be circulating in bats. our data suggest that predict/pdf- cannot infect humans and is not likely to pose a threat to human health, at least in its current form. the spike protein of this virus is distinct from the mers-cov spike, sharing only % amino acid identity, and it appears unable to enter cells that express the functional receptor used by mers-cov (dpp )-or any other receptor expressed by either primate vero cells or human airway epithelial cells. importantly, failure to assemble and release viral particles from the initial infection could also explain our results; however, we suggest that receptor incompatibility is more likely given the steps taken to minimize particle disruption (see materials and methods). these results suggest that adaptation of the spike would be required to permit predict/pdf- replication in human airways. while we did not examine the specific binding properties of the related virus neocov, the high amino acid sequence identity with predict/pdf- indicates that it shares a similar phenotype and is most likely also refractory for human infections. our data suggest that rna recombination is the mechanism that underlies the observed difference in receptor binding. recombination can occur at high frequency during mixed coronavirus infection, allowing different viral lineages to exchange specific functional motifs or even entire genes ( , , ) . phylogenetic incongruence was noted in subunit of the spike protein, and breakpoints were observed in this same region by multiple recombination detection algorithms. it is also parsimonious with the high purifying selection observed across the genome of c viruses (which argues against receptor adaptation via drift or selection) and with previous reports citing recombination in association with host switching for other coronaviruses ( ) ( ) ( ) . given that the recombination is observed in both predict/pdf- and neocov, we support the previous suggestion by corman et al. ( ) that it was the mers-cov that acquired a new spike. given also that the predict/pdf- spike does not use dpp and is seemingly not competent for human infection, we further suggest that the recombination event was the critical factor driving the emergence of mers-cov. what is less clear is whether this recombination occurred in bats or an intermediate host. lineage c strains that use dpp have been reported in bats ( , ) , and there is also evidence of positive selection in the bat dpp that would indicate the existence of a large diversity of (as-yet-unknown) dpp -competent strains ( ) . just as detailed metagenomics studies have revealed the presence of several severe acute respiratory syndrome (sars)-like bat covs that can use the human angiotensin converting enzyme receptor and/or replicate efficiently in human cells ( , , ( ) ( ) ( ) , it seems likely that subsets of diverse mers-cov-like bat coronaviruses will also exist which are preprogrammed to efficiently use the human dpp receptor. this would support the hypothesis that the recombination occurred in bats; however, the mers-cov spike seems to have adapted and acquired a preference for human dpp over the bat homologue ( , ) making it difficult to conclude with certainty that the mers-cov spike has bat origins. increased surveillance will be required to understand the full diversity of spike phenotypes circulating in bats or in intermediate hosts such as camels. in recent years, global surveillance efforts such as the usaid emerging pandemic threats predict program have advanced our understanding of the viral diversity that exists in wildlife ( ) . while this knowledge can be useful for proving the existence of novel viruses ( , , ( ) ( ) ( ) ( ) ( ) , quantifying overall viral diversity ( , ) , and measuring infection prevalence within a population, it does not provide information on their specific zoonotic threat. given that no single correlate of pathogenicity or virulence has been determined for any viral family ( , ) and that it is not possible to determine risk through phylogenetic data alone ( ), the approach used here is an important tool in characterizing the zoonotic potential of viral sequences detected in wildlife. doing so on a large scale (for example, as part of projects like usaid predict) will also provide critical information on host and geographic variation in key viral traits, like potential host tropism, which are currently missing from most risk-based models forecasting hot spots of disease emergence. a bat (id otba - ) was trapped on february in the nkuringo area of kisoro district in southwestern uganda. the bat was caught with a mist net ( . -mm mesh; avinet, inc.) according to established protocols and was released unharmed postsampling. standard morphometric measurements (weight and forearm length) and photographs were obtained to aid species identification, which was confirmed by dna barcoding of the cytochrome b (cytb) and cytochrome oxidase subunit (co ) mitochondrial dna genes ( ) . approximately l of whole blood was collected into edta. oral and rectal swabs were also collected in duplicate (one into viral transport medium and one dry). specimens were stored temporarily on gel packs and frozen in liquid nitrogen in the field within h of collection and then transferred to Ϫ °c for storage until testing. samples were transferred to the center for infection and immunity at columbia university for viral discovery and characterization. coronavirus discovery by consensus pcr. total nucleic acid (tna) was extracted using the roche magna pure platform according to the manufacturer's instructions. tna was reverse transcribed into cdna using superscript iii (invitrogen) according to the manufacturer's instructions. two broadly reactive consensus pcr assays targeting partial and nonoverlapping regions of the coronavirus orf b (containing the rdrp) were performed ( , ) . bands of the expected size were excised from % agarose, cloned into strataclone pcr cloning vector, and sequenced to confirm detection. sequencing and bioinformatic processing. total rna extract was dnase treated (dnase i; ambion, life technologies, inc.) and reverse transcribed using superscript iii (invitrogen, life technologies, inc.) with random hexamer primers. the cdna was rnase h treated before second-strand synthesis with klenow fragment ( = to = exonuclease) (new england biolabs). the resulting double-stranded cdna was sheared to -bp (average) fragments using a covaris focused ultrasonicator e , according to the manufacturer's standard settings, and used for library construction using the kapa hyper library preparation kit (kapa biosystems, roche), again according to the manufacturer's instructions. the final library was quantified using an agilent bioanalyzer and pooled to allocate million reads on the illumina hiseq platform. the q -filtered fastq files were used to generate quality control reports using prinseq software (v . . ) ( ) and were further filtered and trimmed. host background levels were determined by mapping the filtered reads against a bat reference database using bowtie mapper (v . . , http://bowtie -bio.sourceforge.net) ( ) . the host-subtracted reads were de novo assembled using mira assembler (v . ) ( ) . contigs and unique singletons were subjected to homology search using megablast against the genbank nucleotide database. sequences that showed poor or no homology at the nucleotide level were screened by blastx against the viral genbank protein database. viral sequences from blastx analysis were subjected to another round of blastx homology search against the entire genbank protein database to correct for biased e values and taxonomic misassignments. the genome of predict/pdf- was mapped with bowtie against the filtered data set to visualize depth and coverage in integrated genomics viewer. genetic and phylogenetic analyses. sequences were analyzed and edited using geneious (version . . ). full genome and individual gene sequences were aligned with clustalw, and maximum likelihood phylogenetic trees were constructed in paup* ( bootstraps). models of nucleotide substitution were selected using jmodeltest. nucleotide sequence similarity between mers-like viruses was assessed using simplot v . . ( ) with a sliding window size of bp, a step size of nucleotides, and , bootstrap replicates using gap-stripped alignments and the f (maximum likelihood) distance model. the full-genome alignment was scanned for recombination using seven different algorithms (rdp, geneconv, bootscan, maxchi, chimaera, siscan, and seq) implemented in rdp (v . ) ( ). structural modeling. predicted binding differences between dpp and either mers or uganda were determined by structural analysis. the crystal structure demonstrating the interactions between dpp and mers spike binding domain has previously been reported ( ) , and the crystal structure is pdb id kr . we created a homology model of the region of the uganda spike protein homologous to the mers spike binding domain based on the kr structure in association with dpp . we first aligned the amino acid sequences for kr ( ) and the uganda spike using clustal omega ( ). we then used modeller ( ) to create predicted structural coordinates for the uganda spike based on the coordinates of kr . because modeller requires the two sequences to be the same length, we introduced gaps in the sequences where appropriate to maintain the best sequence identity between the amino acid sequences. numbering is based on mers-cov amino acid residues. we then imported the predicted crystal structure for uganda and the known dpp -mers structure into pymol ( ) for visualization and comparative analysis. hydrogen bonding interactions were predicted by selecting the known dpp and kr or the homologous dpp and uganda interaction sites and using the "find polar interactions" function within pymol. generation of a mers-cov recombinant virus. previously, we reported the isolation of recombinant mers-cov that was derived from a cdna clone ( ) . to reconstitute a mers genome expressing the predict/pdf- cov spike, new e and f plasmids were ordered synthetically (bio-basic) to contain the predict/pdf- spike ectodomain; these plasmids were named mers-uganda e and f. mers orf and orf overlap, so to maintain a functional replicase sequence and signal sequence for spike, the first amino acids of the mers spike were retained and the predict/pdf- sequence was fused in frame downstream of the mers-cov s glycoprotein signal peptidase domain beginning at its th amino acid. in short, the sequence of the mers spike coding for amino acids to was replaced with the sequence of the predict/pdf- spike coding for amino acids to , so that following processing, an intact spike glycoprotein was expressed during virus infection. the e and f plasmids were sequence verified prior to the assembly of full-length recombinant dnas. the mers a through f inserts (containing the uganda s gene) were restriction digested, resolved on . % agarose gels, visualized, excised, and purified using a qiaquick gel extraction kit (qiagen). the mers a to f inserts were mixed and ligated overnight at °c, phenol-chloroform extracted, and precipitated under isopropyl alcohol. full-length t transcripts were generated in vitro as described by the manufacturer (ambion; mmessage mmachine) with certain modifications ( ) . for mers-cov n transcripts, g of plasmid dna containing the n gene (amplified using forward primer =-atttaggtgacactat agatggcatcccctgctgcacc- = and reverse primer =-ttttttttttttttttttttttctaatcagtgttaaca tcaatcattgg- =) was transcribed by sp rna polymerase with a : ratio of cap analog to gtp. rna transcripts were added to l of vero cell suspension ( . ϫ cells) in an electroporation cuvette, and four electrical pulses of v at f were delivered with a gene pulser ii electroporator (bio-rad). the transfected vero cells were allowed to recover for min at room temperature and then incubated at °c for to days in a -cm flask. virus progeny were then passaged several times in vero cells or primary human airway epithelial cells for h to detect viable viruses. all viruses were maintained under biosafety level (bsl ) conditions with redundant fans, and personnel used powered air-purifying respirators (paprs) and tyvek suits. to detect leader-containing rnas, intracellular rna from wild type and recombinant mers-cov-uganda (rmers-cov-uganda) was reverse transcribed with a primer at the = end of the genome and cdna was isolated for pcr using a reverse primer located in orf and a forward primer located in the leader rna sequence at the = end of the genome ( =-ctatctcacttcccctcgttctc- =). leadercontaining amplicons were sequenced as previously described ( ) . the cdna products were separated and visualized in . % agarose gels. viruses, cells, and infection. wild-type and chimeric covs were cultured on vero e cells, grown in dulbecco modified eagle medium (dmem) (gibco, carlsbad, ca) and % fetal clone serum (hyclone, south logan, ut) along with antibiotic-antimycotic (anti-anti; gibco, carlsbad, ca). growth curves in vero and primary human airway epithelial cells were performed as previously described ( , ) . human lungs were procured under university of north carolina at chapel hill institutional review board-approved protocols. biosafety and biosecurity. reported studies were initiated after the nih and the university of north carolina institutional biosafety committee approved the experimental protocol (project title, generating infectious clones of bat sars-like covs; lab safety plan id, ; schedule g id, ). accession number(s). the near-complete genome sequence for predict/pdf- has been deposited in genbank under accession number kx . isolation of a novel coronavirus from a man with pneumonia in saudi arabia genetic relatedness of the novel human group c betacoronavirus to tylonycteris bat coronavirus hku and pipistrellus bat coronavirus hku middle east respiratory syndrome coronavirus neutralising serum antibodies in dromedary camels: a comparative serological study evidence for camel-to-human transmission of mers coronavirus middle east respiratory syndrome coronavirus in dromedary camels: an outbreak investigation uganda 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respiratory syndrome coronavirus rewiring the severe acute respiratory syndrome coronavirus (sars-cov) transcription circuit: engineering a recombination-resistant genome release of severe acute respiratory syndrome coronavirus nuclear import block enhances host transcription in human lung cells pathways of cross-species transmission of synthetically reconstructed zoonotic severe acute respiratory syndrome coronavirus we thank nancy simmons (american museum of natural history) and carlos zambrana (ecohealth alliance) for help with species identification. we also thank komal jain for bioinformatic assistance (columbia university) and bernard erima, titus tugume, and raymond mayanja (all from makerere university) for processing samples.this study was made possible by the support of the american people through the united states agency for international development (usaid) emerging pandemic threats predict project (cooperative agreement number ghn-a-oo- - - ) and by support from the national institutes of allergy and infectious disease (ai and ai ) and aging (k ag ). human airway epithelial cell cultures were supported by the national institute of diabetes and digestive and kidney diseases (dk ). key: cord- -b p b g authors: leyva-grado, victor h.; ermler, megan e.; schotsaert, michael; gonzalez, ma g.; gillespie, virginia; lim, jean k.; garcía-sastre, adolfo title: contribution of the purinergic receptor p x to development of lung immunopathology during influenza virus infection date: - - journal: mbio doi: . /mbio. - sha: doc_id: cord_uid: b p b g an exacerbated immune response is one of the main causes of influenza-induced lung damage during infection. the molecular mechanisms regulating the fate of the initial immune response to infection, either as a protective response or as detrimental immunopathology, are not well understood. the purinergic receptor p x is an ionotropic nucleotide-gated ion channel receptor expressed on immune cells that has been implicated in induction and maintenance of excessive inflammation. here, we analyze the role of this receptor in a mouse model of influenza virus infection using a receptor knockout (ko) mouse strain. our results demonstrate that the absence of the p x receptor results in a better outcome to influenza virus infection characterized by reduced weight loss and increased survival upon experimental influenza challenge compared to wild-type mice. this effect was not virus strain specific. overall lung pathology and apoptosis were reduced in virus-infected ko mice. production of proinflammatory cytokines and chemokines such as interleukin- (il- ), gamma interferon (ifn-γ), and cc chemokine ligand (ccl ) was also reduced in the lungs of the infected ko mice. infiltration of neutrophils and depletion of cd b(+) macrophages, characteristic of severe influenza virus infection in mice, were lower in the ko animals. together, these results demonstrate that activation of the p x receptor is involved in the exacerbated immune response observed during influenza virus infection. p x receptor (fig. a) . one hour before infection with influenza a/puerto rico/ / h n (pr/ ) virus or a/netherlands/ / h n pandemic (h n pdm) (nl/ ) virus, cells were treated with bbg, bzatp, or az . no significant differences were observed in pr/ virus growth in cells treated with bbg or bzatp compared to the untreated cells (p ϭ . ) (fig. b) . we observed a significant reduction in virus titers in cells pretreated with az and infected with pr/ at h and h postinfection compared to untreated cells (p Ͻ . ) (fig. b) . in cells infected with nl/ virus and treated with bbg or bzatp, no significant differences were observed in virus growth (fig. c) . in cells pretreated with az and infected with nl/ virus, we observed a significant reduction in virus titers at h postinfection compared to untreated cells (p Ͻ . ) (fig. c) . mice lacking p x receptors have a better outcome after influenza virus infection. activation of the p x r has been associated with an increased and sustained inflammatory response. to determine whether this pathway is related to the exacerbated lung immunopathology developed after influenza virus infection, we tested mice lacking the p x r in our mouse model of influenza virus infection. after infection with pr/ virus, we observed a significant reduction in body weight at days and postinfection in the wild-type (wt) mice (p Ͻ . ) ( fig. a) . we also observed increased survival in the virus-infected p x r ko mice ( %) compared to the wild-type mice ( %) (p Ͻ . ) (fig. b) . similarly, in wild-type mice infected with nl/ virus, we p x receptor in influenza immunopathology ® observed a significant reduction in body weight at days to postinfection (p Ͻ . ) (fig. c) . a significant increase in survival was observed in the infected p x r ko mice ( %) compared to the infected wild-type mice ( %) (p Ͻ . ) (fig. d) . reduced histopathology in the lungs of p x r ko mice infected with the nl/ virus. the amount of replicating virus in the lungs is thought to correlate with the degree of lung pathology. therefore, we infected wild-type and p x r ko mice with nl/ virus, and at or days after infection, the lungs were harvested, and viral titers were determined by plaque assay. reduced virus titers were observed in the lungs of the p x r ko mice compared to the wild-type mice, particularly in the samples collected on day postinfection, although these differences were not statistically significant (p ϭ . ) (fig. a) . in a different set of mice, the lungs were collected on day postinfection, and representative sections were prepared for histopathology. in both groups of mice, lesions were characterized as bronchointerstitial pneumonia, although the severity of the lesions differed in the groups (p Ͻ . ) ( table ). in the lungs of the virus-infected p x r ko mice, the bronchiolar region showed mild to moderate epithelial degeneration, characterized by vacuolation and multifocal necrosis of individual epithelial cells with a thin layer of peribronchiolar inflammatory cells, and moderate perivascular inflammation with little to no presence of intraluminal debris (fig. b) . the bronchiolar region of the lungs from wild-type mice exhibited a marked epithelial degeneration characterized by vacuolation, segmental necrosis, and loss of the epithelium, with a thick layer of peribronchiolar inflammatory cells, and mild to marked amounts of intraluminal debris (fig. c) . at the alveolar level, lesions in the ko mice consisted of mild inflammation as well as tissue damage that was characterized by the presence of scattered cell necrosis with mild to moderate infiltration of inflammatory cells (fig. d) . the alveolar region of lungs from wild-type mice exhibited marked alveolar inflammation characterized by multifocal necrosis and marked infiltration by inflammatory cells (fig. e) . reduction of apoptosis in lungs of mice lacking the p x r and infected with influenza a virus. signaling through the p x receptor induces cell apoptosis by activation of caspase cascades, including caspase- , - , and - . to determine the levels of apoptosis after infection with influenza a nl/ , we quantified the number of fig. a and b ). after quantification, we confirmed that the number of cleaved caspase- -positive cells was significantly lower in the lungs of the infected ko mice (p Ͻ . ), (fig. c) , indicating a reduced amount of apoptosis in the lungs of these mice. the production of proinflammatory cytokines is lower in the virus-infected p x r ko mice than in the wild-type mice. to determine whether the reduced pathology and apoptosis observed in the lungs of the p x r ko mice were correlated with decreased cytokine production after infection, we first quantified the amount of cytokines in ex vivo-infected bone marrow-derived macrophages (bmdm) and then in the lungs of both virus-infected p x r ko and wt mice. cultured bmdm, obtained from p x r ko and wt mice, were infected with nl/ virus, and supernatants were collected at and h postinfection. we observed an increased production of cxc a lung pathology was scored on a scale of to . for amount of tissue affected, lung pathology was scored as follows: , none; , Ͻ % of section; , to % of section; , to % of section; , Ͼ % of section. for inflammation and epithelial degeneration, lung pathology was scored as follows: , no lesions; , mild changes with scattered cell necrosis/vacuolation and few/scattered inflammatory cells in the bronchiolar epithelium with minimal perivascular inflammation; , moderate multifocal vacuolation and/or cell necrosis with peribronchiolar and/or perivascular inflammation (Ͻ cell layers thick); , marked, multifocal/segmental necrosis, epithelial loss/effacement and peribronchiolar and/or perivascular inflammation (Ͼ cell layers thick); , severe, coalescing areas of necrosis, parenchymal effacement with confluent areas of inflammation. for intraluminal debris, lung pathology was scored as follows: , none; , mild amounts; , moderate amounts; , marked amounts; , severe amounts. each score value represents the mean of five mice per group. overall pathology scores for the p x ko group that are significantly different (p Ͻ . ) from those of the wild-type group are indicated by an asterisk. ( fig. ) . a trend to significance was observed in the amount of il- in the lungs of the ko mice compared to the lungs of the wt mice (p ϭ . ) (fig. ). similar results were observed in mice infected with a different influenza virus, pr/ , with significantly larger amounts of il- , ifn-␥, cxcl , ccl , and ccl in the lungs of the wt mice (see fig. s in the supplemental material). the percentages of pulmonary activated macrophages and activated lymphocytes were higher in the p x ko mice after infection. we evaluated the myeloid cell population in the whole lungs of p x ko and wt mice infected with nl/ virus and collected on day or day postinfection (p.i.). we observed a higher number of cd b ϩ macrophages in the lungs of virus-infected p x ko mice at both time points compared to the virus-infected wt mice (p ϭ . ) ( fig. a ) with no significant differences in the absolute number of cells between mock-infected or virus-infected wt and p x ko samples. we then evaluated the leukocyte population in the bronchoalveolar lavage fluid (balf) of both p x ko and wt mice and days postinfection with nl/ virus. the total number of cells and the viability in balf were not different between the groups. the percentage of activated macrophages was significantly increased at day p.i. in the p x ko mice than in the wt mice (p Ͻ . ), but not at day p.i. (fig. b) . the percentage of neutrophils in the balf samples was significantly higher at day p.i. in ® the wt mice than in the p x ko mice (fig. b ) (p Ͻ . ). no differences between groups were observed at day p.i. a significantly higher percentage of activated lymphocytes was observed in the ko mice at day p.i. (p Ͻ . ) (fig. b ). no differences in the percentage of activated lymphocytes were observed between groups at day p.i. the percentage of lysed cells in the balf of wt mice tended to be higher, especially at day p.i. (p ϭ . ) (fig. b ). the mechanisms by which the immune response is balanced to achieve influenza virus clearance and tissue repair versus harmful immunopathology are not well understood ( , , ) . in this study, we showed that mice lacking the purinergic receptor p x have a better clinical outcome after influenza a virus infection characterized by an increased survival rate with an overall reduced immunopathology of the lungs compared to the wild-type mice. atp, the main ligand for p x receptor, is usually located intracellularly, although small amounts can be found in the extracellular space ( , , ) . when cells are under stress or damaged by infection, atp is released into the extracellular space and rapidly binds to excitatory receptors such as p x to promote an inflammatory response ( , , ) . activation of the purinergic pathway through p x r encompasses a series of inflammation-related responses, including activation, proliferation, and recruitment of immune cells, as well as secretion of diverse inflammatory mediators, including cytokines and chemokines ( , , ) . the initial evaluation was done in vitro by infecting a cells and treating them with different p x r agonist and antagonists before influenza virus infection to determine the impact on virus growth. we observed differences only in cells treated with the p x r antagonist az . az is a potent cyclic imide ( % inhibitory concentration [ic ], to nm) that selectively inhibits the human p x r ( ) . in contrast, bbg, the other p x r antagonist used in this study, is moderately selective and less potent in blocking the human p x r (ic , nm) ( ). since we treated cells with the same concentration of compound ( m), we speculate that differences in potency may be one of the reasons why we observed some effect in virus growth with one receptor antagonist but not with the other. for the in vivo studies, we first observed that mice lacking the p x receptor (ko mice) had better survival after infection and that survival was independent of the virus strain used. it is generally accepted that survival of the infected host correlates with a reduction of the viral burden in the lung ( , ) . however, when we quantified the virus titers in the lungs of infected mice, we observed that the virus titers in the lungs of virus-infected ko mice were not significantly different from those in the lungs of virus-infected wild-type mice. this lack of correlation between lung virus titers and severity of disease has been previously reported ( , , ) . in early stages of viral p x receptor in influenza immunopathology ® infection, influenza a virus (iav) activates antiapoptotic signals to evade the host defense mechanism. however, in the later stages of infection, iav triggers apoptosis through activation of intrinsic pathways in order to achieve enhanced viral replication and dissemination ( , ) , contributing to tissue damage. we evaluated apoptosis in lungs at day postinfection in what can be considered late in infection, and we observed reduced numbers of apoptotic cells in the ko mice. apoptosis can occur through many mechanisms, including the production of proinflammatory cytokines leading to an increased burden of apoptosis of the alveolar epithelium ( ) , activation of caspase cascades, including caspase- , - , and - ( ) , and the formation and release of reactive oxygen species ( ) . it is interesting that activation of the p x receptor induces some of the aforementioned proapoptotic mechanisms ( , ) ; therefore, the absence of this receptor is causing a reduction in apoptosis during later stages of infection. the extent and severe damage observed in the lung tissues of the virus-infected wt mice can be due to both the cytolytic effect of the virus infection itself and to immunopathology caused by immune cells targeting infected cells. we did not find a significant difference in lung virus titers between the virus-infected ko and wt mice; therefore, the reduced histopathology and apoptosis observed in the ko mice pointed toward differences in immune-mediated pathology. during influenza virus infection, an increased production of proinflammatory cytokines positively correlates with severity of disease in humans, especially during infection with highly virulent strains of the virus ( , , ) . during the recent h n pandemic, patients with severe disease also had high levels of proinflammatory cytokines ( ) . a similar correlation has been observed in animal models of influenza virus infection ( , ) . in the mouse model of influenza, mice with severe clinical signs that succumb to infection have increased levels of proinflammatory cytokines and chemokines in the lungs and develop lung immunopathology ( ) ( ) ( ) . in our studies, we observed that the virus-infected p x r ko mice had lower levels of proinflammatory cytokines such as il- , tnf-␣, ifn-␥, cxcl , and ccl in their lungs at day postinfection. activation of p x receptors by extracellular atp plays a central role in the release of proinflammatory cytokines, and it plays a role on the regulation of macrophages, neutrophils, and effector t cells, important cell mediators during inflammation ( , , , ). therefore, the lower level of cytokines observed in the lungs of the p x r ko mice after influenza virus infection may be explained by the lack of activation of this purinergic signaling pathway in these mice. we observed that il- levels in the lungs of the ko mice infected with pr/ or nl/ were lower than those of the wt mice. il- is considered an anti-inflammatory cytokine in the context of influenza virus infection, and its production relates to reduced immunopathology and increased survival ( ) . this discrepancy may be explained by the time when we measured il- in the lungs, since the main contributors to il- production in the context of influenza infection are cd ϩ effector t cells, a population of cells that is found later in infection ( ) . in addition, increased levels of this cytokine early during infection can hinder the activation of the antiviral immune response ( ) , suggesting that the increased levels observed in the wt mice early during infection may be detrimental. the suppression of cytokine production at the site of infection using drugs ( ) or mice with specific cytokines knocked out ( - ) is not enough to confer full protection against lethality during influenza virus infection. the lack of the receptor in our model does not induce a global reduction in cytokine production after influenza virus infection since we did not observe differences in cytokine levels in serum from the virus-infected ko and wt mice. we also measured the basal levels of il- ␤, il- , tnf-␣, and ccl in the lungs of naive ko and wt mice, and no significant differences were observed (see fig. s in the supplemental material). together, these results suggest that the reduced histopathology and increased survival observed in the ko mice after infection are in part due to a protective immune response at the site of infection rather than ablation of the response. the local increase in cytokines stimulates the infiltration of neutrophils as well as monocytes from the periphery, an essential step for resolution of virus infection ( , ) . in our studies, we observed a reduction in neutrophil influx to the airways of the ko mice at day postinfection that was also evident during the analysis of the lung histology sections. an exacerbated response with an increased influx of highly proinflammatory neutrophils and the prolonged activation of these cells leads to increased tissue damage and lethality ( , , ) . purinergic signaling in the presence of high concentrations of extracellular atp has a critical role in activation of neutrophils for the production of cytokines and in the increased influx and persistence of neutrophils at the site of infection ( ) . furthermore, we observed a significantly smaller amount of cxcl in the lungs of ko mice infected with nl/ or pr/ influenza virus. this chemokine is an important factor for the chemotaxis and activation (increased oxidative burst) of neutrophils, and high levels in the respiratory tract after influenza virus infection correlate with enhanced pulmonary immunopathology ( ) ( ) ( ) . together, these results suggest that purinergic signaling through the p x receptor is important for the recruitment and possible activation of neutrophils in the lungs after influenza virus infection. we observed an increased influx of activated macrophages to the airway space in the ko mice compared to the wt group. in addition, flow cytometry analysis of the lungs in virus-infected mice showed an increased number of cd b ϩ macrophages or inflammatory macrophages in the ko mice compared to the wt mice. these cells can be proinflammatory or immunomodulatory based on the cytokine profile they produce and surface markers they express after activation ( ) . lung accumulation of these inflammatory macrophages with an immunoregulatory phenotype contributes to reduce the development of immunopathology ( ) . furthermore, it has been shown that maintenance of this cell population for a longer time at the site of infection correlates with protection of mice from influenza-induced immunopathology and lethality ( ) . the role of the p x receptor in the activation of macrophages has been previously established in vivo, ex vivo, and in vitro ( ) . activation of the p x r pathway is important for the release of proinflammatory cytokines, increases in cation fluxes, respiratory burst, inhibition of phagocytosis, and apoptosis in different populations of macrophages ( , , ) . our results indicate that not only the number of immune cells infiltrating the lung after infection determines the development of an exacerbated response leading to immune pathology, but and perhaps more important, is the type and activation status of the cells recruited to the site of infection. the histopathology analysis of lung sections clearly showed an increased number of infiltrating cells in the lung parenchyma of the virus-infected wild-type mice compared to the virus-infected ko mice. however, further analysis of the infiltrating cells showed that the number of cd b ϩ macrophages (whole lung) and number of activated macrophages (balf), both associated with protection, were higher in the virus-infected ko mice. furthermore, activation of the p x receptor can modify the protein secretion profile of stimulated macrophages depending on the activation status of the cells (proinflammatory or immunomodulatory), providing evidence that activation of this pathway may function to fine tune the activity of the heterogeneous population of macrophages observed through infection of the lungs ( ) . activation of the purinergic receptor p x signaling by increased levels of extracellular atp caused by influenza virus infection leads to an exacerbated immune response characterized by increased production of proinflammatory cytokines, induction of apoptosis, increased influx of neutrophils in the airways, and development of lung histopathology. this response likely produced an amplified influenza virus-induced immunopathology, leading to an increased morbidity and mortality in the infected mice. on the contrary, these effects were moderated in the p x r ko mice, resulting in a better outcome to infection. our studies demonstrated an important role for this receptor in the development of lung immunopathology during influenza virus infection. these results contribute to a better understanding on how the immune response may be directed to cause the immunopathology observed during influenza infection. furthermore, understanding the role this receptor has in the activation of the immune response opens an opportunity for development of medical interventions aimed to modulate the host response toward a balanced immune response that will clear the virus with minimum lung damage. purinergic receptor agonists and antagonists. adenocarcinomic human alveolar basal epithelial cells or a cells (atcc, manassas, va) were evaluated to determine the presence of the purinergic receptor p x . a cells cultured on cover glass were fixed with % buffered formalin and later incubated with % nonfat milk for min. after the blocking step, the cells were incubated with a polyclonal anti-p x receptor antibody raised in rabbit (origene, rockville, md). this study was carried out in strict accordance with the recommendations in the guide for the care and use of laboratory animals ( ) . female c bl/ or p x receptor knockout mice ( to weeks old) purchased from jackson laboratories (bar harbor, me) were used for all experiments. for virus challenges, mice were anesthetized by intraperitoneal injection of a mixture of ketamine ( mg/kg of body weight) and xylazine ( mg/kg) before intranasal administration of a lethal dose of influenza virus pr/ or nl/ in a volume of l. animals were monitored daily for clinical signs of illness, and body weights were recorded daily for days. upon reaching % of initial body weight, animals were humanely euthanized. lung virus titers. on days and postinfection, groups of mice were euthanized, and the lungs were collected and homogenized (beadblaster ; benchmark scientific) in ml of sterile phosphatebuffered saline (pbs). the lung homogenates were spun at , ϫ g for min to pellet tissue debris, and the supernatants were collected. samples were stored at Ϫ °c until titration was performed by standard plaque assay on mdck cells. histopathology. mice were euthanized on day or postinfection, and their lungs were perfused in situ with ml of % buffered formalin. lung sections were stained with hematoxylin-eosin and evaluated by an experienced comparative pathologist from the center of comparative medicine and surgery in the icahn school of medicine at mount sinai. the following score system (on a scale of to ) was used to analyze the histopathology of the lung sections: , no lesions; , mild changes with scattered cell necrosis/vacuolation and few/scattered inflammatory cells in the bronchiolar epithelium with minimal perivascular inflammation; , moderate multifocal vacuolation with peribronchiolar inflammation (Ͻ cell layers thick); , marked, multifocal/segmental necrosis, epithelial loss/effacement, and peribronchiolar inflammation (Ͼ cell layers thick); and , severe, coalescing areas of necrosis, parenchymal effacement with confluent areas of inflammation. overall pathology scores per group were used for the statistical analysis of the results. additional sections were processed for immunohistochemistry (ihc) and used to determine the amount of the apoptosis-related protein caspase- . ten-micron-thick deparaffinized sections were subjected to heat-induced epitope retrieval by boiling in mm sodium citrate buffer (ph ) for min, followed by a min cool-down. cleaved caspase- antibody (cell signaling technology, inc., beverly, ma) was diluted : , in the following buffer: . m pbs, . % (wt/vol) bovine serum albumin (bsa), . % (wt/vol) sodium azide, . % (wt/vol) n-ethyl-maleimide, and % (vol/vol) glycerol. this antibody specifically recognizes the large fragment ( / kda) of the active protein (cleaved), but not full-length caspase- . cleaved caspase- -positive cells were counted in printed digital images using a transparent template with a rectangular box measured . mm by . mm for photographs taken at a magnification of ϫ in three different fields. an individual blind to the experimental treatments completed the quantification. bone marrow-derived macrophages. bone marrow-derived macrophages (bmdm) were generated by culturing total bone marrow from femurs and tibias of p x receptor ko or wild-type mice as previously described ( ) . collected cells were plated at a density of ϫ cells/well in six-well plates using high-glucose dulbecco modified eagle medium (dmem) supplemented with % fetal bovine serum (fbs), mm penicillin-streptomycin, mm sodium pyruvate, mm hepes, m -mercaptoethanol, and % l cell conditioned supernatant containing macrophage colony-stimulating factor (m-csf). the cells were cultured for days, after which the medium and nonadherent cells were removed, and fresh medium was added every other day until harvest between days and . the cells were purified by adherence. to harvest the cells, we used l of . % trypsin. for infection studies, cells were plated in -well plates and infected with influenza a nl/ virus at a multiplicity of infection (moi) of for h. supernatants of samples were collected at and h postinfection and analyzed for virus titer and cytokine production. cytokine and chemokine quantification. protein levels of cytokines/chemokines were evaluated using a multiplex bead array assay (milliplex; emd millipore, billerica, ma) in cell culture supernatant of bone marrow-derived macrophages infected with influenza nl/ or in lung homogenates and serum samples from mice infected with influenza virus nl/ or pr/ according to the manufacturer's instructions. the assays were run using , beads per set of each of cytokines measured per well in a total volume of l. the plates were read on a luminex magpix platform. for each bead set, Ͼ beads were collected. the median fluorescence intensity of these beads was recorded and used for analysis with the milliplex software using a p regression algorithm. flow cytometry. mice were euthanized by intraperitoneal administration of pentobarbital at the indicated time points. lungs were isolated in hbss buffer (sigma, st. louis, mo), digested with type iv collagenase (worthington biochemical, lakewood, nj) for min with agitation, and then processed into single-cell suspensions by forcing the digested lungs through a -m nylon cell strainer (falcon, corning, ny). single-cell suspensions were stained in pbs supplemented with % bovine serum albumin and mm edta. antibodies against the following immune markers were used for discrimination of immune cell types: cd ( -f ; bd), cd b (m / ; biolegend), cd c (hl ; bd), ly c (hk . ; ebioscience), gr (rb - c ; ebioscience), major histocompatibility complex class ii (mhcii) (m / . . ; bd), and siglecf (e - ; bd). live cells were discriminated from dead cells by using a viability dye efluor (ebioscience). all flow cytometry data were acquired on an lsr-ii flow cytometer (bd) and analyzed using flowjo software (flowjo lcc, ashland, or). cell sorting was performed on a facs aria (bd). bronchoalveolar lavage cytospin. mice were euthanized by intraperitoneal administration of pentobarbital at the indicated time points. leukocytes recruited to the airway space were collected by lavaging the lungs in situ by slowly injecting ml of cold sterile pbs using an input -ml syringe with three-way stopcock and then approximately ml of bronchoalveolar lavage fluid (balf) was collected from lungs using the output -ml syringe. we repeated this procedure three times. cell number and cell viability were determined using trypan blue and an automatic cell counter (countess ii; thermo fisher scientific, waltham, ma). for the cytospin, l of each balf sample containing ϫ cells was spun at rpm for min. the slides were air dried for h and then stained with diff-quick. the slides were evaluated in a blind manner by a trained comparative pathologist. differential cell counts (percentage) were established by counting at least cells at a high magnification (ϫ ) using the following categorization ( ) . undifferentiated alveolar macrophages were defined as relatively large, round to oval cells with eccentric round to oval nuclei and abundant granular cytoplasm, while activated macrophages were larger and contain more cytoplasm and numerous cytoplasmic vacuoles, with some macrophages binucleated. neutrophils ( to m in diameter) were characterized by an eosinophilic cytoplasm and a basophilic lobulated nucleus with violet granules. inactivated lymphocytes were small (Ͻ m) cells with rounded nuclei, dense nuclear chromatin, and little cytoplasm, while activated (reactive) lymphocytes were larger with more basophilic cytoplasm and eccentric nuclei. finally, we also quantified the percentage of lysed cells by the relative proportion of isolated (naked) nuclei. statistical analysis. statistical differences in virus titers and cytokine production between the ko and wt mice were analyzed using student's t test analysis followed by the holm-sidak test. for analysis of survival rates, kaplan-meier curves were compared using the log rank (mantel-cox) test. for analysis of the flow cytometry results, we used a two-way analysis of variance followed by multiple comparisons using the tukey posttest. error bars indicate standard errors of the means. p values of Ͻ . were considered significant. all the analyses were performed using the graphpad software (la jolla, ca). supplemental material for this article may be found at https://doi.org/ . / mbio. - . immunopathology in 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c(ϩϩ)ly g(-) cells with suppressive activity towards t cells accumulate in lungs of influenza a virus-infected mice bacterial colonization dampens influenza-mediated acute lung injury via induction of m alveolar macrophages extracellular atp regulates the proliferation of alveolar macrophages guide for the care and use of laboratory animals rna helicase signaling is critical for type i interferon production and protection against rift valley fever virus during mucosal challenge comparison of bronchoalveolar lavage cytospins and smears in dogs and cats we thank chen wang in the department of microbiology for excellent technical assistance. we thank ying dai in the laboratory of comparative pathology at the center for comparative medicine and surgery for excellent technical assistance. we thank christine wong for critical review of the manuscript.funding for this work, including the efforts of megan e. ermler, michael schotsaert, adolfo garcía-sastre, and victor h. leyva-grado, was provided in part by the center for research on influenza pathogenesis (crip), a niaid-funded center of excellence for influenza research and surveillance (ceirs) (contract hhsn c; principal investigator, adolfo garcía-sastre). key: cord- -mf vh iu authors: de wit, emmie; rasmussen, angela l.; feldmann, friederike; bushmaker, trenton; martellaro, cynthia; haddock, elaine; okumura, atsushi; proll, sean c.; chang, jean; gardner, don; katze, michael g.; munster, vincent j.; feldmann, heinz title: influenza virus a/anhui/ / (h n ) replicates efficiently in the upper and lower respiratory tracts of cynomolgus macaques date: - - journal: mbio doi: . /mbio. - sha: doc_id: cord_uid: mf vh iu in march , three fatal human cases of infection with influenza a virus (h n ) were reported in china. since then, human cases have been accumulating. given the public health importance of this virus, we performed a pathogenicity study of the h n virus in the cynomolgus macaque model, focusing on clinical aspects of disease, radiographic, histological, and gene expression profile changes in the upper and lower respiratory tracts, and changes in systemic cytokine and chemokine profiles during infection. cynomolgus macaques developed transient, mild to severe disease with radiographic evidence of pulmonary infiltration. virus replicated in the upper as well as lower respiratory tract, with sustained replication in the upper respiratory tract until the end of the experiment at days after inoculation. virus shedding occurred mainly via the throat. histopathological changes in the lungs were similar to those observed in humans, albeit less severe, with diffuse alveolar damage, infiltration of polymorphonuclear cells, formation of hyaline membranes, pneumocyte hyperplasia, and fibroproliferative changes. analysis of gene expression profiles in lung lesions identified pathways involved in tissue damage during h n infection as well as leads for development of therapeutics targeting host responses rather than virus replication. overall, h n infection was not as severe in cynomolgus macaques as in humans, supporting the possible role of underlying medical complications in disease severity as discussed for human h n infection (h. n. gao et al., n. engl. j. med. : – , , doi: . /nejmoa ). mitted, albeit inefficiently, via respiratory droplets or aerosols in the ferret model ( ) ( ) ( ) ( ) . in order to allow a better estimation of the effect of a potential h n pandemic, we studied the a/anhui/ / strain of this virus in the cynomolgus macaque model. this model was chosen because it most closely reflects the human physiology and the development of pneumonia, cytokine, and chemokine responses ( ) , and the pattern of attachment of h n virus to respiratory tissues of macaques was recently shown to be more similar to that in humans than that in other frequently used animal models ( ) . upon inoculation with influenza virus a/anhui/ / , cynomolgus macaques developed transient, moderate disease with virus replication in the upper and lower respiratory tracts. the emerging h n influenza virus was more pathogenic than seasonal influenza a virus and most isolates of the pandemic h n virus but not as pathogenic as the spanish influenza virus or highly pathogenic avian influenza (hpai) h n virus in cynomolgus macaques. year-old cynomolgus macaques ( male and female) were inoculated with ϫ % tissue culture infectious doses (tcid ) of a/anhui/ / (h n ) via a combination of ocular, oral, intranasal, and intratracheal inoculation as described previously ( ) . animals started to show signs of disease at day postinoculation (dpi). clinical disease peaked at and dpi (fig. a) and was moderate. six out of animals showed obvious respiratory signs, such as increased respiration rates, abdominal breathing, and coughing (see table s in the supplemental material). nasal discharge and cough were noted in only one animal. hematologic and blood chemical analyses did not reveal any abnormalities during the course of infection (data not shown). radiographic changes in lungs of animals inoculated with a/anhui/ / . at , , , , and dpi, ventral-dorsal and lateral chest x-rays were taken to monitor the development of pneumonia. radiographic changes in the lungs of inoculated animals were observed starting and dpi and were observed in all inoculated animals to various degrees (see table s in the supplemental material). the interstitial infiltration was observed first in the lower right lung lobe and, in individual animals, spread over time to the right middle, left lower, left middle, and right upper lung lobes and developed into severe diffuse interstitial infiltration ( fig. ; also, see table s in the supplemental material). virus shedding in cynomolgus macaques inoculated with a/anhui/ / . clinical exams were performed at , , , , , and dpi, and nasal, oropharyngeal, ocular, and rectal swabs were collected. swabs were initially analyzed for the presence of viral rna by real-time reverse transcription-pcr (rt-pcr). because of the large number of swabs in which viral rna was detected, all nasal, oropharyngeal, and ocular swabs were titrated on mdck cells; rectal swabs were not titrated, as only rectal swabs were positive by pcr. oropharyngeal swabs were positive in virus titration by dpi and remained positive in all animals until the end of the experiment at dpi (fig. b) . not all nasal swabs were positive by virus titration; most of the virus shedding via the nose occurred in animals h n - and h n - (fig. b) . despite the ability of influenza a viruses of the h subtype to cause conjunctivitis, ocular swabs were only sporadically positive by virus titration: only the ocular swabs collected from h n - at , , and dpi, the ocular swab collected from h n - at dpi, and the ocular swab collected from h n - at dpi were positive, with virus titers between . and . tcid /ml (data not shown). during clinical exams, bronchalveolar lavages (bal) were performed, and the samples were analyzed for the presence of infectious virus. virus could be isolated from the bal fluid of all animals at dpi; bal fluid remained positive throughout the experiment, with significant amounts of virus still detected at dpi, ranging from . ϫ tcid /ml to . ϫ tcid /ml (fig. b) . gross lung pathology in cynomolgus macaques inoculated with a/anhui/ / . upon necropsy of animals at dpi, gross lesions were observed in the lungs of all animals. there was variation in the area of the lung affected between animals, but at least two lobes showed gross lesions in all animals, varying from % to % of tissue affected (fig. c ). in line with our observation on x-rays, the right lung lobes were more severely affected than the left lobes; this is likely a result of the intratracheal inoculation and the anatomy of the lung ( ) . by dpi, the area of the lung displaying gross lesions had increased, with % of all three right lung lobes being affected in of animals (fig. c) . virus titers in tissues of cynomolgus macaques inoculated with a/anhui/ / . for each animal, virus titers were determined in tissue samples collected from all lung lobes and lung lesions. in line with our x-ray and gross pathology observations, at dpi virus could be detected in all three lobes of the right lung but not in all three lung lobes of the left lung in all animals (fig. a) . virus was present in the collected lung lesions, but unexpectedly, virus titers were not higher in lung lesions than in the collected lung lobe samples, indicating widespread virus replication throughout the lower respiratory tract. by dpi, the virus titers in the lung lobes and lung lesions had decreased compared to those at dpi, although this decrease was not statistically significant; the number of animals with positive virus titration also decreased (fig. a) . in other tissues of the respiratory tract, i.e., nasal turbinates, oropharynx, trachea, and right and left bronchus, virus titers were generally higher than in the lungs at dpi and dpi (fig. b) . interestingly, although virus titers in the lung lobes and lung lesions decreased between and dpi, virus titers in the other respiratory tract tissues did not. virus could also be detected in the tonsils and mediastinal lymph nodes of infected animals at and dpi (fig. b ). virus could be detected in the conjunctiva of only one animal at and dpi. the remaining tissues that were collected at and dpi (i.e., heart, liver, spleen, kidney, stomach, jejunum, ileum, transverse colon, and brain) were analyzed for the presence of viral rna by real-time rt-pcr. except for the liver, where vrna was detected in out of animals, vrna was detected sporadically in all tissues except in heart (see table s in the supplemental material). since cycle threshold values (c t ) in almost all of these samples were higher than the level at which virus titration is usually successful (c t Ͻ ), virus titration was not attempted on these samples. histopathology of respiratory tissues of cynomolgus macaques inoculated with a/anhui/ / . at dpi, histopathology of the lungs was similar for all animals necropsied and was characterized as mild to marked, acute, bronchointerstitial pneumonia. the pneumonia was characterized microscopically as mild to marked thickening of alveolar septa by fibrin, edema, neutrophils, and macrophages, and the alveoli contained small to large amounts of these same inflammatory components (fig. c ). mul-de wit et al. tifocally, hyaline membranes were observed. the lumens of terminal bronchioles frequently contained fibrin, edema, hyaline membranes, neutrophils, and macrophages, with multifocal necrosis and loss of lining epithelium. inflammation and necrosis of bronchial submucosal glands were frequently noted, with mild, subacute periglandular inflammation or more severe changes that ranged from neutrophils and macrophages within ductular lumens to necrosis of acinar and ductular epithelium, occasionally affecting the entire gland. larger bronchioles and bronchi were generally much less severely affected than terminal airways, with intact, viable lining epithelium and only occasional mild, neutrophilic luminal exudate. at dpi, histopathology of the lungs was characterized as mild to marked, subacute to chronic, bronchointerstitial pneumonia. microscopic changes were again characterized by the presence of neutrophils, macrophages, fibrin, and edema within alveolar septa, alveoli, and terminal bronchioles (fig. d) . additionally, type ii pneumocyte hyperplasia was observed in extensive portions of each lung lobe, and large clumps of alveolar fibrin frequently engulfed neutrophils and macrophages and were lined and infiltrated by fibroblasts. immunohistochemistry (ihc) of sections of lung demonstrated low to moderate numbers of antigen-positive alveolar type i and type ii pneumocytes, macrophages, and epithelium lining bronchioles, bronchi, and bronchial submucosal glands (fig. ) . the numbers of positively stained alveolar pneumocytes and bronchial and bronchiolar submucosal gland epithelial cells were similar at and dpi. the number of positively stained pulmonary macrophages was increased at dpi compared with dpi. positive staining in alveolar and submucosal macrophages was cytoplasmic, most likely indicating active phagocytosis of virus rather than replication of virus in these cells (fig. h ). antigenpositive macrophages were also consistently noted within mediastinal lymph nodes, with increased numbers of these cells being noted at day dpi. low numbers of positively stained macrophages were noted in pharyngeal tonsils, oropharynges, tracheas, and extrapulmonary bronchi. there was more variation between individual animals in histopathological lesions in the remaining tissues. mild, subacute conjunctivitis was noted histologically in one of eight animals at dpi, and low numbers of macrophages positive for influenza a virus antigen were present in the submucosa from that animal; the viral load in the eye swab collected from this animal (h n - ) at dpi was still high ( tcid equivalents/ml), likely indicating active virus replication. influenza a virus-positive cells were noted in the epithelium of the nasal turbinates with minimal inflammation in of animals at dpi ( fig. a and b ). although no mi- table s in the supplemental material. in severe cases of infection at dpi, lesions were characterized by edema, alveolar fibrin (black asterisk), and hyaline membrane formation (arrow). at dpi, edema, organizing fibrin (white asterisk), and type ii pneumocyte hyperplasia (arrowheads) were observed. magnification (c and d), ϫ . de wit et al. ( fig. e and f). minimal to moderate subacute inflammation was noted in at least one of the extrapulmonary bronchi from each animal, and in one animal there was an extensive area of marked inflammation with ulceration. upon ihc staining of the same sections of bronchi, few positively stained epithelial cells and/or macrophages were noted ( fig. g and h) . matching tissue sections were stained with hematoxylin and eosin (h&e) or studied by immunohistochemistry (ihc) using an anti-np monoclonal antibody (ihc; visible as red-brown staining). at dpi, mild to marked thickening of alveolar septa by fibrin, edema, neutrophils, and macrophages was observed (a), and virus antigen was present in type i and type ii (black arrows) pneumocytes (b). inflammation and necrosis of bronchial submucosal glands were frequently noted (c), with virus antigen being present in submucosal gland (open arrowhead) and bronchial epithelium (white arrow). infection of the bronchial lining epithelium was more pronounced by dpi (e and f). cytoplasmic staining of alveolar macrophages indicates active phagocytosis on dpi (h; black arrowheads). (j) virus replication in bronchial submucosal gland epithelium on dpi. magnification, ϫ . inoculated with influenza virus a/anhui/ / . cynomolgus macaques were euthanized at (a and b) and (c to h) days postinoculation, and tissue was collected and stained with hematoxylin and eosin (h&e) or immunohistochemistry (ihc) using an anti-np monoclonal antibody (ihc; visible as redbrown staining). in the rostral nasal cavity (a and b) and nasopharynx (c and d), influenza virus a/anhui/ / mainly replicated in the lining epithelium. in the trachea (e and f), cell debris (arrowhead) with immunopositive staining is visible in a submucosal gland. in the bronchus (g and h), bronchial lumen exudate is visible, with virus replication in the epithelium lining the bronchus. magnification, ϫ . and vascular endothelial growth factor (vegf) levels. at dpi, there was a statistically significant increase in levels of g-csf, il- ra, il- , il- , il- , mcp- , and tnf-␣ (fig. ) ; levels of ifn-␥, il- , il- , mip- ␣, mip- ␤, and vegf increased at that time point, but increases were not statistically significant ( fig. ; also data not shown). by dpi, all levels of cytokines and chemokines had returned to baseline. alterations in host gene expression upon infection with a/anhui/ / . to elucidate global host responses specifically associated with sites of virus-induced airway injury in influenza virus a/anhui/ / -infected macaques, we used microarrays to assess transcriptional profiles induced in lung lesions compared to the adjacent lung tissue. welch's t test (p Ͻ . ; change Ͼ . -fold) identified differentially expressed genes (deg) in the lesions compared to lung samples from the same animals at dpi ( fig. a ; also, see table s in the supplemental material); these genes were differentially expressed in all animals. of those, were upregulated and were downregulated in lesions compared to samples from the right lower lung lobe. using ingenuity pathway analysis (ipa), we assessed the functional significance of this molecular signature and constructed a network of critical molecules based on direct interactions in the ipa knowledge base (ipkb) (fig. b ). as expected, there are numerous transcripts upregulated from functional categories previously observed in cynomolgus macaque models of influenza infection ( ) ( ) ( ) ( ) , including known mediators of antiviral immunity, such as pattern recognition receptors and downstream signaling molecules (tlr and myd ), interferons and interferon-stimulated genes (ifnl ), interferon-regulatory factors (irf , irf , and jak ), and proinflammatory cytokines and inflammatory mediators (il- , nlr family, nlrp , tnfrsf b, tnfrsf b, and tnfsf ). we also identified a number of upregulated genes associated with leukocyte migration and differentiation (cxcl , cxcl , sele, il r, il- , and csf ra). this suggests that molecules that recruit infiltrating effector leukocytes are increased at the site of lesions and is in line with the observed influx of neutrophils and macrophages observed microscopically in the lungs. moreover, hyaluronic acid synthase (has ), a molecule known to be associated with lung injury, was strongly upregulated in the lung lesion samples. we also observed several functional categories of molecules that were downregulated in lesions relative to the adjacent lung tissue (fig. b) . these were associated predominantly with lipid metabolism and adipogenesis regulated by peroxisome proliferator-activated receptor alpha (ppar␣). we also observed downregulation of growth factors, particularly those related to insulin signaling and regulation of glucose levels (insr and igfbp ). by dpi, deg were identified in lung tissue versus lung lesions of animals euthanized at dpi (see fig. s and table s in the supplemental material). due to substantial variability between animals, we were not able to determine any significantly enriched functional categories at this time point. next, the upstream analysis function in ipa was used to identify drugs predicted to act as significant upstream regulators of the key deg identified at dpi. drugs that are predicted to inhibit pathological host responses were selected based on the activation z score. negative activation z scores are predicted to cause opposite or inhibitory effects on significant genes associated with pathology in the lesions. these findings were confirmed using connectivity map (cmap), a resource allowing comparison with transcriptional signatures from multiple cell types treated with a library of chemical and genetic perturbagens ( ) , by determining if the connectivity score was also negative, indicating that the compound would have inhibitory effects on transcriptional signatures associated with lesion formation. we identified ten compounds in ipa (table ) , four of which were perturbagens listed in cmap. we identified two compounds that met our criteria in ipa and cmap, rosiglitazone and simvastatin, predicted to have inhibitory effects on pathological host responses associated with lesions in influenza virus a/anhui/ / -infected animals ( table ) . these molecules are both involved in regulating lipid biosynthesis and metabolism. rosiglitazone is an fda-approved diabetes drug that modulates ppar activity and insulin sensitivity and has been shown to affect rna virus replication ( ) ( ) ( ) , virus-induced inflammation ( , ) , and lung inflammation ( ) ( ) ( ) . simvastatin is a fda-approved statin which lowers blood cholesterol by inhibiting -hydroxy- -methyl-glutaryl coenzyme a (hmg-coa) reductase. it has been shown to reduce lung inflammation in mouse models of airway injury ( - ) and bacterial infections ( ) ( ) ( ) . however, simvastatin was not shown to have a significant effect on influenza a virus pathogenesis in mice ( ) ( ) ( ) . besides rosiglitazone and simvastatin, mainly immunomodulatory drugs were predicted to have inhibitory effects on pathological host responses to a/anhui/ / infection. the ability of rosiglitazone to inhibit a/anhui/ / replication was tested in mdck cells treated with different concentrations of this drug after infection with a/anhui/ / at a multiplicity of infection of . . twenty-four hours after addition of m rosiglitazone, but not at lower concentrations, a statistically significant (p ϭ . ; two-tailed unpaired t test) -fold reduction in virus titers in the supernatant of treated, infected cells compared to mock-treated cells was observed (see fig. s a in the supplemental material); a small cytotoxic effect of the drug was also noticed at this time point in an mts [ -( , -dimethyl- -yl)- -( -carboxymethoxyphenyl)- -( -sulfophenyl)- h-tetrazolium, inner salt] assay (see fig. s b in the supplemental material). at h after treatment, the inhibitory effect of rosiglitazone on a/anhui/ / replication could no longer be detected (data not shown). china has shifted the focus away from hpai h n to h n as one of the main pandemic threats. although much attention has been drawn to the ability of this virus to be transmitted between ferrets, the pathogenicity of h n influenza a virus in ferrets does not match the severity of disease observed in human cases ( ) ( ) ( ) ( ) . the physiology of the cynomolgus macaque lung is more similar to that of humans, and cytokine and chemokine responses to infection in macaques are similar to those in humans ( ) . moreover, it was shown recently that the pattern of attachment of h n influenza a virus to respiratory tissues of cynomolgus macaques is more similar to the attachment pattern in humans than that in other animal models frequently used for influenza a virus pathogenesis studies, including the ferret ( ) . therefore, we studied the h n infection of cynomolgus macaques in detail. in agreement with the observed abundant attachment of the h n influenza a virus to the human upper and lower respiratory tracts ( ) , and the replication of the h n virus in ex vivo cultures of the human upper as well as lower respiratory tract ( ) , h n virus replicated well in the upper and lower respiratory tracts of cynomolgus macaques, as indicated by virus titers in nasal turbinates, oronasopharynges, tracheas, bronchi, and lung tissue samples, reflecting the previously described receptor distribution of influenza virus h n in the macaque respiratory tract ( ) . surprisingly, the virus titers in nasal turbinates, oronasopharynges, tracheas, and bronchi did not decrease between and dpi, unlike the virus titers in the lung samples, which did decrease. this extended virus replication in the upper respiratory tract could result in prolonged virus shedding and thus an increased risk of virus transmission. the histopathology of the lungs in fatal human h n cases was similar to, albeit more severe than, that observed in cynomolgus macaques, with diffuse alveolar damage, infiltration of polymorphonuclear cells, formation of hyaline membranes, and, later after onset of symptoms, pneumocyte hyperplasia and fibroproliferative changes ( ). however, clinically and histopathologically, h n infection was not as severe in cynomolgus macaques as it has been described in humans, where h n has a high case fatality rate. although viral characteristics could cause differences in disease severity between humans and macaques, another possible explanation for the discrepancy in disease severity could be the high percentage of human cases with underlying medical complications ( ) . since some of the h n influenza virus strains isolated from human cases have an r k substitution in na that renders them partially resistant to treatment with neuraminidase inhibitors ( , ( ) ( ) ( ) , it is important to identify drugs that either directly inhibit virus replication in the host or reduce the severity of disease and the level of lung injury after infection. by analyzing the gene expression profiles in lung lesions compared to adjacent infected, nonlesional lung tissue, we were able to predict drugs reported to act as upstream regulators of some of these genes that may thus play a role in the development of lung lesions during h n infection. one of the predicted drugs, rosiglitazone, modestly reduced a similar data analysis resulted in the identification of a compound that reduced replication of the middle east respiratory syndrome coronavirus in vitro ( ) ; however, the validity of this approach has not yet been tested in vivo. thus, this analysis of the gene expression profile hints at new avenues of treatment to explore in in vivo models, rather than revealing novel treatments directly applicable in the clinic. although it is difficult to compare studies investigating the pathogenicity of different influenza a viruses in the macaque model, because of different inoculation routes and doses used and because sampling schemes do not overlap between studies, the h n infection in macaques in our study was similar to that described previously ( ) . in both studies, animals developed transient clinical signs, with virus replication in the upper as well as lower respiratory tract and similar histopathological lesions in the lower respiratory tract. more importantly, h n infection seemed to be clinically more severe than most infections with isolates of pandemic h n ( ); virus shedding from the throat was higher in h n -infected animals, and a larger area of the lung was affected with gross lesions ( ) . inoculation of cynomolgus macaques with seasonal h n influenza a virus resulted in infection with mild or even asymptomatic disease ( , ) . cynomolgus macaques inoculated via the same route and with the same dose of spanish influenza virus developed lethal disease ( ) , and hpai h n influenza a virus inoculated into macaques at a -fold-lower dose caused more severe disease than h n influenza a virus in this study ( ) . thus, the emerging h n influenza virus is more pathogenic than seasonal influenza a virus and most isolates of the pandemic h n virus but not as pathogenic as the spanish influenza virus and hpai h n virus in cynomolgus macaques. however, the pathogenicity of the h n virus may decrease if the virus adapts further to solely using ␣ , -linked sialic acids as the receptor for entry, as pandemic influenza viruses to date have done ( ) ( ) ( ) ( ) . exclusive attachment to ␣ , -linked sialic acids would most likely result in a shift to replication mainly in the upper respiratory tract of humans, likely resulting in less severe disease, as has been described for the pandemic h n virus ( ) and upon adaptation of hpai h n virus to efficient transmission via respiratory droplets or aerosols ( ) . all animal experiments were approved by the institutional animal care and use committee of the rocky mountain laboratories and performed following the guidelines of the association for assessment and accreditation of laboratory animal care, international (aaalac), by certified staff in an aaalac-approved facility. cells. madin-darby canine kidney (mdck) cells were cultured in eagle's modified essential medium (emem) (gibco) supplemented with % fetal calf serum (fcs), iu/ml penicillin, g/ml streptomycin, mm glutamine, . mg/ml sodium bicarbonate, and nonessential amino acids. virus. a/anhui/ / (passage e /e ) was obtained from the centers for disease control in atlanta, ga, and passaged once in mdck cells. animal study and sample collection. eight cynomolgus macaques ( males, females; age, years; to kg) were inoculated with ϫ tcid of a/anhui/ / via a combination of intratracheal ( ϫ tcid ; ml), intranasal ( ϫ tcid ; l/nostril), oral ( ϫ tcid ; ml), and ocular ( ϫ tcid ; l/eye) routes. the animals were observed twice daily for clinical signs of disease and scored using a previously described clinical scoring system ( ) . at , , , , and days postinoculation, clinical exams were performed on anesthetized animals, and lateral x rays were taken and analyzed by a veterinarian. nasal, oral, urogenital, and rectal swabs were collected in ml dulbecco's modified essential medium (dmem) with u/ml penicillin and g/ml streptomycin; bronchoalveolar lavages (bal) were performed using ml sterile saline solution; blood was collected for hematology, blood chemistry analysis, and peripheral blood mononuclear cell (pbmc) isolation. the total white blood cell count, lymphocyte, platelet, reticulocyte, and red blood cell counts, hemoglobin and hematocrit values, mean cell volume, mean corpuscular volume, and mean corpuscular hemoglobin concentrations were determined from edta-containing blood with the hemavet fsϩ hemoglobin analyzer (drew scientific). pbmc were isolated by centrifugation over a histopaque gradient (sigma) as per the manufacturer's recommendation. at and dpi, macaques were euthanized, and samples of the conjunctivas, right and left eyes, nasal turbinates, tonsils, oronasopharynges, tracheas, right and left bronchi, all six lung lobes, lung lesions, mediastinal lymph nodes, hearts, livers, spleens, kidneys, stomachs, jejuna, ilea, transverse colons, olfactory bulbs, cerebella, brain stems, and bone marrow were collected. the percentage of the lung surface area affected by gross lung lesions was quantitated for each lung lobe, ventrally and dorsally, by a board-certified veterinary pathologist at the time of necropsy. histopathology and immunohistochemistry. histopathology and immunohistochemistry were performed on macaque tissues. after fixation for days in % neutral buffered formalin and embedding in paraffin, tissue sections were stained with hematoxylin and eosin (h&e). to detect influenza a virus antigen, immunohistochemistry was performed using an anti-np monoclonal hb- antibody (evl, the netherlands) as a primary antibody. rna extraction. rna was extracted from swabs, bal fluid, and whole-blood samples using the qiaamp viral rna kit (qiagen) according to the manufacturer's instructions. tissues were stored at Ϫ °c until further processing; tissue samples ( mg) were homogenized in rlt buffer, and rna was extracted using the rneasy kit (qiagen). quantitative real-time rt-pcr. a one-step real-time rt-pcr targeted at the matrix gene of influenza a virus was performed using the quantifast probe kit (qiagen) according to instructions of the manufacturer using the primers and probe described in reference . virus titrations. viruses were titrated by endpoint dilution in mdck cells. mdck cells were inoculated with tenfold serial dilutions of culture supernatants. one hour after inoculation, cells were washed with phosphate-buffered saline (pbs) and supplemented with infection medium (emem supplemented with iu/ml penicillin, g/ml streptomycin, mm glutamine, . mg/ml sodium bicarbonate, nonessential amino acids, and g/ml trypsin). three days after inoculation, the supernatants of infected cell cultures were tested for agglutination activity using turkey red blood cells as an indicator of infection of the cells. infectious titers were calculated from replicates by the spearman-karber method ( ) . serum cytokine and chemokine analysis. serum samples for analysis of cytokine/chemokine levels were inactivated with gamma radiation ( megarads) according to standard operating procedures. concentrations of granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, ifn-␥, il- ␤, il- receptor antagonist, il- , il- , il- , il- , il- , il- , il- / (p ), il- , il- , il- , mcp- , mip- ␣, mip- ␤, soluble cd ligand (scd l), transforming growth factor ␣, tnf-␣, vascular endothelial growth factor (vegf), and il- were measured on a bio-plex instrument (bio-rad) using the nonhuman primate cytokine milliplex -plex map kit (millipore) according to the manufacturer's instructions. microarray data and functional analysis. rna was extracted using qiagen micro-rneasy spin columns per the manufacturer's protocol. low-yield samples were concentrated using the rna clean and concentrator kit (zymo research, irvine, ca). as multiple lesions were collected from each animal, equal masses of rna from these samples ( or samples per animal) were pooled to investigate comprehensive signatures from multiple pathological sites within the same individual. probe labeling was carried out using the agilent low-input processing protocol and hybridized to agilent rhesus macaque ϫ k microarrays (agilent technologies, santa clara, ca) using the manufacturer's one-color analysis protocol. for comparisons of differentially expressed genes (deg) in infected lungs and lung lesions, raw array data were uploaded to genedata analyst . (genedata inc., san francisco, ca). data were normalized using the quantile normalization method, and the log ratio expression was calculated relative to the mean probe values of the right lower lung lobe samples per time point (lesion versus lung comparisons). statistically significant deg were identified using welch's t test (p Ͻ . ; fold change, Ն . ). hierarchical clustering of deg was performed by the unweighted average method (unweighted pair group with arithmetic mean [upgma]) using spotfire decisionsite . . (tibco, somerville, ma). analysis of functional enrichment was performed using ingenuity pathway analysis (ipa) software (ingenuity systems, redwood city, ca), and upstream drug efficacy predictions were made using both the upstream analysis function of ipa and connectivity map (broad institute, cambridge, ma). drugs predicted to inhibit pathological host responses were selected based on the activation z score. negative activation z scores are predicted to cause opposite or inhibitory effects on significant genes associated with pathology in the lesions, and in this study we sought compounds with activation z scores less than Ϫ and a p value less than . . these findings were confirmed using connectivity map (cmap) ( ) . cmap assigns enrichment scores ranging between Ϫ and , and we accepted compounds with cmap enrichment scores less than Ϫ . . these are compounds inducing transcriptional profiles that are negatively connected with the gene expression signature associated with lung pathology, confirming that these drugs may induce a transcriptional profile that is inhibitory to tissue damage and lesion formation. antiviral assay. confluent mdck cells in -well culture plates were infected in triplicate with influenza virus a/anhui/ / at a multiplicity of infection of . . after h at °c, cells were washed once with pbs, and infection medium containing rosiglitazone ( to m) was added to the cells. cells were incubated for h; supernatant was then harvested, stored at Ϫ °c for subsequent virus titration, and replaced with fresh infection medium containing rosiglitazone. supernatant was again collected at h after infection and stored at Ϫ °c for subsequent virus titration. to determine a potential cytotoxic effect of rosiglitazone, mdck cells were simultaneously plated in -well culture plates and treated with rosiglitazone ( to m). after h incubation, cytotoxicity was tested using the celltiter aqueous one-solution cell proliferation assay (mts) (promega) according to the manufacturer's instructions. microarray data accession number. raw microarray data have been deposited in ncbi's gene expression omnibus database (gse ) and are also available to the public at http://viromics.washington.edu. supplemental material for this article may be found at http://mbio.asm.org /lookup/suppl/doi: . /mbio. - /-/dcsupplemental. figure s , tif file, . mb. figure s , tif file, . mb. human infection with a novel avian-origin influenza a (h n ) virus confirmed human cases of avian influenza a(h n ) reported to who. world health organization clinical findings in cases of influenza a (h n ) virus infection clinical, virological, and histopathological manifestations of fatal human infections by avian influenza a(h n ) virus past, present, and possible future human infection with influenza virus a subtype h avian influenza a virus (h n ) associated with human conjunctivitis and a fatal case of acute respiratory distress syndrome genetic analysis of novel avian a(h n ) 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influenza virus pathogenesis of influenza a (h n ) virus infection in a primate model receptor specificity in human, avian, and equine h and h influenza virus isolates early alterations of the receptor-binding properties of h , h , and h avian influenza virus hemagglutinins after their introduction into mammals a two-amino acid change in the hemagglutinin of the influenza virus abolishes transmission transmission and pathogenesis of swine-origin a(h n ) influenza viruses in ferrets and mice avian-type receptor-binding ability can increase influenza virus pathogenicity in macaques airborne transmission of influenza a/h n virus between ferrets thoracic radiography as a refinement methodology for the study of h n influenza in cynomologus macaques (macaca fascicularis) practical considerations for high-throughput influenza a virus surveillance studies of wild birds by use of molecular diagnostic tests beitrag zur kollektiven behandlung pharmakologischer reihenversuch [a contribution to the collective treatment of pharmacological experimental series key: cord- - yfs pa authors: almazán, fernando; dediego, marta l.; sola, isabel; zuñiga, sonia; nieto-torres, jose l.; marquez-jurado, silvia; andrés, german; enjuanes, luis title: engineering a replication-competent, propagation-defective middle east respiratory syndrome coronavirus as a vaccine candidate date: - - journal: mbio doi: . /mbio. - sha: doc_id: cord_uid: yfs pa middle east respiratory syndrome coronavirus (mers-cov) is an emerging coronavirus infecting humans that is associated with acute pneumonia, occasional renal failure, and a high mortality rate and is considered a threat to public health. the construction of a full-length infectious cdna clone of the mers-cov genome in a bacterial artificial chromosome is reported here, providing a reverse genetics system to study the molecular biology of the virus and to develop attenuated viruses as vaccine candidates. following transfection with the cdna clone, infectious virus was rescued in both vero a and huh- cells. recombinant mers-covs (rmers-covs) lacking the accessory genes , a, b, and were successfully rescued from cdna clones with these genes deleted. the mutant viruses presented growth kinetics similar to those of the wild-type virus, indicating that accessory genes were not essential for mers-cov replication in cell cultures. in contrast, an engineered mutant virus lacking the structural e protein (rmers-cov-Δe) was not successfully rescued, since viral infectivity was lost at early passages. interestingly, the rmers-cov-Δe genome replicated after cdna clone was transfected into cells. the infectious virus was rescued and propagated in cells expressing the e protein in trans, indicating that this virus was replication competent and propagation defective. therefore, the rmers-cov-Δe mutant virus is potentially a safe and promising vaccine candidate to prevent mers-cov infection. importance since the emergence of mers-cov in the arabian peninsula during the summer of , it has already spread to different countries, infecting around persons and showing a mortality rate higher than %. this article describes the development of the first reverse genetics system for mers-cov, based on the construction of an infectious cdna clone inserted into a bacterial artificial chromosome. using this system, a collection of rmers-cov deletion mutants has been generated. interestingly, one of the mutants with the e gene deleted was a replication-competent, propagation-defective virus that could only be grown in the laboratory by providing e protein in trans, whereas it would only survive a single virus infection cycle in vivo. this virus constitutes a vaccine candidate that may represent a balance between safety and efficacy for the induction of mucosal immunity, which is needed to prevent mers-cov infection. likely have not required hospital assistance. in fact, recent data suggest that mild respiratory illness might also be part of the clinical spectrum of mers-cov infection ( ) . in addition to mild or acute respiratory illness, other reported clinical symptoms are abdominal pain and diarrhea, fever, and in some cases, renal failure ( ) . many hospitalized cases occurred in persons with chronic underlying medical conditions or immunosuppression ( , ) . the virus loads are highest in lower respiratory tract samples, although low concentrations of viral rna can also be found in stool, urine, and blood samples ( ) . the genome of mers-cov includes more than , nucleotides and contains at least predicted open reading frames (orf a, orf b, s, , a, b, , e, m, and n), of which seem to be expressed from a nested set of eight mrnas ( , ) . interestingly, the partial genome sequences of three independent mers-cov isolates reveal that it evolved following a strict molecular clock model ( ) . a functional receptor of mers-cov is dipeptidyl peptidase (dpp- ) from both human and bat ( ) . this receptor binds to a -residue region in the spike (s) protein of mers-cov ( , ) , a domain different from the receptor-binding site of other betacoronaviruses ( ) . infection of human airways by mers-cov prevents the induction of interferon-regulating factor (irf- )mediated antiviral alpha/beta interferon (ifn-␣/␤) responses. however, mers-cov was markedly more sensitive to the antiviral state established by ectopic ifn than severe acute respiratory syndrome cov (sars-cov) ( , , ) . soon after mers-cov emergence, a diagnostic assay was designed ( ) . similarly, antivirals inhibiting virus replication, such as cyclosporine a, ifn-␣, or ribavirin, have been described ( , , ) . in contrast, reliable vaccines have not yet been developed, although the s protein and the receptor-binding site within this protein induce neutralizing antibodies and, in principle, could serve as a subunit vaccine ( ) . covs infect respiratory and enteric mucosal areas, and thus, induction of mucosal immunity is necessary to protect these tissues from infection. live attenuated viruses are expected to elicit mucosal immunity more efficiently than nonreplicating antigens, which elicit reduced secretory immune responses. live attenuated viruses can be generated by the deletion of genes conferring virulence, a procedure that requires the availability of a reverse genetics system for mers-cov. in this article, we describe the construction of an infectious cdna clone of mers-cov using a bacterial artificial chromosome (bac). using this clone, recombinant mers-cov (rmers-cov) deletion mutants were constructed lacking genes nonessential for virus replication. in addition, we deleted the structural envelope (e) protein gene, because in previous work from our laboratory, deletion of the e gene in two other covs led to mutants that were either replication-competent, propagation-defective viruses or attenuated viruses ( ) ( ) ( ) . all deletion mutants efficiently replicated and spread in cell cultures except the one in which the e gene was deleted, which was replication competent but propagation defective. this virus was propagated in cells by providing e protein in trans. therefore, this deletion mutant missing the e gene can serve as the basis for a safe vaccine candidate. an infectious cdna clone was assembled as a bac under the control of the cytomegalovirus (cmv) immediate-early pro-moter, based on the genome sequence of the mers-cov-emc strain (genbank accession number jx ) ( ) . to this end, the same approach described for the generation of other cov infectious cdna clones ( ) ( ) ( ) ( ) was used. this system allows the efficient intracellular production of viral rna from the cdna clone without the need for in vitro ligation and transcription steps. the bac clone carrying the mers-cov infectious cdna was generated in several steps (fig. ) . after selection of appropriate restriction sites in the viral genome (fig. a) , the intermediate plasmid pbac-mers- = = (fig. b) was generated as the backbone to assemble the full-length cdna clone. this plasmid contained the first nucleotides of the viral genome fused to the cmv promoter, a multicloning site containing the restriction sites selected in the first step (bamhi, stui, swai, and paci), and the last , nucleotides of the genome, followed by a nucleotide (nt) poly(a) stretch, the hepatitis delta virus (hdv) ribozyme, and the bovine growth hormone (bgh) termination and polyadenylation sequences. finally, the full-length mers-cov infectious cdna clone (pbac-mers fl ) was assembled by sequential cloning of four chemically synthesized overlapping dna fragments (mers- to mers- ) into the plasmid pbac-mers- = = (fig. c) . the full-length clone sequence was identical to that reported for the mers-cov-emc strain ( ) , with the exception of a silent point mutation (t to c) introduced in the cdna at position , (fig. c ). this mutation, which eliminates an additional swai restriction site at position , , was introduced to facilitate the cloning process and was used as a genetic marker to identify the virus recovered from the cdna clone. the assembled infectious cdna clone was stable during its propagation in escherichia coli dh b cells for more than generations, as determined by restriction endonuclease analysis (data not shown). rescue of infectious rmers-cov from the cdna clone in vero a and huh- cells. infectious viruses were recovered from the full-length cdna clone, using susceptible vero a and huh- cells, with titers of around pfu/ml at h posttransfection (h.p.t.). the recovered viruses were cloned by three rounds of plaque purification, and their phenotypic and genotypic properties were determined. viruses rescued from both cell lines (rmers-cov) induced a clear cytopathic effect (cpe), characterized by the induction of cell fusion, which was more apparent in huh- cells (fig. ). in addition, the replication of the rmers-cov was confirmed by indirect immunofluorescence microscopy using a nucleocapsid (n) protein-specific antibody, showing a cytoplasmic staining pattern in both cell lines (fig. ) . to further confirm the identity of the rmers-cov, the fulllength genome sequences of two independent clones rescued in vero a and huh- cells were analyzed. the recombinant viruses rescued in huh- cells presented the same sequence as the cdna clone, including the genetic marker at position , . however, in the case of the viruses rescued in vero a cells, several changes in the region of the accessory genes were detected in both clones. one of them presented a deletion of nucleotides (from nucleotide , to , ) that disrupted gene b and eliminated the transcription-regulating sequence (trs) and the first amino acids of gene . the second clone presented a -base insertion at position , that changed the reading frame and promoted the expression of a truncated gene . taking these data into consideration, two new virus clones rescued in vero a cells were sequenced, and only one of them presented the wild-type sequence, suggesting that the mers-cov was more stable in huh- cells. therefore, huh- cells were selected for further work. rescue of infectious rmers-covs lacking accessory genes , a and b, and . the availability of the pbac-mers fl infectious clone opened the door to investigate the importance of accessory genes , a, b, and for mers-cov replication. to this end, cdna clones with genes (pbac-mers-⌬ ), a and b (pbac-mers-⌬ ab), or (pbac-mers-⌬ ) deleted were constructed from pbac-mers fl (fig. a) . the expression of gene was abrogated by deletion of its trs and coding sequence, with the exception of the = last nucleotides, containing the a trs, in order to preserve the expression of gene a. in the case of genes a and b, the majority of both coding sequences was deleted, except for the last nucleotides of gene b, which overlap the gene trs. finally, the expression of gene was avoided by deletion of its trs and complete coding sequence. infectious viruses were recovered in huh- cells from plasmids pbac-mers-⌬ , pbac-mers-⌬ ab, and pbac-mers-⌬ with virus titers similar to that of the parental rmers-cov (around pfu/ml). after one passage on fresh cell monolayers, the recombinant viruses were cloned by three plaque isolation steps and their genetic structure was confirmed by sequencing. all the deletion mutant viruses (rmers-cov-⌬ , rmers-cov-⌬ ab, and rmers-cov-⌬ ) were identical to the parental virus (rmers-cov) in terms of cpe and plaque morphology (data not shown). the growth kinetics of these viruses were also similar, reaching maximum virus titers at h postinfection (h.p.i.) (fig. b ). in the case of rmers-cov-⌬ ab, the viral titer was around -fold lower than that obtained from the parental virus (fig. b ). these data indicated that the proteins encoded by genes , a, b, and were not essential for mers-cov replication in cell cultures. generation of a rmers-cov mutant lacking the structural e protein gene. based on published data showing that the deletion of cov e protein resulted in either replication-competent, propagation-defective viruses ( ) or attenuated viruses ( , , ) , a cdna clone with the e gene deleted (pbac-mers-⌬e) was constructed from pbac-mers fl . the expression of the e gene was abrogated by the deletion of its trs and coding sequence, with the exception of the = last nucleotides, in order to preserve the expression of gene m (fig. a ). to recover infectious virus, bhk cells were transfected with pbac-mers-⌬e or the full-length cdna clone pbac-mers fl . six h.p.t. the transfected cells were overlayed on vero a cell monolayers, and at h.p.t., the supernatants were harvested and serially passaged three times on fresh huh- cells. infectious rmers-cov was recovered with titers of around pfu/ml, whereas visible plaques were not detected for rmers-cov-⌬e virus throughout these passages (data not shown). since cpe was observed at passages and , the cell supernatants from the different passages were titrated in huh- cells by limiting dilution. in contrast to the wild-type virus, which was recovered with high titers (around ϫ % tissue culture infection dose [tcid ]/ml), the rmers-cov-⌬e was detected only at passage with apparent low titers (around ϫ tcid /ml) (fig. a ). this apparent low titer was most probably due to the transfer of detached cells transfected with the pbac-mers-⌬e. these cells were taken with the supernatant used to infect the next cell monolayer and formed syncytia with the nontransfected cells, giving the impression of virus production. in fact, rmers-cov-⌬e virus was lost at subsequent passages in several independent experiments performed in two different cell lines, vero a and huh- cells (data not shown). the presence of viral proteins was analyzed by immunofluorescence microscopy in huh- cells infected with either rmers-cov or rmers-cov-⌬e from passage . as expected, e protein was detected in cells infected with rmers-cov but not in those infected with rmers-cov-⌬e (fig. b ). viral n protein was detected in the cytoplasm of both rmers-cov-and rmers-cov-⌬e-infected cells (fig. b) . interestingly, whereas the n protein was detected all over the cell monolayer in rmers-cov-infected cells, it was only detected in small syncytia in cells infected with rmers-cov-⌬e. altogether, these data suggested that e protein was required for efficient virus propagation. previous reports from our laboratory showed that deletion of the transmissible gastroenteritis coronavirus (tgev) e gene leads to a propagation-defective virus that can only spread from cell to cell by expression of the e protein in trans ( , ) . to analyze whether rmers-cov-⌬e could also be complemented in cells transiently expressing e protein, the rescue of rmers-cov-⌬e and of rmers-cov as a control was analyzed in huh- cells that did not express e protein (e Ϫ ) and in cells transiently expressing the e protein (e ϩ ). the transfection efficiencies in e ϩ cells varied between and % in each independent experiment. infectious rmers-cov was rescued from both e ϩ and e Ϫ cells with virus titers of around ϫ tcid /ml and ϫ tcid /ml, respectively (fig. a ). in contrast, rmers-cov-⌬e was rescued in e ϩ cells with titers of around ϫ tcid /ml but not in control e Ϫ cells, in which the virus was not detectable from passage (limit of detection, tcid /ml) (fig. a) . these data indicated that the e protein was necessary for either viral rna synthesis or virus propagation. to evaluate the role of the e protein in viral rna synthesis, the level of genomic rna (grna) was evaluated by quantitative reverse transcription-pcr (rt-qpcr) at each passage. viral grna was detected for rmers-cov in both e Ϫ -and e ϩ -expressing cells, as expected. however, mers-cov-⌬e viral rna was detected at high levels in e ϩ cells at passages , , , and , whereas it was only detected at similar levels in e Ϫ cells at passage , suggesting that mers-cov-⌬e was a replication-competent virus (fig. b) . to further confirm these data, viral rna synthesis was analyzed in a single-cycle infection. e Ϫ cells were infected with either rmers-cov or rmers-cov-⌬e grown in e ϩ cells. at h.p.i., the levels of grna and subgenomic mrna (sgmrna n) were evaluated by rt-qpcr (fig. ). similar levels of grna and sgmrna n were detected in cells infected with both viruses, indicating that e protein was not required for efficient viral replication and transcription. overall, these data indicated that rmers-cov-⌬e was a replicationcompetent, propagation-defective virus. the emergence of mers-cov represents a public health threat that requires further research to understand the virus biology and provide the basis for the development of control strategies. this paper describes for the first time the construction of a reverse genetics system for mers-cov, using bacs as vectors. the recombinant viruses described in this work were rescued using a combination of synthetic biology and reverse genetics techniques, as previously described for other covs ( ). this system constitutes a valuable molecular tool for the rational design and launching of attenuated viruses that may serve as efficient and safe vaccine candidates. in addition, the infectious cdna clone will be useful to study the role of specific viral genes in virus-host interactions in the context of the complete viral cycle. a full-length cdna copy of mers-cov-emc was generated from synthetic fragments cloned downstream from the cmv promoter in a bac. the bac-based strategy allows the efficient and reproducible intracellular production of viral rna, since it is first synthesized in the nucleus by the cellular rna polymerase ii (pol ii) and then amplified in the cytoplasm by the viral replicase encoded in the rna itself ( , ) . the mers-cov infectious cdna was stably maintained in bacteria for more than generations, allowing the easy and direct manipulation of the viral cdna for molecular studies. in addition, this bac-based system allows for the generation of viral replicons that may be used for the screening of drugs affecting viral rna synthesis ( , ) . the full-length sequence of rmers-cov, recovered from the infectious cdna clone, was completely identical to that published for the original mers-cov-emc isolate ( ) , except for the silent point mutation introduced as a genetic marker. therefore, both viruses should have the same biological properties. in fact, the growth kinetics and cpe caused by both viruses were similar when the same multiplicity of infection (moi) was used to infect huh- cells ( ) . interestingly, the rmers-cov sequence seemed more stable in huh- cells than in vero a cells. however, the data presented in this article are statistically very limited to definitively conclude that virus genome stability depends on the cell type used. based on the preliminary data presented here, it would be interesting to analyze the evolution of the mers-cov sequence in different cell types, including human respiratory epithelial cells. the = third of the mers-cov genome contains a set of accessory genes encoding proteins with no similarity to other viral or mammalian known proteins ( ) . in general, cov accessory genes are not essential for virus growth in vitro ( ) ( ) ( ) ( ) . the reverse genetics system described in this article was used to study the importance of these proteins in cell culture. mers-cov genes , a, b, and were each found to be dispensable for virus replication in tissue cultures. interestingly, some of the rmers-cov viruses recovered from vero a cells contained mutations in the accessory gene genome region that would prevent the expression of any of these genes. similar results were previously reported for the original mers-cov-emc isolate after passage in vero cells ( ) . these data suggested an apparent lack of selection pressure on mers-cov accessory genes during passages in cell culture and reinforced the dispensability of these genes for virus growth in vitro. although not essential in tissue culture, these mers-cov accessory genes could have an important role in virus-host interaction in vivo, leading to attenuated phenotypes. cov accessory genes have been associated with the modulation of viral virulence ( ) . among all covs, sars-cov contains the largest number of accessory genes, and it has been proposed that these genes may have important contributions to its high virulence ( , ) . to date, mouse hepatitis virus (mhv) ns and a, tgev , and sars-cov b and proteins have been implicated in the modulation of innate immune responses, using different mechanisms to influence virus virulence ( , ( ) ( ) ( ) ( ) . rmers-cov-⌬e was a replication-competent, propagationdeficient virus and was only efficiently disseminated in cells expressing the e protein in trans. in the presence of transiently ex-pressed e protein, rmers-cov-⌬e yielded maximum progeny viral titers of around tcid /ml. this modest yield could be improved by the generation of cell lines stably expressing the e protein. in fact, a direct relation between viral titers and the amount of e protein expressed was previously observed for tgev ( ) . however, high expression levels of e protein could induce apoptosis, as described for mhv e protein expression ( ) . to overcome this potential adverse effect in the case of mers-cov, an inducible system for e protein expression would have to be established. rmers-cov-⌬e did not spread in cells in the absence of e protein, thus constituting a single-cycle replicative virus. however, infected cells produced syncytia, which suggests good expression of viral s protein. in addition, high levels of n protein were observed by immunofluorescence. these data suggested that the high expression levels of viral proteins might serve as potent immunogens to elicit a protective immune response. in the case of sars-cov, it has been shown that nonreplicating sars-cov-like particles bearing the e, s, and membrane (m) proteins induced immune responses that were protective against sars in mice ( , ) . in addition, sars-cov inactivated viruses induced adaptive immunity that protected against challenge ( ) ( ) ( ) ( ) . the potential of rmers-cov-⌬e as a vaccine candidate is reinforced by previous observations indicating that a sars-cov lacking the e gene (sars-cov-⌬e) is attenuated and induces protection in hamsters, transgenic mice, and conventional aged mice ( ) ( ) ( ) . mers-cov infects mucosal areas in the lungs and, probably, the enteric tract. mucosal immunity in a specific tissue, such as in lung infections with mers-cov, is optimally induced by local stimulation. therefore, immunization with live attenuated forms of rmers-cov-⌬e virus grown in a packaging cell line providing the e protein in trans may be a convenient option, particularly in comparison with purified mers-cov antigens, such as the s protein, that could serve as a subunit vaccine. vaccines based on live attenuated viruses may present biosafety problems associated with the possibility of reversion to virulent phenotypes or causing disease in immunocompromised individuals. in this sense, the use of rmers-cov-⌬e would be a safer option, as it does not propagate in the absence of e protein expression, preventing straightforward reversion to virulence. to increase the biosafety of a rmers-cov-⌬e-based vaccine, additional safety guards could be included, such as the previously de-scribed attenuating mutations in distant genomic locations, like those encoding the nsp ( , ) or nsp ( ) replicase proteins, or by introducing genomic rearrangements ( ) . overall, we consider rmers-cov-⌬e a promising vaccine candidate that should be further developed. rmers-cov-⌬e could also be used as the starting point to generate an inactivated vaccine in case of an urgent need to control the disease. in order to guarantee the absence of virulent viruses after an incomplete chemical inactivation due to clump formation, potential noninactivated viruses would be propagation defective and, therefore, attenuated. rmers-cov-⌬e, in which one of the nonessential accessory proteins was deleted, could be considered a marker vaccine, as it will allow the sera of field-infected patients to be distinguished from sera of vaccinated patients, based on the lack of antibodies specific for nonessential viral proteins ( ) . the rmers-cov-⌬e could also be considered a viral replicon, as its genome self amplifies in infected cells but infection is not efficiently spread from cell to cell. the construction of a minimal replicon is also possible with reduced effort using the infectious clone, a project that is currently in progress in our laboratory. therefore, the introduction of a reporter gene, such as green fluorescent protein, within this replicon could easily generate a useful tool for mers-cov antiviral drug screening. in this paper, we describe for the first time a reverse genetics system of mers-cov engineered on bacs, which has allowed the generation of the first modified live vaccine candidate to protect against mers-cov. furthermore, this reverse genetics system is a useful tool for the identification of viral genes involved in pathogenesis and the associated signaling pathways. drugs inhibiting these pathways would be potential antivirals. baby hamster kidney cells (bhk- ) were obtained from american type culture collection (atcc ccl- ). human liverderived huh- cells were kindly provided by r. bartenschlager (university of heidelberg, germany). african green monkey kidney-derived vero a cells were kindly provided by a. carvajal (university of leon, spain). in all cases, cells were grown in dulbecco's modified eagle's medium (dmem) supplemented with mm hepes, % nonessential amino acids (sigma), and % fetal bovine serum (fbs) (biowhittaker). virus titrations were performed on vero a or huh- cells following standard procedures and using closed flasks or plates sealed in plastic bags. for plaque assays, infected cells were overlaid with dmem containing . % low-melting agarose and % fbs, and at h.p.i., cells were fixed with % formaldehyde and stained with crystal violet. for % tissue culture infectious dose (tcid ) assays, cpe was recorded at h.p.i. all work with infectious virus was performed in biosafety level facilities by personnel wearing positive-pressure air-purifying respirators (highefficiency particulate air-mate). plasmids and bacteria strains. plasmid pbelobac ( ), kindly provided by h. shizuya (california institute of technology, pasadena, ca), was used to assemble the mers-cov infectious cdna clone. this plasmid is a low-copy-number plasmid (one to two copies per cell) based on the e. coli f factor ( ) that allows the stable maintenance of large dna fragments in bacteria. e. coli dh b (gibco/brl) cells were transformed by electroporation using a micropulser unit (bio-rad) according to the manufacturer's instructions. bac plasmid and recombinant bacs were isolated and purified using a large-construct kit (qiagen), following the manufacturer's specifications. construction of a full-length cdna clone of mers-cov. based on the data of the full-length sequence of the mers-cov-emc strain (genbank accession number jx ) ( ), a mers-cov infectious cdna clone was assembled in bac using a three-step strategy. in the first step, the restriction sites bamhi (genomic position ), stui (genomic positions , and , ), swai (genomic position , ), and paci (genomic position , ), present in the viral genome, were selected (fig. a) . second, the intermediate plasmid pbac-mers- = = was constructed as the backbone for assembly of the full-length cdna clone (fig. b) . to generate this plasmid, two dna fragments were generated by chemical synthesis (bio basic, inc.). the first fragment contained the cmv promoter fused to the first nucleotides of the viral genome flanked by sfoi and bamhi sites, and the other one contained a multicloning site with the restriction sites selected in the first step (bamhi, stui, swai, and paci) followed by the last , nucleotides of the viral genome joined to a -nt poly(a), hdv ribozyme, and bgh termination and polyadenylation sequences. the first dna fragment was cloned into pbelobac Ϫstui (a pbelobac without the stui restriction site) and digested with sfoi and bamhi to generate the plasmid pbac-mers- =, and then the plasmid pbac-mers- = = was generated by cloning the second dna fragment, digested with bamhi and sfii, into pbac-mers- = digested with the same restriction enzymes. finally, the third step was the assembly of the full-length cdna clone (pbac-mers fl ) by sequential cloning of four overlapping dna fragments (mers- to mers- ) into the multicloning site of the intermediate plasmid pbac-mers- = = (fig. c) . the overlapping dna fragments flanked by the appropriate restriction sites were generated by chemical synthesis (bio basic, inc.). in the case of fragment mers- , a silent mutation (t to c) was introduced at position , in order to eliminate the swai restriction site at position , and to use it as a genetic marker. the genetic integrity of the cloned dnas was verified throughout the assembly process by extensive restriction analysis and sequencing. construction of mers-cov cdna clones lacking accessory genes , a, b, and . the deletion of gene was generated by pcr-directed mutagenesis using the plasmid puc-mers- (a puc plasmid containing the mers- fragment spanning nucleotides , to , of the mers-cov genome) as the template and the oligonucleotides mers-s-tth i-vs ( = tgctatttgacaaagtcactatagctgatc =, where the restriction site tth i is underlined) and mers-s-paci-rs ( = cccttaattaactgagtaaccaacgtcaaaaagattcacact attagtgaacatgaaccttatgcggctcgaggtcgtattcc =, where the restriction site paci is underlined). the pcr product, including the deletion (from nucleotides , to , ), was digested with tth i and paci and cloned into the same sites of puc-mers- , leading to puc-mers- -⌬ . to generate pbac-mers-⌬ , the swai-paci digestion product from puc-mers- -⌬ was cloned into the same restriction sites of pbac-mers fl (fig. a) . the deletions of genes a, b, and were introduced by pcr-directed mutagenesis, using as a template the plasmid pbac-mers- = (a bac plasmid containing the mers- = fragment spanning nucleotides , to , of the mers-cov genome). for deletion of genes a and b, overlapping pcr fragments were amplified using oligonucleotides del ab-vs ( = gaactctatggattacggttgtctccatacggtc =) and del ab-rs ( = gaccgtatggagacaaccgtaatccataga gtt =). the final pcr product was amplified with outer oligonucleotides t and sa rs ( = caaacagtggaatgtagg =), digested with paci and nhei, and cloned into the same restriction sites of pbac-mers- =, leading to pbac-mers- =-⌬ ab that contains a deletion spanning nucleotides , to , of the mers-cov genome. for gene deletion, a pcr fragment lacking gene (nucleotides , to , ) was amplified using oligonucleotides sa vs ( = gttaattaacga actctatggattacg =, where the restriction site paci is underlined) and del -sandi-rs ( = cacgggacccatagtagcgcagagctgct gttaaaatcctggatg =, where the restriction site sandi is underlined), digested with paci and sandi, and cloned in the same sites of pbac-mers- =, leading to pbac-mers- =-⌬ . to generate plasmids pbac-mers-⌬ ab and pbac-mers-⌬ , the paci-rsrii digestion products from plasmids pbac-mers- =-⌬ ab and pbac-mers- =-⌬ were cloned in the same sites of pbac-mers fl (fig. a) . all cloning steps were checked by sequencing of the pcr fragments and cloning junctions. construction of a mers-cov cdna clone lacking the structural e gene. the pbac-mers-⌬e, encoding a mers-cov lacking the e gene, was constructed from the full-length plasmid pbac-mers fl . to this end, the sandi-rsrii dna fragment ( , bp) from pbac-mers fl was exchanged with a chemically synthesized (bio basic, inc.) sandi-rsrii dna fragment with a deletion from nucleotides , to , that included the trs core sequence and the first nucleotides of the e gene (fig. a) . the genetic integrity of the cloned dna was verified by restriction analysis and sequencing. recovery of recombinant viruses from the cdna clones. to recover infectious virus, bhk cells were grown to % confluence in a . -cm flask and transfected with g of the infectious cdna clone using g of lipofectamine (invitrogen) according to the manufacturer's specifications. at h.p.t., cells were trypsinized, plated over a confluent monolayer of either vero a or huh- cells grown in a . -cm flask, and incubated at °c for h. the cell supernatants were harvested and passaged once on fresh cells, and the recovered viruses were cloned by three rounds of plaque purification, following standard procedures. virus genome sequencing. the complete genome sequence of each rescued recombinant mers-cov was determined by sequencing overlapping rt-pcr fragments of . kb covering the full-length viral genome. reverse transcription and pcrs were performed with specific oligonucleotides using thermoscript reverse transcriptase (invitrogen) and the expand high-fidelity pcr system (roche), respectively, following the manufacturers' recommendations. the genomic =-and =-terminal sequences were determined using the =/ = race (rapid amplification of cdna ends) kit (roche) according to the manufacturer's specifications. sequence assembly and comparison with the consensus sequence of the mers-cov-emc strain were performed with the seqman and megalign programs (lasergene, madison, wi). for the generation of huh- cells transiently expressing e protein, cells were nucleofected with the plasmid pcdna -e (expressing the mers-cov e protein under the cmv promoter) by using a d nucleofector device (lonza) and the buffer and program recommended by the manufacturer. for the construction of plasmid pcdna -e, the e gene was amplified by pcr using pbac-mers fl as the template and the specific oligonucleotides e -ecori-vs ( = gtgctggaattcgccgccatgttacccttt gtccaagaacgaa =, restriction site ecori is underlined) and e -xhoi-rs ( = cgcccagctcgagttaaacccactcgtcaggtgg =, restriction site xhoi is underlined) and cloned into the plasmid pcdna (invitrogen) digested with ecori and xhoi. analysis of viral rna synthesis by rt-qpcr. total intracellular rna was extracted from transfected or infected cells with the rneasy miniprep kit (qiagen) according to the manufacturer's specifications. in the case of transfected cells, the residual dna was removed from samples by treating g of each rna with u of dnase i (roche) in l for min at °c, and dna-free rnas were repurified using the rneasy miniprep kit (qiagen). viral rna synthesis was quantified by rt-qpcr. total cdna was synthesized with random hexamers from ng of total rna using a high-capacity cdna reverse transcription kit (invitrogen). using this cdna, the viral rna synthesis was analyzed using two custom taqman assays specific for mers-cov grna (forward primer = gcacatctgt ggttctcctctct =, reverse primer = aagcccaggccctactat tagc =, and mgb probe = tgctccaacagttacac =) and sgmrna n (forward primer = cttcccctcgttctcttgca =, reverse primer = tcattgttatcggcaaaggaaa =, and mgb probe = ctttgattttaacgaatctc =). data were acquired with an applied biosystems real-time pcr system and analyzed with abi prism software, version . . . the relative quantifications were performed using the cycle threshold ( Ϫ⌬⌬ct ) method ( ) . to normalize for differences in rna sampling, the expression of eukaryotic s rrna was analyzed using a specific taqman gene expression assay (hs _s ; applied biosystems). generation of polyclonal antisera specific for mers-cov n and e proteins. rabbit polyclonal antisera (pab) specific for mers-cov n and e proteins were purchased from biogenes. in brief, peptides ntgrs-vyvkfqdskppl (corresponding to e protein amino acids to ) and aaaknkmrhkrtst (n protein amino acids to ) were synthesized and used to immunize two rabbits with each peptide according to the company's standard protocol. the polyclonal antisera obtained were evaluated by enzyme-linked immunosorbent assay (elisa) using the synthetic peptides, leading to titers ranging from : , to : , in all cases. indirect immunofluorescence assay. vero a and huh- cells were grown to % confluence on glass coverslips and infected with the recombinant mers-covs. at h.p.i., cells were fixed either with % paraformaldehyde in phosphate-buffered saline (pbs) at room temperature for min or with methanol at Ϫ °c for min. for n protein immunodetection, paraformaldehyde-fixed cells were permeabilized with . % saponin in pbs containing % fbs for min and incubated with mers-cov n protein pab (dilution : ) in pbs containing % fbs at room temperature for min. for e protein immunodetection, methanol-fixed cells were incubated with mers-cov e protein pab (dilution : ) in pbs containing % fbs overnight at °c. coverslips were washed times with pbs and incubated at room temperature for min with goat anti-rabbit antibody conjugated to alexa fluor (invitrogen) diluted : in pbs containing % fbs. nuclei were stained using dapi ( =, =-diamidino- -phenylindole) ( : , sigma). to fully inactivate the samples' infectivity, methanol-fixed cells were treated with % paraformaldehyde in pbs as described above. finally, coverslips were mounted in prolong gold antifade reagent (invitrogen) and analyzed on a leica sp confocal microscope. images were acquired with the same instrument settings and analyzed with leica software. novel coronavirus associated with severe respiratory disease: case definition and public health measures isolation of a novel coronavirus from a man with pneumonia in saudi arabia update: severe respiratory illness associated with middle east respiratory syndrome coronavirus (mers-cov)-worldwide middle east respiratory syndrome coronavirus (mers-cov): announcement of the coronavirus study group human betacoronavirus c emc/ -related viruses in bats full-genome deep sequencing and phylogenetic analysis of novel human betacoronavirus detection of alpha and betacoronaviruses in multiple iberian bat species pneumonia from human coronavirus in a macaque model ksa mers-cov investigation team. . hospital outbreak of middle east respiratory syndrome coronavirus evidence of person-to-person transmission within a family cluster of novel coronavirus infections first cases of middle east respiratory syndrome coronavirus (mers-cov) infections in france, investigations and implications for the prevention of human-to-human transmission clinical features and virological analysis of a case of middle east respiratory syndrome coronavirus infection clinical features and viral diagnosis of two cases of infection with middle east respiratory syndrome coronavirus: a report of nosocomial transmission mers-coronavirus replication induces severe in vitro cytopathology and is strongly inhibited by cyclosporin a or interferon-alpha treatment genomic characterization of a newly discovered coronavirus associated with acute respiratory distress syndrome in humans dipeptidyl peptidase is a functional receptor for the emerging human coronavirus-emc the receptor binding domain of the new mers coronavirus maps to a -residue region in the spike protein that efficiently elicits neutralizing antibodies molecular basis of binding between novel human coronavirus mers-cov and its receptor cd human cell tropism and innate immune system interactions of human respiratory coronavirus emc compared to those of severe acute respiratory syndrome coronavirus tropism of and innate immune responses to the novel human betacoronavirus lineage c virus in human ex 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viral cell tropism recovery of a neurovirulent human coronavirus oc from an infectious cdna clone the small envelope protein e is not essential for murine coronavirus replication absence of e protein arrests transmissible gastroenteritis coronavirus maturation in the secretory pathway synthetic recombinant bat sars-like coronavirus is infectious in cultured cells and in mice the nucleoprotein is required for efficient coronavirus genome replication comparative analysis of twelve genomes of three novel group c and group d coronaviruses reveals unique group and subgroup features coronavirus gene counteracts host defenses and modulates virus virulence the group-specific murine coronavirus genes are not essential, but their deletion, by reverse genetics, is attenuating in the natural host engineering the transmissible gastroenteritis virus genome as an expression vector inducing lactogenic immunity severe acute respiratory syndrome coronavirus group-specific open reading frames encode nonessential functions for replication in cell cultures and mice coronaviruses post-sars: update on replication and pathogenesis accessory protein a is a major antagonist of the antiviral action of interferon against murine coronavirus severe acute respiratory syndrome coronavirus open reading frame (orf) b, orf , and nucleocapsid proteins function as interferon antagonists cell type-specific type i interferon antagonism influences organ tropism of murine coronavirus alphacoronavirus protein modulates host innate immune response transmissible gastroenteritis coronavirus gene is not essential but influences in vivo virus replication and virulence induction of apoptosis in murine coronavirus-infected cultured cells and demonstration of e protein as an apoptosis inducer chimeric coronavirus-like particles carrying severe acute respiratory syndrome coronavirus (scov) s protein protect mice against challenge with scov immune responses against severe acute respiratory syndrome coronavirus induced by virus-like particles in mice comparative evaluation of two severe acute respiratory syndrome (sars) vaccine candidates in mice challenged with sars coronavirus a double-inactivated whole virus candidate sars coronavirus vaccine stimulates neutralising and protective antibody responses immunogenicity and protective efficacy in monkeys of purified inactivated vero-cell sars vaccine immunogenicity, safety, and protective efficacy of an inactivated sars-associated coronavirus vaccine in rhesus monkeys a live attenuated sars coronavirus is immunogenic and efficacious in golden syrian hamsters immunization with an attenuated severe acute respiratory syndrome coronavirus deleted in e protein protects against lethal respiratory disease complete protection against severe acute respiratory syndrome coronavirus-mediated lethal respiratory disease in aged mice by immunization with a mouse-adapted virus lacking e protein severe acute respiratory syndrome coronavirus nsp suppresses host gene expression, including that of type i interferon, in infected cells severe acute respiratory syndrome coronavirus evades antiviral signaling: role of nsp and rational design of an attenuated strain a live, impaired-fidelity coronavirus vaccine protects in an aged, immunocompromised mouse model of lethal disease coronaviruses maintain viability despite dramatic rearrangements of the strictly conserved genome organization vaccines to prevent severe acute respiratory syndrome coronavirusinduced disease complete nucleotide sequence of two generations of a bacterial artificial chromosome cloning vector cloning and stable maintenance of -kilobase-pair fragments of human dna in escherichia coli using an f-factor-based vector analysis of relative gene expression data using real-time quantitative pcr and the (-delta delta c(t)) method we thank n. m. beach for critical reading of the manuscript. we also thank milagros guerra for skillful technical assistance. key: cord- -hqrd e p authors: rozell, daniel j. title: assessing and managing the risks of potential pandemic pathogen research date: - - journal: mbio doi: . /mbio. - sha: doc_id: cord_uid: hqrd e p nan h n virus experiments occur in special facilities (bsl- ϩ) and, using the erasmus mc facility as an example, he estimated that the risks are much lower due to extra physical barrier biosafety measures, lab personnel vaccinations, and available antiviral therapeutics. thus, he estimated the risk of a laboratory-acquired infection (lai) to be less than ϫ Ϫ per person-year. taking into account that any infected lab worker would have already been vaccinated against a homologous h n virus, would be taking antiviral medication, and would be quarantined, fouchier estimated that a lab-induced pandemic would occur every billion years-more than twice the known age of the universe. he concluded with the observation that there have been no confirmed influenza virus lais or releases in decades, which suggests current measures are sufficient. a reply by lipsitch and inglesby ( ) questioned fouchier's claim that virology labs are safer than other bsl- labs. they also noted that fouchier's calculations incorrectly accounted for the uncertainty associated with observed events ( ) . furthermore, the assumption of events was claimed to be unreasonable, because viral lais have occurred in non-u.s. facilities ( ) . in separate comments, klotz ( ) argued that fouchier's calculations were based on the wrong method of calculating the elapsed time of escape for an lai and that the estimate for an lai was too low. a reply by fouchier ( ) argued that klotz did not provide "scientific justification" for higher estimates. within this debate among competing risk estimates, there appears to be disagreement as to not only what constitutes the appropriate methodology, but also what constitutes evidence. for example, fouchier ( ) does not believe that recent laboratory errors (most notably at the cdc) constitute relevant data, because either the errors did not result in lais, the pathogen was not an engineered avian influenza virus, or the work was not conducted specifically in a bsl- ϩ laboratory. however, critics contend that these errors demonstrate the general failure of laboratory safety procedures upon which fouchier's calculations depend. adding to this concern is a study ( ) that estimated a % to % probability that a laboratory escape event would go undetected. likewise, investigative reporting on u.s. labs ( ) suggested that laboratory accident records are poorly tracked, generally underreported, and difficult for the public to access. a review of these various assessments suggests that the most useful contribution of a single independent quantitative risk as-sessment may be to standardize the language of the debate. it is difficult enough to assess the quality of the data and validity of assumptions of each risk assessment. further comparisons are made nearly impossible because of use of inconsistent units (e.g., escape probability, risk per lab-year, and risk per worker-year) and different treatments of uncertainty (e.g., point estimates versus % confidence intervals). by using a single rba as a starting point, hopefully the various stakeholders will at least be able to argue using the same mathematical framework. despite the nih request for a comprehensive quantitative riskbenefit analysis, there is acknowledgment that this may not be possible. during the nrc symposium ( ), both baruch fischhoff and ronald atlas discussed the difficulty of estimating benefits from the gof research or, more generally, any basic research, due to its unpredictable and serendipitous nature. likewise, the public health benefits of gof research are difficult to estimate because they are conditioned on factors outside the laboratory ( ) . that is, while the risk of accidental release is largely controlled by laboratory conditions, the beneficial use of any discovered knowledge depends on the existing public health system, which varies widely among communities, regions, and nations. for example, year into the influenza pandemic, there was still only enough vaccine for one-quarter of the world's population ( ) . further complicating a benefits analysis are the multiple ways in which evidence can be interpreted. for example, during the nrc symposium, it was widely acknowledged that genetic analysis of ppps currently could not predict the resulting phenotype ( ) . critics of gof research argued that this lack of predictive ability severely limits the benefits of this line of research for any practical therapeutic purposes (e.g., vaccine design). however, proponents argued that this lack of knowledge was the very reason that gof research should continue. thus, an argument against the current practical value of the research is being interpreted by others as a supportive argument from the perspective of basic science. in this case, interpretation of a benefit is a subjective value judgment. the gof controversy includes many other value-laden debates regarding risks, benefits, and assessment methodologies. for example, proponents argue that gof research has a unique scientific value ( ) , while critics argue that the scientific value may be no greater than that of safer alternatives, which should be considered an opportunity cost in an rba ( ) . the debate also extends to disagreements regarding: the practical value of gof experiments to policy makers ( , ) , how we should count and compare the various ways of valuing research (e.g., intrinsic value versus instrumental value) ( , ) , and how publication criteria should compare public health risk(s) to scientific merit ( , ) . considerable disagreement even exists regarding ancillary effects, such as the impact of the various moratoria and regulations on the decisions of young scientists to work in virology ( ) ( ) ( ) . the gof controversy even includes debates over definitions. as discussed at the nrc symposium ( ), gof research is already widely used for multiple beneficial and largely benign purposes, including increasing vaccine yields ( ) , expanding genomic sequence surveillance databases ( ) , and creating animal models of human viral infections to aid further research. furthermore, naturally arising gof mutations are common in research labs that work with rna viruses. because the current state of science is unable to predict what genomic changes will increase danger, we cannot be sure what experiments will result in new undesirable traits. proponents of the moratorium argue that the wording was specific enough that only federally funded projects were affected and that public health surveillance and vaccine development activities were exempt ( ) . rather, proponents accuse critics of the moratorium of attempting to widen the definition of what might be banned in hopes of weakening support for any restrictions. this is not the only debate over terminology. it has been argued that the use of the term "pandemic" itself is an "apocalyptic rhetorical device" ( , ) that preempts any reasonable discussion of risks and benefits by appealing to our innate fear of rare but catastrophic events. however, this assumes that the risk of a pandemic is actually rare, despite considerable disagreement among informed scientists regarding the likelihood of such an event. ironically, labeling the use of "pandemic" as rhetorical sophistry may itself be a rhetorical trick if it is used to dismiss a category of serious claims without due consideration of merit. ultimately, the purpose of summarizing and critiquing some of the arguments within the gof/ppp debate is to emphasize the many epistemic and ethical value judgments inherent to rba and to provide evidence for prior claims that a consensus-building quantitative assessment is unlikely ( ). this naturally leads us to wonder if there is a better alternative. fischhoff suggests that, rather than use rba to only inform the eventual policy decision, it should instead be used to improve research design ( ). lipsitch and galvani ( ) made the same argument for improving gof/ppp research design, but in the context of responsible research principles. they argued that most gof/ppp experiments are not ethically justifiable because they do not meet the criterion of yielding humanitarian benefits not attainable by safer alternatives. one approach to improving research design is to use the design principle of inherent safety ( ) ( ) ( ) , which focuses on attempting to eliminate material hazards in research and manufacturing. in contrast, conventional risk management generally focuses on reducing the likelihood of an accident through safety procedures and equipment. the formal inherent safety concept is frequently used in the chemical and nuclear engineering communities but it has not been widely adopted by scientists and engineers in other fields ( ) . while this idea seems to be common sense, it is a departure from most previous work on biosafety and biosecurity ( , ) , which was focused on improving risk management through formalized processes and training. the continued emphasis on these methods is unfortunate, given the generally poor record of implementation ( , ) . an additional benefit of the inherent safety concept is its ability to address security concerns ( ) . for example, a traditional safety measure, such as removing all ignition sources near an explosive material, is of little security value; malevolent actors will bring their own ignition source. likewise, terrorists are attracted to hazards that already instill public dread. inherently safe design makes terrorism more difficult by removing the exploitable hazard. because safety has traditionally been the concern of engineers at the production level, the r&d community often fails to consider these principles in the early stages of research when the most impact can be made ( ) . however, inherent safety in research is sometimes recognized in hindsight. a cdc report ( ) that summarized an internal review of the june exposure of laboratory workers to potentially viable bacillus anthracis at a cdc bio-letter to the editor terrorism response lab noted that an avirulent strain could have been used as a substitute in the experiment. it is also interesting that in its list of responses, the report focused primarily on revised biosafety protocols and procedures. a reference to reducing the hazard (i.e., inherent safety) was made only within the fifth of eight recommendations. the calls for inherently safe design appear to have yielded some consensus from the opposing camps in the gof/ppp controversy. one sign during the nrc symposium was provided by yoshihiro kawaoka, a principal investigator of one of the two original studies that started the debate ( ) , who endorsed the idea that some research could be conducted with alternative techniques, such as loss-of-function studies, use of less-pathogenic viruses, and phenotypic analyses ( ) . proponents of inherently safe ppp research have also been buoyed by recent successes. for example, langlois et al. ( ) showed that species-specific microrna targeting can be used to conduct relevant animal model ppp research that still poses low risks to humans. as michael imperiale stated, "you can develop safer approaches to do these types of experiments; it just needs a little bit of imagination on the part of researchers" ( ) . as summarized here, many of the disagreements within the gof/ppp debate involve epistemic and ethical value judgments that suggest that definitive quantitative risk-benefit analysis is not possible. this does not devalue rba; it is still useful as a tool for engaging experts and the public in a conversation about riskbenefit tradeoffs. however, if calls for rba become knee-jerk responses to what are essentially quantitatively intractable technological risk problems, everyone will be disappointed. rba works best when expectations are realistic. when data are plentiful and there are no moral or cultural differences among the stakeholders, rba can generate "answers" for policy formulation. however, for emerging technologies and controversial research where data are sparse and uncertainty is high, putting a number on a subjective quantity only engenders suspicion. the question of whether the benefits of gof/ppp outweigh the risks is unlikely to be resolved by an independent formal rba. however, this question may become less relevant if safer approaches can achieve the same goals. that is, inherently safe design may be the best compromise solution for the gof/ppp controversy. because the inherent safety principle will not be invoked unless a risk is perceived, the appropriate next step is to regard the eventual results of the rba as a tool for risk exploration, which then inspires more inherently safe research. over the long term, changing the biosafety/biosecurity culture in the life sciences to emphasize inherent safety principles will help avoid similar heated controversies in the future. risks and benefits of gain-of-function experiments with pathogens of pandemic potential, such as influenza virus: a call for a science-based discussion mbio addresses the pause in gain-offunction (gof) experiments involving pathogens with pandemic potential (ppp) the h n moratorium controversy and debate conducting risk and benefit analysis on gain-of-function research involving pathogens with pandemic potential moratorium on research intended to create novel potential pandemic pathogens the unacceptable risks of a man-made pandemic the consequences of a lab escape of a potential pandemic pathogen. front public health : monitoring select agent theft, loss and release reports in the united states- - airborne transmission of influenza a/h n virus between ferrets potential risks and benefits of gain-of-function research: summary of a workshop studies on influenza virus transmission between ferrets: the public health risks revisited studies on influenza virus transmission between ferrets: the public health risks revisited probability of adverse events that have not yet occurred: a statistical reminder strengthening risk governance in bioscience laboratories comments on fouchier's calculation of risk and elapsed time for escape of a laboratory-acquired infection from his laboratory comments on fouchier's calculation of risk and elapsed time for escape of a laboratory-acquired infection from his laboratory containing the accidental laboratory escape of potential pandemic influenza viruses inside america's secretive biolabs great expectations-ethics, avian flu and the value of progress perspective: ill prepared for a pandemic improving pandemic influenza risk assessment an epistemological perspective on the value of gain-of-function experiments involving pathogens with pandemic potential can limited scientific value of potential pandemic pathogen experiments justify the risks? mbio reply to "can limited scientific value of potential pandemic pathogen experiments justify the risks valuing knowledge: a reply to the epistemological perspective on the value of gain-of-function experiments valuing knowledge: a reply to the epistemological perspective on the value of gainof-function experiments an avian h n gain-of-function experiment of great concern the decision to publish an avian h n influenza virus gain-offunction experiment vagueness and costs of the pause on gain-of-function (gof) experiments on pathogens with pandemic potential, including influenza virus a brain drain due to increased regulation of influenza virus research is highly speculative reply to "a brain drain due to increased regulation of influenza virus research is highly speculative influenza gain-of-function experiments: their role in vaccine virus recommendation and pandemic preparedness use of highly pathogenic avian influenza a (h n ) gain-of-function studies for molecularbased surveillance and pandemic preparedness the apocalypse as a rhetorical device in the influenza virus gain-of-function debate ethical alternatives to experiments with novel potential pandemic pathogens what you don't have, can't leak inherently safer plants how to make inherent safety practice a reality developments in inherent safety: a review of the progress during - and opportunities ahead biosafety risk assessment methodology biosafety controls come under fire can biosecurity be embedded into the culture of the life sciences promoting inherent safety are we too risk-averse for inherent safety? report on the potential exposure to anthrax experimental adaptation of an influenza h ha confers respiratory droplet transmission to a reassortant h ha/h n virus in ferrets microrna-based strategy to mitigate the risk of gain-of-function influenza studies key: cord- - aszklx authors: duprex, w. paul; casadevall, arturo title: falling down the rabbit hole: atrip toward lexiconic precision in the “gain-of-function” debate date: - - journal: mbio doi: . /mbio. - sha: doc_id: cord_uid: aszklx nan blow-by-blow account of how we ended up at the point where the u.s. government instigated a "pause" on new and a voluntary cessation of ongoing/funded virology experiments. however, to understand where we are demands some appreciation of how virology began, the types of questions virologists ask, and the approaches that they use. long before virology existed as a discipline or before scientists knew what viruses were, people observed what administration of filtered contagium vivum fluidum did in a natural host. transmission experiments in plants and animals led to reproducible diseases, and such approaches were foundational in showing the absolute requirement of living cells for virus growth. eggs (although not of the humpty variety) were pivotal, as they provided a novel cellular substrate for the culture of influenza viruses. subsequent development of in vitro cell culture systems opened the door for virologists to bring dangerous pathogens into the laboratory. what is important to appreciate is that these approaches established a basic principle in virology: the use of unnatural cell substrates to culture viruses. as viruses are obligate intracellular pathogens that infect bacteria, plants, animals, and humans, the very act of introducing a virus into a new environment will drive evolutionary adaptations. changes in the genome during tissue culture adaptation lead to alterations in the phenotype as functions are gained and lost. this fact seems to have been lost in the ongoing debate into the creation of novel viruses, and it is both simplistic and incorrect to apply the term "gain of function" to a small subset of influenza transmission experiments. how did we find ourselves in this lexiconic morass? by not appreciating the history of virology, by not recognizing that "gain of function" is a term used by geneticists, not virologists, and by not nipping usage of this misnomer in the bud from the outset. at this juncture, it is reasonable to ask, "is it possible to extract ourselves from this rabbit hole?" it could be argued that expending any effort on such semantics is unwise and that it is impossible to let go of gof. however, we contend that this is a defeatist approach, and rather than complain about the problems of the past will suggest more-precise terminology which could be used in the future. in fact, changes in the common lexicon in recent years show that terminology can indeed be altered, as evidenced by the switch from "stewardess" to "flight attendant," "handicapped" to "disabled," and "third world" to "underresourced," if there is a need for better usage. in writing this opinion piece, we reached out to a number of colleagues vested in this debate, some of whom are grappling with what it means for their studies to be "paused" and others of whom see the value in halting experiments which might lead to the generation of potentially problematic pathogens. what follows is a synthesis of our thoughts, which have been enhanced and altered by their comments and concerns. it is by no means meant to be prescriptive, and our goal is to provoke an inclusive discussion since this is long overdue. two common mistakes involve confusing dual-use research (dur) with dual-use research of concern (durc) and equating gof experiments with durc. the first is easy to address, as durc simply represents a subset of dur (fig. a ). durc is defined as "life sciences research that, based on current understanding, can be reasonably anticipated to provide knowledge, information, products, or technologies that could be directly misused to pose a significant threat with broad consequences to public health and safety, agricultural crops and other plants, animals, the environment, or national security" ( ) . currently, durc focuses on select agents, viruses, bacteria, and toxin (fig. b) , and includes human, animal, zoonotic, and synthetically generated pathogens, such as variola major virus, foot-and-mouth disease virus, the reconstructed influenza virus, and ebola virus (fig. c ). three have been eradicated from general circulation, although rinderpest virus would be very straightforward to reconstruct using reverse-genetics approaches published years ago, synthetic biology, and wild-type genome sequences which are in the public domain ( , ) . although influenza virus has not been eradicated by vaccination, reverse genetics was used to synthesize the h n influenza virus responsible for the pandemic ( ) . based on a predication that there are approximately , mammalian viruses ( ), the list of durc-relevant viruses represents a mere . %. furthermore, the "gof"-research of concern represents a minuscule fraction of the virological research portfolio and is usually only thought of as influenza transmission studies (fig. d, red circle) . interestingly, severe acute respiratory syndrome (sars) and middle east respiratory syndrome (mers) viruses are not on the durc list, even though work with these is also subject to the "pause." this demonstrates that in practice, durc is wider than a list of pathogens and illustrates the broader impact on microbiological research of this debate. gain-and loss-of-function experiments are foundational approaches of microbial genetics. they span all of microbiology, not just the pathogens on the durc list. selection processes are used to alter wild-type phenotypes making this word a more appropriate descriptor for any change in function. it covers both loss conducted for legitimate purposes that generates knowledge, information, technologies, and/or products that could be utilized for both benevolent and harmful purposes. (b) dual-use research of concern (durc) is a small subset of dur involving life sciences research that, based on current understanding, can reasonably be anticipated to provide knowledge, information, products, or technologies that could be directly misapplied, posing a significant threat with broad potential consequences to public health and safety, agricultural crops and other plants, animals, the environment, materiel, or national security. the agents and toxins listed are subject to select-agent regulations. (c) the seven viruses of concern listed include circulating, synthetically generated, and eradicated agents. (d) although spanning all of microbiological research, much of the durc debate has focused on virology, where selection processes of circulating and synthetically generated agents have been used to enhance transmission. although selection is a mainstay of microbial genetic research and gain (green shading)and loss (red shading)-of-function experiments are commonplace if the durc list is strictly adhered to, there is a very small number of durc projects (red circle). furthermore, gain of function is often accompanied by loss of function and vice versa (yellow shading). (e) alterations in phenotype which give cause of concern can include loss and gain of function. editorial and gain and recognizes an important part of any selection process; there is often a trade-off such that as one function is acquired or enhanced, another function is concomitantly lost or reduced. this is illustrated for all of virology (fig. d) . for example, a human virus passaged in animal cells can adapt to a different receptor and/or intracellular factors and can infect more cell types than the wild-type virus (gain of function). however, these and other adaptive mutations can lead to a decrease in virulence as the process of pathogenesis is altered (loss of function). live attenuated vaccines can be placed in the yellow section, as by definition a key goal of the selection process is loss of virulence, but a consequence of passage is a gain of infectivity and a change in tropism. this example illustrates the fundamental problem of focusing exclusively on one function and one virus. most of the discussion has centered on the gain of transmission of influenza leading to a situation where the proverbial "tail is wagging the dog." this is why we need to define not just one but all of the "f's." accepting that a selection process leads to an alteration in phenotype and produces a pathogen with different functions is straightforward, as this is a common approach in microbiology. agreeing which functions to focus on is also relatively easy, as the durc policy is clear. from the seven categories of experiments requiring oversight, six identify functions of concern and one focuses on (i) reconstructing pathogens which have been eradicated or (ii) reconstituting viruses no longer in general circulation. synthetic reconstruction from published sequences or reconstitution from archived clinical samples is simple to regulate at the level of an institutional biosafety committee (ibc) or institutional review entity (ire). it is expected that any such body would point out that work with rinderpest or smallpox virus as part of an nih application is not permissible. however, more in-depth discussions are required between review committees and a researcher who seeks permission to perform microbiological experiments which aim to alter one or more of the six functions of concern. increases in transmission, range, infectivity, pathogenesis, resistance to a therapeutic agent, and disruption of immunity are the functions which demand attention (fig. e ). for some, gain of function causes the most concern, although even for the influenza transmission studies, it is simplistic to focus on only one phenotype/ one function as a range or on infectivity changed during selection of mammal-adapted avian influenza viruses. once again, this highlights why "gof" is such a vague term. conversely, loss of function, such as the ability to be neutralized by an antibody, inhibited by a drug, or detected by a diagnostic assay, also raises significant concern. an experiment that uses one or more of the pathogens and produces, aims to produce, or can be reasonably anticipated to alter (a) transmission (t), range and resistance (r), infectivity/ immunity (i), and pathogenesis (p) is what concerns us; this can be condensed to "atrip." this simple acronym precisely identifies the functions of concern, moves away from the current preoccupation with enhancement, and does not assume that dual use is pertinent from the outset. although potentially applicable to all of microbiology, it should be used to identify only the small subset of experiments with prescribed pathogens which may produce data relevant for biosecurity (fig. d, red circle) . focusing on alterations to explicit phenotypes will help scientists think holis-tically during experimental design and assist members of the ibc/ ire during application evaluation. we consider this a simple and pragmatic resolution to the lexiconic muddle which, if adopted to describe experiments of concern for any pathogen requiring durc oversight, would be workable across all of microbiology. from the outset of this debate, microbiologists were concerned that "creep" would occur and that the list of durc-relevant agents would inevitably expand. many in the community predicted a "slippery slope" and waited for additional oversight. the current "pause," which added studies on mers and sars coronaviruses and low pathogenicity avian influenza viruses to the list, proves that their concerns were legitimate. many believe that calls for increased regulation and risk-benefit analyses are knee-jerk reactions which are not based on scientific evidence ( ) . they argue that opponents of the research do not recognize the functionality of existing biocontainment practices or appreciate the extensive risk mitigation practices currently in place to permit microbiologists to work with dangerous pathogens safely. the subtle shift in focus from biosecurity to biosafety issues also leads to confusion and once again illustrates why using precise terminology is critical. microbiologists should not be complacent and must be open to suggestions and approaches from other fields. meaningful, quantifiable, evidence-based assessments are not a threat to research. there is a responsibility to communicate clearly when undertaking an atrip experiment and justifying to an ibc/ ire why altering transmissibility or range or making a resistant mutant is necessary. to conclude, let us return full circle, something which is useful when we are puzzled. when humpty met alice, he asked the intrepid adventurer "tell me your name and your business?" on face value, this is a much more reasonable approach to dialog from the cantankerous egg, but as usual, in wonderland, things are never as they seem. '"my name is alice, but-" . . . "it's a stupid name enough!" humpty dumpty interrupted impatiently. "what does it mean?" "must a name mean something?" alice asked doubtfully. "of course it must," humpty dumpty said with a short laugh!' ( ) . does the name atrip help, or does it just add more complexity to this muddle? it would satisfy colleagues who demand precision and agree that names should mean something. it specifies the altered functions that cause concern, does not assume dual-use potential from the outset, but still prompts investigators and overseers to consider the possibility. it would act as a filter. microbial genetics experiments often involve selection processes; not all alter specific phenotypes, far fewer use pathogens on the current list, and even fewer involve creation of agents which are novel in any meaningful sense of the word. this is a scientific and rational approach to nomenclature which cuts through the "gof"-smog. it stands as a straw man ready to be knocked down. it is meant to provoke debate and help microbiology set the terms rather than be subject to terms. we commend it to the community for discussion, and if it is found useful, we encourage its adoption by scientists, governments, and the media. it might be atrip, but hopefully it is a trip in the right direction. through the looking glass, and what alice found there united states government policy for oversight of life sciences dual use research of concern rescue of synthetic genomic rna analogs of rabies virus by plasmid-encoded proteins the plowright vaccine strain of rinderpest virus has attenuating mutations in most genes characterization of the reconstructed spanish influenza pandemic virus a strategy to estimate unknown viral diversity in mammals risks and benefits of gain-of-function experiments with pathogens of pandemic potential, such as influenza virus: a call for a science-based discussion the views expressed in this article do not necessarily reflect the views of the journal or of asm we are very grateful to julie pfeiffer, ut southwestern medical center, texas, for her rendering of the gain-of-function/loss-of-function venn diagram that we incorporated into the figure associated with this editorial and for her many constructive comments and useful suggestions. thanks go to a large number of colleagues who responded to questions about terminology and critiqued and canned suggestions which never should have made it into print as we flew some lexiconic kites in their faces. finally, we are grateful to lewis carroll, a veritable purveyor of nonsense and master of a rollicking trip. key: cord- -o sy zi authors: baric, ralph s.; crosson, sean; damania, blossom; miller, samuel i.; rubin, eric j. title: next-generation high-throughput functional annotation of microbial genomes date: - - journal: mbio doi: . /mbio. - sha: doc_id: cord_uid: o sy zi host infection by microbial pathogens cues global changes in microbial and host cell biology that facilitate microbial replication and disease. the complete maps of thousands of bacterial and viral genomes have recently been defined; however, the rate at which physiological or biochemical functions have been assigned to genes has greatly lagged. the national institute of allergy and infectious diseases (niaid) addressed this gap by creating functional genomics centers dedicated to developing high-throughput approaches to assign gene function. these centers require broad-based and collaborative research programs to generate and integrate diverse data to achieve a comprehensive understanding of microbial pathogenesis. high-throughput functional genomics can lead to new therapeutics and better understanding of the next generation of emerging pathogens by rapidly defining new general mechanisms by which organisms cause disease and replicate in host tissues and by facilitating the rate at which functional data reach the scientific community. m icrobial genome sequencing efforts continue at an increasing rate, resulting in an expanding catalogue of genes of unknown function for important pathogens (fig. ) . in some cases, up to % of the annotated genes in fully sequenced microbial genomes have no known or predicted function. in many cases, these uncharacterized genes are highly conserved and are implicated in the pathogenic process through either their timing of expression or requirement for microbial replication. such data indicate that these genes execute important, general biological functions. the interpretation of existing and emerging microbial genomics data will require the scientific community to uncover new paradigms hidden within these sequences. this point is highlighted by a recent publication by hutchison et al. focused on building a minimal genome of mycoplasma mycoides ( ) . the genes in this genome include many required for known essential functions, such as dna replication, transcription, and translation, but the genome also contains genes (~ %) of unknown function. likewise, the known coding capacity of dna viruses has been doubled over the past few years, an achievement made possible by using novel high-throughput sequencing and proteomic methods. the discovery of these genes of unknown function not only promises to affect our understanding of microbial pathogenesis but also provides an undiscovered wealth of new therapeutic targets for antibiotic, antiviral, and vaccine development for improved global and economic health. unlike large-scale genome-sequencing or structural-genomics efforts, the functional annotation of uncharacterized genes is not well developed technologically, and therefore, the scientific community cannot rely on a well-defined, mature set of experimental approaches. simple deletion or overexpression of an uncharacterized gene often fails to yield any discernible phenotype in standard laboratory contexts. in the same vein, a purely biochemical approach that relies on purification and in vitro biochemical characterization of uncharacterized genes often fails to yield fruitful functional data. furthermore, while sequence-based bioinformatic analyses and computational models may provide clues about functional genetic interactions, such predictions are often limited by our current biological knowledge and databases. as a consequence, functional annotation is incorrect in many cases because unvalidated information is propagated across species. in cases where gene function is experimentally assigned and validated, the information is often not broadly propagated or may not apply to another organism. therefore, a successful functional annotation endeavor requires a multipronged approach that involves open collaboration among scientists with expertise in genetics, bioinformatics, molecular biology, biochemistry, cell physiology, host-microbe interactions, and data management. such a program is outside the scope of traditional funding mech- anisms and requires integrated teams of experts and new highthroughput experimental methodologies. the niaid has implemented a program aimed at assigning functions to open reading frames (orfs) and small noncoding rnas (ncrnas) that have been discovered by large-scale sequencing efforts to begin addressing this gap in our understanding of bacterial and viral gene function. this timely initiative from the niaid is highly significant because it is the institute's first attempt to incorporate functional annotation into its genomics and advanced technologies program, which is focused on developing genomics, proteomics, and bioinformatics resources to advance our understanding of infectious and immune-mediated diseases. the niaid functional genomics (fg) program builds on previous functional annotation efforts, such as combrex ( ) , and brings together multidisciplinary groups to work on shared goals. its mission is to probe new biology in pathogenic bacteria and viruses by assigning functions to new genes. the fg program is unique in its approach in that it enables principal investigators to follow up on "omics" and phenotypic screening data with targeted experiments to establish gene function. most pathogens are evolving constantly, and such an integrated approach will provide the database to allow us to work toward predicting new emerging infectious diseases and respond to new pandemics. projects across the niaid fg program are driven by the idea that the assignment of gene function must be rooted in experimentation in vivo and in vitro. the challenge to the experimentalist is to develop systematic approaches to gene function discovery that have a reasonable chance of success over a large number of genes. fg centers are taking a variety of approaches to uncover the functions of protein-coding genes and small ncrnas. some approaches, which perhaps will have the highest yield, require at least some prediction of function, while others presuppose no specific functional knowledge. current state-of-the-art technologies, such as genome synthesis, next-generation nucleic acid sequencing, ribosomal profiling, high-resolution mass spectrometry, metabolomics, and molecular and systems modeling, enable high-throughput approaches to characterize genes of unknown function. though these technologies are clearly valuable, they are most effectively leveraged when combined with functional assays, such as measurements of microbial permeability and antimicrobial susceptibility or assays of host response to infection, which have been scaled up through recent developments in robotics, whole-genome synthesis, and high-content cellular imaging. in this commentary, we provide an overview of our coordinated efforts that are aimed at filling the gaps in our understanding of microbial gene function. these efforts are restricted to a few organisms and technologies but could be attempted on a much larger scale. such functional analyses could provide the scientific basis for rational approaches to the development of new therapeutic products to combat future and current gaps in the treatment of bacterial and viral infection. improving the quality of genome maps. most protein-coding genes are annotated by either their similarity to known genes or generalized guidelines for identifying translational start sites. such predictions are often incorrect. for example, we have demonstrated that a protein highly homologous to a secreted bacterial amidase is actually an intracellular regulator of peptidoglycan synthesis ( ) . moreover, predictive algorithms to define the locations of non-protein-coding genes (e.g., small ncrnas, out-of-frame orfs, etc.) are generally poor. defining the boundaries of genes of unknown function is an important component of experimental functional annotation. we are using combinations of directed biochemical, computational, and next-generation-sequencing approaches to produce improved maps of gene boundaries in bacterial and viral pathogens. these efforts enable improved functional genetic and biochemical experiments. leveraging existing protein structure data. molecular structure data can provide tremendous insight into the biochemical functions of proteins. a high-quality structural model enables one to map conserved residues on the molecule and to develop hypotheses regarding active-site chemistry, ligand binding sites, protein docking sites, etc. we are taking advantage of the large amount of structural data available in the pdb to build homologybased protein models. in addition, we are using hidden markov model (hmm)-based approaches to build predictive structural models in cases where homology in the pdb is low. experimental structures and high-confidence structural models generated by the fg program are available to the community and have been used to develop and test specific functional hypotheses in vitro and in vivo and to define protein function ( ). high-throughput protein production for functional biochemical analysis. structural models of proteins of unknown function are informing functional biochemical hypotheses that are being tested in vitro. specifically, we are leveraging existing high-throughput structural genomics infrastructure to produce expression clones of proteins of unknown function. this approach is yielding milligram quantities of many targets of unknown function, which are being assayed for a range of biochemical activities and are being used to produce antibodies for cellular studies. one specific use for these purified proteins is in activitybased metabolomic profiling ( ), an unbiased approach to finding substrates and products for putative enzymes. defining gene function by biochemical association. a wellestablished approach to define function is to test for physical associations of proteins and rnas with other proteins or transcripts of known function. we are employing such biochemical association strategies. for protein-coding genes, we are measuring protein-protein associations using quantitative proteomic methods. for example, using a proteomics approach, we reported that kaposi's sarcoma-associated herpesvirus (kshv) viral interferon regulatory factor (virf ) can bind the cellular interferonstimulated gene (isg ) e ligase, herc . interaction of virf with herc inhibits the conjugation of isg to cellular proteins, thereby dampening the ifn response to the virus ( ). using a functional genomics screen, coupled with synthetic genome design, we demonstrated that the s glycoprotein genes of several severe acute respiratory syndrome coronavirus (sars-cov)-like bat coronaviruses can bind human receptors for entry and replicate efficiently in primary human airway epithelial cells and that they are resistant to existing sars vaccines and immunotherapeutics ( , ) . our early results suggest that an efficient way to discover function using this approach is to target protein complexes of known function and define associated proteins of unknown function. these efforts have required us to develop new bioinformatics methods to understand the complex data produced by these experiments. prediction of ncrna targets. ncrnas represent a particular challenge in bacterial and virus research, as they can vary tremen-perspective dously among different bacterial species and viruses and little is known about their functions. we have generated catalogues of the ncrnas for the organisms (for example, in mycobacteria) ( ) that we study and are applying large-scale methods to identify their targets. these methods start with bioinformatics, though the predictive algorithms are not yet particularly robust. in addition, we are applying cross-linking-, ligation-, and sequencing-based approaches to systematically link small ncrnas of unknown function to their transcript targets. thus, while many of our approaches to investigating ncrnas are still at the developmental stage, defining catalogues of ncrnas provides the community with a road map of targets for downstream studies and analyses. we are also using next-generation-sequencing technologies, such as transcriptome sequencing (rnaseq) and selective =hydroxyl acylation analyzed by primer extension (shape) analysis of rna, to investigate rna structure-function correlations. paired with these analyses, we are investigating the rna transcriptome to correlate conserved and unique rna structure elements with pathogen virulence factors, identify previously uncharacterized and/or rare translation initiation sites, and associate protein structure elements with virus biology, pathogenesis, and host range. genetic approaches: identifying phenotypes. we are using multiple strategies to identify phenotypes that are associated with the deletion or overexpression of target genes of unknown function. for bacterial pathogens, we have initially focused on genes that, when disrupted, produce measurable growth phenotypes or alterations in cellular barrier function under specified conditions, including antibiotic treatment. these assays are facilitated by wholegenome-mutant defined mutant libraries that allow more efficient high-throughput screening for specific phenotypes ( ) . we are assaying the growth of mutant strains in axenic culture and in cell and animal infection models. the biolog screening platform provides a high-throughput approach to phenotype identification. we are growing individual deletion strains in parallel with wild-type control strains under~ , defined conditions. differences between strains identify specific medium conditions under which a particular gene is required for wild-type growth and have provided clues for target gene function ( ) . in a particular case, a functional genetic study of mutants harboring deletions of genes of unknown function is informing the development of a new live attenuated brucella abortus vaccine strain ( ) . in addition, we are applying genome-wide screening approaches to find genes that interact genetically to modify phenotypes. in our studies of viral pathogens, we have developed a highthroughput, multiarmed screening program to investigate both rna and dna virus-encoded host evasion functions. to accomplish this, we are applying modular cloning technology to easily shuttle candidate genes of unknown function into replicon expression and/or lentiviral vectors for alternate applications, such as the expression of toxic or otherwise challenging proteins and antibody generation. using these screening assays, we are defining viral counterdefense mechanisms mediated by known, novel, and noncanonical viral orfs and ncrnas, whose products modulate the host innate immune response and cellular defense machinery to the virus's advantage. screening assays include assessments of beta interferon (ifn-␤) antagonism, nf-b, toll-like receptor, and apoptosis modulation, inflammasome signaling, cgas-sting and p pathway modulation, cellular localization, global protein synthesis, and mtor inactivation. for example, we have reported multiple herpesviral proteins that modulate the cgas-sting dna sensing pathway and middle east respiratory syndrome coronavirus (mers-cov) and closely related bat merslike virus phosphodiesterase proteins that antagonize rnase l activation during infection ( ) ( ) ( ) . in parallel, we also seek to identify viral entry proteins that program efficient infections across multiple species ( , ) . our synthetic approach allows us to rapidly test hypothetical proteins-proteins whose expression has not yet been verified in the context of infection. genes of interest are then deleted or mutated using reverse genetic platforms, and virus mutant growth is examined both in primary human targets, such as airway epithelial cells and various immune cells, and during infection. although the four centers funded by the fg program have different specific goals, we share approaches and are experiencing common challenges, which we are working to solve together. shared solutions drive research forward. for example, each center includes a small rna (srna) discovery project. there is no generally agreed upon approach to elucidating srna gene function. it has been useful to compare experiences and potential solutions across centers. srna discovery projects as a group are converging on approaches that may work generally in bacteria. in addition, the data management groups at the four centers share common priorities for public dissemination of data and were able as a group to begin the process of defining new priorities for updating capabilities for the patric and vipr brc public resources. an example of this collaboration is the specification of the mode and format for transfer of transposon-sequencing (tn-seq) experimental data and biolog phenotyping data to patric, along with the computational tools to analyze these data ( ) . this effort will standardize the process of data transfer from independent centers and will define the format for public display of these and other data sets generated by fg centers. another interaction is occurring with the seattle structural genomics center for infectious diseases and the center for structural genomics of infectious diseases. using validated overexpression platforms and novel uncharacterized and/or hypothetical orfs, the collaboration is designed to determine the structures of high-priority candidate proteins that antagonize host antimicrobial defense pathways in the host. it is likely in the future that the data we generate will be used by the systems biology centers to further create more complex models of host-pathogen interactions. as described above, we have used a multipronged investigation strategy to directly evaluate unknown and hypothetical genes from a diverse array of pathogens to characterize the biological functions encoded by these genes (table ) . a particular strength of this approach is that this systematic workflow, which can be adapted to all pathogens with sequenced genomes, can ensure rational, directed, and rapid response times for vaccine and therapeutic design in answer to emerging and reemerging epidemics. the work of this program is defining a future blueprint to perform functional analysis of new pathogens as they emerge and to more rapidly respond to the need for knowledge of emerging organisms. design and synthesis of a minimal bacterial genome the combrex project: design, methodology, and initial results a cytoplasmic peptidoglycan amidase homologue controls mycobacterial cell wall synthesis structural asymmetry in a conserved signaling system that regulates division, replication, and virulence of an intracellular pathogen activity-based metabolomic profiling of enzymatic function: identification of rv c as a mycobacterial -hydroxy- -oxoadipate synthase kaposi's sarcoma-associated herpesvirus viral interferon regulatory factor interacts with a member of the interferon-stimulated gene pathway a sars-like cluster of circulating bat coronaviruses shows potential for human emergence sars-like wiv -cov poised for human emergence leaderless transcripts and small proteins are common features of the mycobacterial translational landscape resources for genetic and genomic analysis of emerging pathogen acinetobacter baumannii wrpa is an atypical flavodoxin family protein under regulatory control of the brucella abortus general stress response system brucella abortus ⌬rpoe confers protective immunity against wild type challenge in a mouse model of brucellosis modulation of the cgas-sting dna sensing pathway by gammaherpesviruses evasion of innate cytosolic dna sensing by a gammaherpesvirus facilitates establishment of latent infection middle east respiratory syndrome coronavirus ns b protein inhibits host rnase l activation transit-a software tool for himar tnseq analysis r.s.b. and b.d. were supported by nih grant ai , s.c. was supported by nih grant ai , s.i.m. was supported by nih grant ai , and e.j.r. was supported by nih grant ai . this work, including the efforts of ralph s. baric and blossom damania, was funded by hhs | nih | national institute of allergy and infectious diseases (niaid) (ai ). this work, including the efforts of sean crosson, was funded by hhs | nih | national institute of allergy and infectious diseases (niaid) (ai ). this work, including the efforts of samuel miller, was funded by hhs | nih | national institute of allergy and infectious diseases (niaid) (ai ). this work, including the efforts of eric j. rubin, was funded by hhs | nih | national institute of allergy and infectious diseases (niaid) (ai ). key: cord- - i x f authors: klotz, lynn c. title: danger of potential-pandemic-pathogen research enterprises date: - - journal: mbio doi: . /mbio. - sha: doc_id: cord_uid: i x f nan t he research goal in a number of laboratories is to make highly pathogenic avian influenza (hpai) viruses contagious to humans via respiratory aerosols. for instance, the h n influenza virus has been made contagious to ferrets ( ), the animal model often used as a proxy for humans. concern over escape from a laboratory of a deadly human-contagious virus (e.g., influenza, severe acute respiratory syndrome [sars] , and middle east respiratory syndrome [mers] viruses) prompted the u.s. government to hold back funding for this research "until a robust and broad deliberative process ( ) is completed that results in the adoption of a new usg gain-of-function research policy." this discussion is now under way in the united states and is to be completed in . in relation to this discussion, mbio published three letters to the editor in a debate between dr. ron fouchier and me. defending the safety of his work in the first letter ( ), dr. fouchier calculated that it would likely take more than a million years for an escape from his lab through a laboratory-acquired infection (lai). intuitively, this million-year claim seems dubious. i questioned the equation used in the fouchier calculation and the extremely low probability of escape that he employed in the calculation, and i outlined an alternative approach ( ). fouchier's response to my comments was then published in mbio ( ) . it is clear that he did not understand my methodology. in calculations of the probability of a community lai ("e"), dr. klotz further assumes that transmission studies in the erasmus mc facility will be performed for a period ("y") of million years. i am hopeful that our research enterprise will have reached solid conclusions on determinants of airborne transmission a bit sooner. rhetorical quip aside, neither his million-year result nor my questioning of it implies or assumes in any way that research must be performed for a given number of years. my questioning and my alternative approach were simply a comment on his approach. elsewhere in my comments ( ) , i assumed that the research enterprise will be concluded in years, as does he. in order to respond to fouchier's misunderstanding and to expand on the general usefulness of my approach, i provide here two simple equations for estimating both the likelihood of escape and the elapsed time to an escape. the equations may be employed for a single lab and for a "research enterprise" of many labs. the conclusion is that the likelihood of an escape may be uncomfortably high and that the elapsed time to an escape may be uncomfortably short. dr. fouchier uses the simplistic formula y ϭ /p to calculate the likely number of years elapsed, y, before an escape occurs, where p is the probability of escape from his lab in year. his -million-year calculation ( ) is misleading, as it does not account for the fact that research will proceed for more than year, and it was not expanded to calculate the number of years that elapse before an escape occurs for the many labs in the research enterprise. my approach, embodied in equation , may be used to calculate the probability, e, that at least one escape will occur for an n-lab research enterprise conducting research for y years (at least one is likely exactly one, as the probability that two or more escapes will occur is extremely small, and if an escape does occur, the whole n-lab research enterprise would almost certainly be shut down), and equation may be used to calculate the number of years that elapse, y, before an escape. solving equation for y gives derivations of equations and may be found in the appendix. in equation , probability e may be viewed as how much escape risk we are willing to tolerate, that is, the value of e that is too high a risk for an n-lab y-year research enterprise. is an e of . %, %, or % too high? the level of risk that we are willing to tolerate is subjective. since a lab escape may result in an uncontrollable disease outbreak with thousands to millions of deaths, even a % chance, e ϭ . , seems much too high. following fouchier's focus on elapsed years as a measure of biosafety, only elapsed years will be calculated here. the results, presented in table , are for a -lab research enterprise; is the number of nih-funded labs that have been identified as subject to the research pause. (originally labs were identified for the funding pause. that number was subsequently reduced to .) while some of these labs do not focus on developing mammalcontagious influenza viruses, there are likely many labs throughout the world not funded by the nih that are, so the number seems a reasonable guess for the size of the research enterprise. presented in table are the same calculations but for a single lab (n ϭ ), such as fouchier's lab. three quite different probabilities for escape, p , are employed in the calculations in the tables. the highest probability (p ϭ . ) is a minimum estimate calculated ( ) from cdc statistics ( ) for undetected or unreported lais in biosafety level (bsl ) labs. this probability is likely higher than that for lais in bsl labs that have extra biosafety precautions in place (called bsl ϩ), such as fouchier claims for his lab. the probability (p ϭ . ) is times lower and is an estimate for differences between bsl and bsl ϩ labs. the final probability (p ϭ ϫ Ϫ ) is fouchier's estimate ( ) . to make his estimate, fouchier itemizes the various safety measures in his lab and generously reduces the probability for each safety measure but admits that it is a guess: "the magnitude of this increase in safety is not known." finally, the discussion here of probabilities has been restricted to lais, but there are other routes of escape, such as mechanical failure and removal of live virus from bsl ϩ containment accidentally or for hostile purposes. from table , it is clear that the research enterprise is unsafe when an intermediate small p equal to . and a risk tolerance (e) of % are employed. the number of years that we would need to wait to exceed the % chance of an escape is only . years, well within the estimated years for the research to be completed. if p is equal to . , as calculated from the cdc statistics, there would be a % chance of an escape in less than a year (y ϭ . year). if p is really as low as fouchier suggests, we would need to wait years to reach a % chance of escape, an elapsed time that would appear to make the research enterprise safe in some researchers' thinking, but risk equals likelihood times consequences, and consequences such as fatalities could be very high for a human-contagious influenza virus with a high case fatality rate. this would lead to an intolerable number of fatalities even using fouchier's low p estimate. potential fatalities for the enterprise and the fatality burden for each lab in the enterprise were quantified for his very low p of ϫ - in my published criticism ( ) of fouchier's million-year calculation. the conclusion there was that each lab in the enterprise would carry the potential burden of over fatalities per year. "to put this fatality burden number in perspective, no institutional review board tasked with assessing human subject research would approve a proposed research project with potential fatalities per year." turning to the number of years that elapse before an escape occurs for a single lab in table , for a % chance of escape with the intermediate p , it would take years of research to exceed the % risk tolerance. for fouchier's low p , it would take , years to reach a % chance of an escape, making the research seem quite safe. i suspect that most researchers in the enterprise use this reasoning to justify the safety of their own lab, but this kind of thinking is flawed, as argued over years ago by the philosopher immanuel kant for his "categorical imperative," which is the cornerstone of his moral reasoning: "act only according to that maxim whereby you can at the same time will that it should become a universal law without contradiction." the "it" in the quote is labs in the research enterprise. at the risk of trivializing kant's complicated moral reasoning, a few examples should make the categorical imperative argument clearer. is it really acceptable for a manufacturing company to dump mercury in the ocean, because that one factory's output would not be enough to pose any danger to us when we consume fish? is there nothing wrong if i buy a gas guzzler that gets miles per gallon and has faulty emissions control, since my car's individual contribution to climate destruction is minimal? "what if everyone researched live smallpox?" has been implicitly answered according to kant's categorical imperative by everyone agreeing to limit research to two places. clearly, the magnitude of the basic probability (p ) is critically important in assessing the risk of the research enterprise, and its magnitude is a point of contention. finding a good estimate of this basic probability should be a major focus in gryphon scientific's risk-benefit assessment. fouchier has other criticisms of my comments that i feel should be addressed. his response is problematic in several ways. in addressing the problems, i will quote frequently from my comments and from his response to make sure that it is clear what was said. the biggest problem is that dr. fouchier does not once address my calculation of potential fatalities and fatality burden that employs his low probability of an undetected or unreported lai escaping from his laboratory. instead, he chooses to argue against my peripheral comments that his probability is likely much too low. his focus unfortunately draws attention away from my calculation that finds intolerable numbers of potential fatalities and the fatality burden. i number specific comments below to keep each point separate. the cdc's shipping of an h n virus-contaminated sample to the usda and similar incidents show the importance of not underestimating human error, especially if one considers the influenza lab at the cdc to be one of the top federal labs in the country. although biosecurity measures have improved greatly over the years, human nature has not. laboratory accidents will happen, and laboratory workers will get infected, not realize it or not admit it, and so take the infection home. the achilles' heel in fouchier's argument is that no number of safety procedures can provide for human error. while the history of escapes should make us worry that the probability may be very high, here the difference between fouchier and me is moot since i employ his low probability in my calculations. . dr. fouchier writes ( ) dr. klotz proposes to multiply the low likelihood of lais by , based on an estimated laboratories involved in the "whole research enterprise" for years, and assumes that part of this research enterprise may lack the rigorous safety practices in place at erasmus mc. both assumptions are wrong, to the best of my knowledge; just over a handful of laboratories have worked on airborne transmission of avian influenza viruses, each of which has rigorous safety practices in place. our disagreement here is because we define "research enterprise" differently. i defined it as research on potential pandemic pathogens with nih funding (influenza and sars category pathogens) and labs otherwise funded. ( ) another key aspect is that dr. klotz estimates the likelihood of onward transmission from a case of lai as . ( %), in contrast to my justification for an adjusted likelihood of Ͻ ϫ - , based on the specific conditions under which the research is performed, without providing a rationale for that important deviation. i certainly do provide a rationale for the % likelihood of lai ( ) through references and (risk assessment studies). summarizing the literature, lipsitch and inglesby estimate the probability that a community lai leads to a global spread (pandemic) to be to %. this range is consistent with the to % range found by merler and coworkers ( ) and with the to % range found in a focused risk assessment ( ) for infection spread beginning on crowded public transportation. furthermore, there is a rather arcane subject in probability, branching theory, which allows prediction of the likelihood of uncontrolled spread of any pathogen based on its observed reproductive number (r o ) value and the variance to mean of the r o . a large variance to mean could occur due to superspreaders, for instance, some people infected with sars virus. for a wide range of r o values, lipsitch and coworkers have calculated the probability of uncontrolled spread (see fig. a in their study [ ] ). for a single infected individual with an r o of , the probability of an uncontrolled outbreak ranges from % (spread of r o s) to % (uniform r o ). thus, the pandemic likelihood from a single infected individual is potentially large. i suspect that future risk assessments will confirm that once a highly contagious potentially pandemic pathogen escapes, the probability of an uncontrolled outbreak is significant, leading again to a focus on the probability of a laboratory escape as the important factor. fouchier mentions vaccination and antivirals ( ) as factors that reduce onward transmission. antivirals would not be prescribed for undetected lais. vaccines may reduce viral replication in the index case, but active virus may still be present when the infected person leaves the laboratory, potentially infecting unvaccinated persons. the annual flu vaccine is sometimes less than % effective, so it is unclear if vaccinated laboratory workers are protected by the laboratory vaccine strain. i would classify vaccination and antivirals, effective or not, as inside laboratory measures. but if an lai escapes, clearly these measures were not effective in preventing the undetected or unreported lai. again, we come back to the probability of escape from a laboratory as a key challenge in this debate. once an undetected or unreported lai from a highly contagious pathogen escapes, it is out of fouchier's control. its global spread will depend on the reproductive number, r o , and other factors external to fouchier's laboratory. fouchier claims ( ) that "the viruses are ferret adapted rather than human adapted," which could lead to a lower r o in humans. among the different mutated viruses presumably under development in his laboratory, some could be highly transmissible and deadly in humans. we will never know, for testing them on humans is, fortunately, unethical. of course if one escaped. . . . the argument of being ferret adapted and not human adapted is misleading. first, it cannot be proved. second, fouchier's own work may have already brought an avian h n virus far closer to successful replication in humans. if such a virus escaped from his laboratory, it may well adapt within the individuals in the early transmission chain and then take off in a big way. dr. fouchier and the field do not have the knowledge to know just how short of a successful virus they have engineered. that is why they are doing this work. . dr. fouchier concludes ( ) the following. finally, dr. klotz describes the (apocalyptic) scenario of an influenza pandemic with million fatalities based on a % casefatality rate in % of the world's population. these numbers not only ignore the scientifically justifiable counterarguments raised before ( ) but also are at odds with the documented influenza pandemics of the past. in my view, the "gain-of-function" debate has suffered from the apocalyptic scenarios that are provided as factual whereas they provide estimates that are far beyond the observed worst cases ( ) . it is estimated that the pandemic influenza virus infected % of the world's population. the h n "spanish" flu killed perhaps % of its victims. the h n avian influenza virus, the subject of fouchier's research, kills about % of those who are infected through direct contact with poultry. the scenario i use as an example represents a combination of these three real events. while this scenario has not yet and may never occur in nature, it is a possible scenario perhaps more likely from a laboratory escape. since the consequences of most scenarios, even one on a par with seasonal influenza-several hundred thousand deathswould be catastrophic and unacceptable, it behooves us to be exceedingly careful in deciding which potential pandemic pathogen research should be allowed. for much of this research, the potential risk far outweighs the potential benefits. to a lab escape. let p be the yearly probability of escape of a pathogen from a single lab. the first question to be asked is "what is the probability of at least one escape from one of the n labs conducting research on the pathogen for y years?" the probability of no escapes in y years for a single lab is ( Ϫ p ) y . for y years and n labs, the probability of no escapes is ( Ϫ p ) yn . the probability of at least one escape in y years from one of the n labs (e) is solving equation for y will allow this question to be answered. log( Ϫ e) ϭ log( Ϫ p ) yxn ϭ y ϫ n ϫ log( Ϫ p ), where y ϭ ( ⁄ n) ϫ [log( Ϫ e) ⁄ log( Ϫ p )] checking the limit for equation , if there is no likelihood of escape, p is equal to , log( ) is equal to , and as expected, y is equal to ϱ. another observation about equation is that the number of years of research, y, which must elapse before we reach our risk tolerance is inversely proportional to the number of labs, n. airborne transmission of influenza a/h n virus between ferrets government gain-of-function deliberative process and research funding pause on selected gain-of-function research involving influenza, mers, and sars viruses. u.s. department of health and human services studies on influenza virus transmission between ferrets: the public health risks revisited comments on fouchier's calculation of risk and elapsed time for escape of a laboratory-acquired infection from his laboratory comments on fouchier's calculation of risk and elapsed time for escape of a laboratory-acquired infection from his laboratory the consequences of a lab escape of a potential pandemic pathogen. front public health : monitoring select agent theft, loss and release reports in the united states- - categorical imperative, on wikipedia, the free encyclopedia transmission dynamics and control of severe acute respiratory syndrome acknowledgment i thank simon wain-hobson for comments and contributions to the text. key: cord- -upcf q authors: oudshoorn, diede; rijs, kevin; limpens, ronald w. a. l.; groen, kevin; koster, abraham j.; snijder, eric j.; kikkert, marjolein; bárcena, montserrat title: expression and cleavage of middle east respiratory syndrome coronavirus nsp - polyprotein induce the formation of double-membrane vesicles that mimic those associated with coronaviral rna replication date: - - journal: mbio doi: . /mbio. - sha: doc_id: cord_uid: upcf q betacoronaviruses, such as middle east respiratory syndrome coronavirus (mers-cov), are important pathogens causing potentially lethal infections in humans and animals. coronavirus rna synthesis is thought to be associated with replication organelles (ros) consisting of modified endoplasmic reticulum (er) membranes. these are transformed into double-membrane vesicles (dmvs) containing viral double-stranded rna and into other membranous elements such as convoluted membranes, together forming a reticulovesicular network. previous evidence suggested that the nonstructural proteins (nsp’s) , , and of the severe acute respiratory syndrome coronavirus (sars-cov), which contain transmembrane domains, would all be required for dmv formation. we have now expressed mers-cov replicase self-cleaving polyprotein fragments encompassing nsp - or nsp - , as well as coexpressed nsp and nsp of either mers-cov or sars-cov, to characterize the membrane structures induced. using electron tomography, we demonstrate that for both mers-cov and sars-cov coexpression of nsp and nsp is required and sufficient to induce dmvs. coexpression of mers-cov nsp and nsp either as individual proteins or as a self-cleaving nsp - precursor resulted in very similar dmvs, and in both setups we observed proliferation of zippered er that appeared to wrap into nascent dmvs. moreover, when inactivating nsp - polyprotein cleavage by mutagenesis, we established that cleavage of the nsp /nsp junction is essential for mers-cov dmv formation. addition of the third mers-cov transmembrane protein, nsp , did not noticeably affect dmv formation. these findings provide important insight into the biogenesis of coronavirus dmvs, establish strong similarities with other nidoviruses (specifically, the arteriviruses), and highlight possible general principles in viral dmv formation. in the first luminal loop of nsp ( , , ) . when both these sites were mutated in mhv nsp ( , ) , the virus was attenuated in cell culture and dmv formation was impaired, suggesting that nsp plays a critical role in coronaviral ro formation. the combined membrane-spanning regions of these proteins (i.e., including all luminal loops and flanking transmembrane domains) are commonly referred to as tm , tm , and tm , respectively. nsp , nsp , and nsp are nonconventional transmembrane proteins in the sense that they are derived from a polyprotein and do not contain n-terminal signal sequences for cotranslational membrane insertion. it is currently unknown how their membrane insertion is accomplished and whether polyprotein cleavage precedes (or is perhaps required for) translocation across the er membrane. to a certain extent, nsp , nsp , and nsp of the distantly related arteriviruses (also members of the order nidovirales) can be considered equivalent to coronavirus nsp , nsp , and nsp , in terms of their relative position in the replicase polyprotein and their membrane-spanning properties. for arteriviruses, expression of nsp and nsp alone was necessary and sufficient for the formation of double-membrane structures strikingly resembling the dmvs observed in infected cells ( ) . coexpression of nsp reduced the size of the induced dmvs but did not change their overall architecture ( ) . in the case of coronaviruses, it was recently reported that the transient coexpression of sars-cov nsp , nsp , and nsp led to the formation of dmvs ( ) . cells coexpressing ( ) , and nsp was detected with anti-v monoclonal antibody. (c) constructs expressing mers-cov nsp or nsp or a gfp control were transfected into t cells, which were metabolically labeled with [ s]methionine-cysteine from to h posttransfection. lysates were immunoprecipitated with the indicated antibodies, separated on an sds-page gel, and visualized using phosphorimaging. bands not corresponding to expected protein size in the western blot are indicated with asterisks. the~ -kda band in the nsp ip was also observed in the western blot. nsp bands in ip were fuzzy likely due to the relatively high hydrophobicity of the protein. (d) huh- cells were transfected with the indicated plasmids, and localization of mers-cov nsp and nsp was analyzed using immunofluorescence labeling and confocal microscopy at h posttransfection. nsp was detected with anti-sars-cov-nsp serum, and nsp was detected with anti-v monoclonal antibody. nsp and nsp alone contained so-called maze-like bodies (mlbs), consisting of paired er membranes (zippered er) and some circular profiles that were interpreted as cross sections of double-membrane tubules. therefore, it was concluded that nsp is essential for the biogenesis of sars-cov dmvs, whereas nsp and nsp can mediate the pairing of membranes that are likely an intermediate in dmv formation ( ) . in the current study, we examined the role of mers-cov nsp's in betacoronavirus ro biogenesis. using em and et, we found that mers-cov nsp and nsp , either coexpressed from separate plasmids or expressed as a self-cleaving polyprotein fragment (nsp - ), are essential and sufficient for the formation of dmvs that assemble into an rvn. addition of the third transmembrane subunit of the mers-cov replicase, nsp , did not alter the overall morphology of the induced dmvs. when nsp - polyprotein processing was prevented by mutagenesis, this blocked the formation of dmvs while membrane pairing did still occur, strongly suggesting that proteolytic processing coordinates dmv formation in time and/or space. to compare our results for mers-cov with the previous work on sars-cov ( ) , we used et to analyze the three-dimensional ( d) structure of the maze-like bodies induced upon coexpression of sars-cov nsp and nsp and were thus able to conclude that the circular profiles observed in that setting in fact correspond to dmvs rather than tubules. this established that, also in the case of sars-cov, coexpression of nsp and nsp suffices to induce dmv formation. together, our results provide important new insights regarding the biogenesis of coronavirus ros and demonstrate the conservation of certain principles underlying ro formation, both among the coronaviruses and in comparison to more distantly related members of the order nidovirales. mers-cov nsp and nsp colocalize in the perinuclear region of the cell. to study whether the transmembrane nsp's of mers-cov are able to induce dmv formation, we expressed nsp and nsp from a cag promoter ( ) either by cotransfection of cells with plasmids encoding individual proteins or by transfection with a single plasmid encoding a self-cleaving nsp - polyprotein fragment ( fig. a ; table s ). constructs were codon optimized for expression in human cells, potential splice sites were eliminated, and the encoded proteins were equipped with hemagglutinin (ha), myc, or v tags at their termini. the constructs were transfected into t cells to verify protein expression and processing (fig. b) . the wild-type nsp - polyprotein was fully cleaved into mature nsp and nsp , as was previously described ( ) . as a control, a mutant in which the nsp /nsp cleavage site was inactivated (g a/g a; ggϾaa) ( ) was included to generate the noncleaved precursor. interactions between nsp and nsp were previously shown to occur for mhv and sars-cov ( , ) , and we assessed whether this was also the case for the corresponding mers-cov proteins. to this end, t cells were transfected with a construct expressing ha-nsp -myc or nsp -v or cotransfected with both constructs. expression products were labeled metabolically with [ s]methionine and [ s]cysteine and subsequently immunoprecipitated with either ha-or v -specific antibodies (fig. c ). upon immunoprecipitation with the ha-specific antiserum, nsp -v was brought down when ha-nsp was present (left panel). conversely, when using the v -specific antibody, ha-nsp was coimmunoprecipitated when nsp -v was present (right panel). these findings demonstrated that these two mers-cov proteins interact and further supported the notion that this is a common feature of coronaviruses. when using immunofluorescence microscopy, separate expression of nsp or nsp in huh- cells yielded a reticular labeling pattern, with some more-intense foci in the perinuclear region of the cell, suggesting that-in the absence of the other-either protein localized at least partially to the er (fig. d ). this reticular pattern (but without the foci) has been described previously upon transient expression of mhv and sars-cov nsp ( , ) , whereas full-length sars-cov nsp was reported to localize to foci similar to those that we observed ( ) . when coexpressing mers-cov nsp and nsp or when expressing the self-cleaving nsp - polyprotein, the reticular pattern was much less pronounced and the two proteins mainly colocalized in foci in the perinuclear region (fig. d , lower panels). this was in agreement with the finding that mers-cov nsp and nsp interact and suggested that this interaction strongly promotes their recruitment to the foci in the perinuclear region. mers-cov nsp and nsp are required and sufficient to induce dmv formation. the next step was to determine whether nsp and nsp could induce the formation of double-membrane structures similar to those observed during infection. as a reference, mers-cov-infected huh- cells were analyzed by em. the membrane structures that were previously described in high-pressure frozen and freeze-substituted vero cells infected with mers-cov ( ) were readily apparent at h postinfection (p.i.) in chemically fixed huh- cells ( fig. a) . numerous dmvs were found (red asterisks), often adjacent to areas containing cm. the dmv interior appeared electron translucent, a difference from cryofixed samples ( ) that can likely be attributed to the different sample preparation method, as the contents of cov-induced dmvs are easily lost upon chemical fixation ( , , ) . occasionally some smaller circular profiles were observed that seemed similar in size to the spherules recently described for the gammacoronavirus infectious bronchitis virus (ibv) (red arrows) ( ) . none of these structures was found in mock-infected control samples (fig. b) . when huh- cells expressed either nsp or nsp , areas containing modified membranes were observed, which likely corresponded to the foci observed in fluorescence microscopy (fig. d) . in nsp -expressing cells (fig. c) , we detected large regions, usually several micrometers in diameter, of disordered membrane bodies (dmbs), which were similar to those previously observed after sars-cov nsp expression ( ) . the membrane structures clustering in these dmbs were reminiscent of the surrounding er cisternae, with which they were frequently connected, suggesting that dmbs consisted of clustered er-derived membranes. upon expression of mers-cov nsp , large clusters of modified single membranes (msm) were observed (fig. d ), but these structures seemed more irregular than those induced by nsp (fig. c) . the expression of sars-cov nsp did not result in changes in intracellular membrane morphology ( ) , in contrast with our present observations following mers-cov nsp expression. whether this reflects differences between the experimental setups used or an actual difference between these viral proteins remains to be determined. when mers-cov nsp and nsp were expressed in the same cell, either by cotransfection or by expression of the self-cleaving nsp - polyprotein, a remarkably different set of membrane structures was observed ( fig. e and f) . a combination of circular double-membrane profiles (red asterisks) and paired membranes (red arrows) was present in both cases, suggesting that the combined expression of mers-cov nsp and nsp is sufficient to induce dmv formation. there was no apparent difference between the structures resulting from coexpression of nsp and nsp and those resulting from expression of the self-cleaving nsp - polyprotein ( fig. e and f), but in both cases the circular profiles were significantly smaller than the ones observed in mers-cov-infected cells (average diameters of and nm, respectively, versus nm in infection) (see fig. s in the supplemental material). these membrane modifications were frequently found in all the samples analyzed. in order to further investigate how the frequency of em-positive cells compared to the transfection efficiency, a quantitative analysis was carried out on samples of cells expressing mers-cov nsp - . immunofluorescence microscopy showed that approximately % of the transfected cells were positive for expression of mers-cov nsp and nsp (n ϭ ), while around % of the cell sections (n ϭ ) contained doublemembrane structures. both dmvs and zippered er clustered together in all the em positive cell sections, although at slightly different ratios (see fig. s for a gallery). as the em analysis was based on one random section per cell (~ nm thick) that may not always capture the region with membrane modifications, it was not surprising that the fraction of positive cells observed in em was smaller than that observed in whole cells using light microscopy. the numbers above in fact strongly suggest that the formation of dmvs and zippered er is induced in most, if not all, cells expressing mers-cov nsp and nsp . while the circular profiles observed were suggestive of double-membrane vesicle formation, they could also correspond to cross sections of double-membrane tubular structures. to resolve this issue, we obtained d reconstructions of these membrane structures using et ( fig. ; movies s and s ), which confirmed that genuine dmvs were indeed formed upon expression of either nsp plus nsp or nsp - of mers-cov. the distinctive feature that unambiguously identifies a vesicle in a tomogram is a circular profile that is largest at the vesicle's equator and decreases in diameter when moving up or down from that plane through successive tomographic slices until, if the vesicle is fully contained in the section, it disappears. indeed, many profiles like this were observed in the tomograms (fig. a , red asterisks; movies s and s , green dots). we found no openings connecting the dmv interior and the cytosol, similar to what was observed previously upon tomographic analysis of coronavirus-infected cells ( , ) . the tomograms corroborated the structural similarity between the membrane structures induced by cotransfection with nsp and nsp constructs and by expression of the self-cleaving nsp - polyprotein. the electron density of the dmv interior seemed similar to that of the surrounding cytoplasm, and in this sense, it was different from that of dmvs in mers-cov-infected cells (compare to fig. a ), which is likely due to the absence of other viral proteins and double-stranded rna. in some cases, dmvs appeared to be contained in a larger double-membrane structure (fig. b , red asterisks). such structures have not been observed in coronavirus-infected cells. the paired membranes were often continuous with er cisternae (fig. b ) and resembled the so-called zippered er that has also been observed in ibv-infected cells ( ) , although they have not been documented so far for betacoronavirus-infected cells. these paired membranes may represent an intermediate of dmv biogenesis. further supporting this explanation, structures in which the zippered er seemed to transform into a nascent dmv could readily be observed in the tomograms (fig. b , red arrows). we also observed dmv-dmv, dmv-zippered er, and dmv-er connections (fig. c , red arrows), whereas completely isolated dmvs were in fact rare. in summary, while the described differences between nsp - -expressing and mers-cov-infected cells suggest that other viral components may modulate the process of dmv formation and would be required to form the full array of membrane structures observed during infection, our results establish that mers-cov nsp and nsp are sufficient to trigger all the membrane-remodeling steps required for inducing dmv formation, likely through the transformation of er membranes into an rvn consisting of dmvs and modified er. mers-cov nsp does not alter dmv morphology. the dmvs induced by expression of mers-cov nsp and nsp largely mimicked those observed during infection. overviews of reconstructed tomograms (available as movies s and s , respectively) for both conditions. some of the fully reconstructed closed dmvs are indicated with red asterisks. (b) zippered er curving into putative intermediates during dmv biogenesis (indicated with red arrows) is shown. two dmvs that are enclosed within other dmvs are indicated with red asterisks. (c) examples of connections between dmvs and (zippered) er (indicated with red arrows). all the images are virtual -nm-thick slices from the reconstructed tomograms. bars, nm. mers-cov nsp and nsp suffice to induce dmv formation ® however, the additional rvn elements that have been observed in this and previous studies of coronavirus-infected cells (cm and spherules) were not detected. to investigate whether nsp , the third transmembrane subunit of the coronavirus replicase, plays a role in their formation or affects dmv formation, we aimed to extend the expressed polyprotein fragment to include nsp and nsp . in addition to pl pro cleaving the nsp /nsp site, this should lead to processing of the nsp /nsp and nsp /nsp junctions by the nsp -based m pro , an assumption based on sequence conservation and studies performed with other coronaviruses ( ), as the kinetics of mers-cov polyprotein processing in cell-based assays have not been documented in any detail. remarkably, however, when the "regular" nsp - polyprotein was expressed, efficient processing of the nsp / site was achieved, but nsp's located downstream of this junction were retained in processing intermediates due to poor cleavage of the nsp / and nsp / junctions, as observed by immunoprecipitation (ip) analysis ( fig. a and b) . this prompted us to design a set of alternative polyprotein constructs to investigate and optimize the proteolytic autoprocessing of the nsp - region (see table s ). efficient cleavage at all sites was observed only for an engineered polyprotein (nsp - -gfp- ) in which green fluorescent protein (gfp) had been inserted between two copies of the nsp / cleavage site (fig. a ). immunoprecipitation analysis established that this nsp - -gfp- polyprotein was processed into four separate nsp's and gfp (fig. b) . consequently, this construct could be used to evaluate the effect of expressing nsp in addition to nsp and nsp . when huh- cells expressed the "regular" nsp - polyprotein, which was barely cleaved at the nsp / junction (fig. b) , we no longer detected the dmvs previously observed upon nsp - expression. instead, large areas of highly organized and curved membrane structures were seen (fig. c) , which were connected to surrounding er cisternae (fig. c, red arrows) . in contrast to the large single-membrane clusters observed in nsp -or nsp -expressing cells (fig. c and d) , they consisted of double membranes (fig. c , black arrow in the inset). the geometric pattern in these large areas containing double-membrane structures is typical of cubic membranes ( ) , which can result from overexpression and/or misfolding of er proteins, leading to protein and membrane aggregation. in contrast, when huh- cells expressed the engineered nsp - -gfp- polyprotein, which was almost fully processed (see above), cubic membranes were not observed and we found instead putative dmvs together with zippered er (fig. d) , structures very similar to the ones found in cells expressing just nsp and nsp (compare with fig. e and f) . also, the average size of these dmvs ( nm) was comparable to that of dmvs induced by nsp - expression ( nm) (fig. s ). circular profiles (putative dmvs) were detected in out of cell sections analyzed; however, none of these regions contained cm or spherules. this suggests that, while nsp and nsp are necessary and sufficient to induce the rearrangement of intracellular membranes into dmvs, the presence of (cleaved) nsp does not suffice to trigger the formation of the additional membrane structures typical of mers-cov infection. other viral components that are present during mers-cov replication, such as viral rna or other viral proteins, might thus be required for the formation of convoluted membranes and spherules. cleavage of the mers-cov nsp /nsp junction is essential for dmv formation. to gain more insight into the biogenesis of coronavirus dmvs, we set out to determine the role of the nsp /nsp cleavage event. we surmised that the membrane modifications induced by an uncleaved nsp - polyprotein could differ from those triggered by the (cleaved or coexpressed) nsp and nsp subunits. we transfected huh- cells with plasmids encoding nsp - carrying either a mutated nsp /nsp cleavage site (ggϾaa) or a catalytic site mutation in the nsp pl pro domain (c a) that inactivates the protease ( ) . in both cases, only the uncleaved nsp - precursor was observed (fig. a) . interestingly, dmvs were no longer found and instead we detected concentric structures consisting of zippered er that mostly lacked the pronounced curvature present in dmvs ( fig. b and c) . cotransfection of the cells with a plasmid encoding the active pl pro domain restored the nsp /nsp cleavage in the nsp - c a mutant polyprotein but not in the nsp - polyprotein with the mutated cleavage site (fig. a) . accordingly, expression of pl pro together with the nsp - cleavage site mutant (fig. d ) did not alter the structures observed. in contrast, when transcleavage of the nsp /nsp site, by coexpression of pl pro with the nsp - c a polyprotein, was achieved, dmv formation was at least partially restored and resulted in a mixture of abundant dmv and zippered er profiles (fig. e ), as observed before (fig. e and f) . expression of pl pro by itself did not have a membrane-remodeling effect. these results clearly showed that the nsp - precursor is able to induce the membrane pairing required to form zippered er but that cleavage of the nsp /nsp junction is essential for the formation of dmvs. sars-cov nsp and nsp are also sufficient to induce dmv formation. recently, angelini et al. reported that, in the case of sars-cov, nsp , nsp , and nsp are all required for the formation of dmvs when these proteins are transiently expressed as individual subunits ( ) . in their two-dimensional ( d) imaging study, coexpression of sars-cov nsp and nsp , in the absence of nsp , led to the formation of so-called maze-like bodies (mlbs), large clusters of double-membrane structures that were interpreted as closely packed double-membrane tubules, not vesicles. since our mers-cov tomography data (fig. ) established that dmv formation can be triggered just by coexpression of nsp and nsp , the interpretation of angelini et al. suggested that these subunits of mers-cov and sars-cov differ in their ability to induce dmv formation in the absence of nsp . to address this issue, we coexpressed nsp and nsp of either virus, using the same experimental setup previously used for sars-cov by angelini et al. ( t cells trans-fected using lipofection), and employed et for a comparative analysis in d. coexpression of sars-cov nsp and nsp led to the formation of mlbs very similar to those observed by angelini et al. ( ) , with areas of zippered er, often clustered as regularly spaced profiles, and circular double-membrane profiles ( fig. a and b) . the latter were postulated to be cross sections of double-walled tubules, of which the regularly spaced zippered er profiles would then represent longitudinal sections ( ) . the fact that the spacing between clustered zippered er profiles roughly coincided with the diameter of the circular profiles supported this interpretation; however, angelini et al. also acknowledged that et would be required for its validation. to determine whether the circular profiles in the mlbs represented tubular or vesicular structures, we now used et to analyze several mlbs, two of which are shown in fig. a and b. in one of those images, zippered er is the dominant structure ( fig. a; movie s ) , whereas the other mainly contained circular double-membrane profiles ( fig. b; movie s ) . in both tomograms, we could detect multiple double-membrane profiles that increase and decrease in diameter when progressing through the tomogram and ultimately disappear (marked with green dots in the tomogram movies), indicating that they represent vesicles rather than tubules. in fact, no tubular structures were observed in the tomograms. the presumed longitudinal views of tubular structures turned out to consist of zippered er winding through the mlb. for mers-cov, coexpression of nsp and nsp in t cells led to the formation of numerous circular double-membrane profiles together with some zippered er (fig. c and d) , which strongly resembled what we had observed in huh- cells previously. taken together, our et results make it clear that, in the case of sars-cov as well, coexpression of nsp and nsp suffices for the induction of dmv formation and strongly suggest that this is a common feature among betacoronaviruses. the generation of membranous organelles that support their replication machinery is a universal mechanism among positive stranded rna viruses infecting eukaryotes. the formation of these ros is induced by viral proteins ( , , ) , which are largely uncharacterized in most instances, and appears to be also reliant on host factors, some of which have been identified as important players ( , ) . in this study, focusing on betacoronaviruses, we sought to identify the viral proteins required to induce the formation of dmvs, the most prominent membrane structure formed during coronavirus infection. using et, we found that coexpression of nsp and nsp of either sars-cov or mers-cov was required and sufficient to trigger the formation of erderived dmvs. moreover, the d architecture of these membrane structures was similar to what has been observed during betacoronavirus infection ( ) . the dmvs formed upon coexpression of nsp and nsp were closed, with no detectable opening connecting the dmv interior and the surrounding cytosol, whereas their outer membrane generally was continuous with those of other dmvs and/or with (modified) er. our data importantly alter the conclusions of an earlier sars-cov study ( ) , which was based on the transient coexpression of sars-cov nsp and nsp from separate plasmids and the d imaging of the resulting membrane structures. the observation of maze-like bodies and circular double-membrane profiles, which were interpreted to represent tubular structures, led these authors to conclude that coexpression of sars-cov nsp and nsp was not sufficient for dmv formation. using et, we could now show that the circular profiles observed in these maze-like bodies are in fact dmvs (fig. a and b) , suggesting that the basic capability of nsp and nsp to induce dmv formation probably is a common feature of betacoronaviruses. these findings also highlight the importance of d analysis as a tool to ascertain and characterize the ultrastructure of membranous viral ros. interestingly, in the case of the arterivirus equine arteritis virus (eav), nsp and nsp mers-cov nsp and nsp suffice to induce dmv formation ® were found to be required and sufficient for dmv formation ( , ) . at least to a certain extent, these proteins can be considered the functional equivalents of coronavirus nsp and nsp , respectively, as they share a number of features like the presence of multiple membrane-spanning domains and a papain-like protease in the upstream protein that cleaves the junction between the two subunits. they also occupy comparable positions in the replicase polyproteins, suggesting that there may also be similarities in terms of the relative order in which these subunits are synthesized, released, and targeted to the membranes that they transform ( ) . these functional similarities and the potential to trigger dmv formation may thus be shared by these proteins of all coronaviruses and arteriviruses and possibly extend to other branches of the order nidovirales, like the poorly studied ronivirus and mesonivirus families. our findings also shed more light on dmv biogenesis, for which two models have been proposed that are not mutually exclusive. the first has been termed "double budding," where a vesicle would first bud into the er lumen and then bud out again to acquire a second membrane. the alternative model is based on "wrapping": membranes would first pair or "zipper" and then curve and finally form a closed dmv after a membrane fission event ( , ) . the frequent observation of zippered er after coexpression of nsp and nsp and multiple em images in which zippered er seemed to wrap into a dmv (e.g., fig. b ) suggest that this structure is a dmv precursor. interestingly, whereas the uncleaved nsp - precursor was able to induce the pairing of er membranes, dmv formation occurred only upon cleavage of the nsp /nsp junction, which strongly suggests that membrane pairing is an early step in dmv formation. our findings contrast with what was observed previously for arteriviruses, where cleavage of the nsp / junction was not required for the formation of dmvs in an expression system ( ) . together, our observations favor the wrapping model for dmv formation, and even though the existence in parallel of a double budding mechanism cannot be formally ruled out, the current data add to the mounting evidence pointing toward double-membrane wrapping as the central mechanism for dmv formation. for the distantly related arteriviruses ( , ) and the unrelated picornaviruses ( , ) , putative wrapping intermediates have been also described, indicating that this might be a common mechanism of dmv biogenesis among positive stranded rna viruses. several steps are required for dmv biogenesis: pairing of membranes, membrane curvature (both positive and negative), and fission ( , ) . in the wrapping model for dmv biogenesis, membrane pairing is an early step that may be mediated directly by interactions between the viral proteins inducing dmv formation. the interaction(s) between nsp and nsp that we described here for mers-cov may be sufficient to facilitate membrane pairing. similar observations have been made for sars-cov and mhv ( , , ) . the most likely candidate regions for this kind of interactions are the luminal loops of nsp and nsp ( , ) that are located in the tm and tm regions, respectively, as these could interact with their counterparts on the opposite side of the er cisterna, thus inducing membrane pairing. this view is partly supported by studies on mhv and sars-cov for which a truncated nsp lacking the region upstream of tm coexpressed with nsp was sufficient to induce membrane pairing but not the formation of dmvs ( , ) . this suggests that, although the cytosolic n-terminal region of nsp is required for complete dmv formation, the tm region (together with nsp ) may be sufficient to induce membrane pairing. in principle, the liberation (by pl pro -mediated cleavage of the nsp /nsp junction) and presumed membrane insertion of the hydrophobic n-terminal domain of nsp may be an important determinant of the ultimate transmembrane configuration of this protein, potentially with direct implications for the transformation of zippered er into dmvs. however, we found no major differences between dmvs induced by expression of self-cleaving mers-cov nsp - and those induced by coexpression of nsp and nsp , suggesting that nsp is properly inserted in the membrane when individually expressed. the concentric zippered er observed after expression of the uncleaved nsp - polyprotein could then reflect an intermediate stage in which the lack of nsp /nsp cleavage prevents proper membrane remodeling. the proximal (and largest) luminal loop of nsp contains an n-linked glycosylation site (n in mers-cov), similar to the glycosylation site(s) in sars-cov and mhv ( , , ) . analysis of the use of that site in proteolytically processed nsp and the uncleaved nsp - precursor could provide insight into the sequence of events leading to the membrane insertion of mers-cov nsp . although our data establish that expression of nsp and nsp suffices for coronaviral dmv formation, the precise role in this process-if any-of the nsp transmembrane subunit remains unclear. our mers-cov data suggest that, compared to cells expressing nsp and nsp only, coexpression of cleaved nsp does not affect dmv formation, nor does it lead to the formation of additional structures like cm or spherules. however, coexpression of sars-cov nsp , nsp , and nsp was previously reported to induce cm formation in addition to dmvs ( ) . additionally, expression of nsp alone resulted in the formation of small single-membrane vesicles near the microtubule organizing center ( ) , which suggested that nsp may have membrane proliferation and vesiculation abilities that could play a role in ro formation. when mers-cov nsp was retained in an unprocessed nsp - precursor, dmvs were no longer formed and membrane clusters that resemble cubic membranes appeared (fig. c ). it has been proposed that the cm formed by coronaviruses are in fact a form of cubic membranes ( ) . this might be related to observations that, compared to dmvs, cm are mostly formed relatively late in infection ( , , ) when viral proteins or polyprotein fragments accumulate. it is conceivable that such accumulation could lead to aggregation, misfolding, and/or impaired polyprotein processing resulting in the formation of cubic membranes. in other words, there could be a link between the status of polyprotein processing in the nsp - region and the membrane structures formed. along the same lines, the observation that blocking the cleavage at the nsp / junction impedes dmv formation (see above) directly implicates polyprotein processing in the control of membrane remodeling, possibly by facilitating conformational changes required for specific interactions with the membrane and/or between nsp and nsp . for an unrelated dmv-forming virus, hepatitis c virus (hcv), it was recently shown that dmv formation became less efficient when the proteolytic cleavage of the ns b/ a site in the viral polyprotein was accelerated, which similarly suggests a role for the polyprotein processing in dmv formation ( ) . the existence of different proteolytic processing intermediates containing nsp is well documented for arterivirus infection ( ) , although the role of the different precursors in dmv formation has not been studied in depth so far. unfortunately, the kinetics of polyprotein processing by m pro in mers-cov and sars-cov are still largely unknown. m pro 's enzymatic activity has mainly been assessed using recombinant nsp and peptide substrates in vitro ( , , , ) , but an analysis of the kinetics in a large (or larger) polyprotein setting is mostly lacking. most information on the processing of the coronavirus nsp -to-nsp region is derived from studies on other coronaviruses, such as mhv, ibv, and human coronavirus e (hcov- e) (the latter two being a gamma-and an alphacoronavirus, respectively) ( , , ) . an in-depth analysis of the kinetics of polyprotein maturation during coronavirus infection, the identification of nsp -containing processing intermediates, and the investigation of their possibly distinct roles in membrane remodeling could help to further unravel the mechanisms underlying the formation of the coronavirus ros. cells, viruses, and antibodies. huh- cells (kindly provided by ralf bartenschlager, heidelberg university) were grown in dulbecco's modified eagle's medium (dmem; lonza) supplemented with % (vol/vol) fetal calf serum (fcs; bodinco), mm l-glutamine (paa laboratories), and nonessential amino acids (paa laboratories). t cells (kindly provided by the virgin lab, washington university school of medicine in st. louis, mo) were cultured in dmem with % (vol/vol) fcs. all cell culture media contained u/ml penicillin and g/ml streptomycin. infection of huh- cells with mers-cov (emc/ strain kindly provided by ron fouchier, erasmus medical center, the netherlands [ , ] ) was performed as previously described ( ) . primary antibodies used were mouse anti-ha (clone ha.c ; abcam), mouse anti-␤-actin (clone ac- ; sigma), and mouse anti-v (clone f f ; thermo fisher). a rabbit serum recognizing sars-cov-nsp that cross-reacts with mers-cov nsp has been previously described ( , ) . a polyclonal rabbit serum was used against a combination of two mers-cov nsp peptides, sglvkmshpsgdveac (amino acids to of pp a) and cpadqlsdpnydalli (amino acids to ), which was produced by eurogentec. plasmid construction and transfection. human codon-optimized coding sequences of mers-cov nsp - were designed using geneart, ordered from thermo fisher in four fragments, and subsequently assembled in low-copy-number vector pacnr ( ) using conventional cloning. the precise parts of mers-cov pp a used for polyprotein constructs are outlined in table s in the supplemental material. the nsp construct included the c-terminal aa of nsp to prevent the n-terminal hydrophobic region of nsp from acting as a signal sequence, which could result in improper membrane insertion. in all constructs with a c-terminal myc or v tag, the c-terminal glutamine of the viral sequence was omitted to prevent the removal of the tag by m pro . the sars-cov nsp gene (frankfurt strain, pp a amino acids to ) was synthesized by bio basic inc. (ontario, canada). coding sequences were transferred to the pcaggs expression vector (addgene) for expression. pcaggs-sars-nsp was described previously ( ) . t cells were transfected using lipofectamine (thermo fisher) according to the manufacturer's instructions. huh- cells were transfected using a nucleofector b device (lonza) with nucleofector kit t (lonza) in ϫ cells and g of plasmid dna per transfection. cotransfections were carried out with equimolar amounts of plasmids. western blotting. cells were lysed in ϫ laemmli sample buffer ( mm tris-hcl, ph . , % [vol/vol] glycerol, % [wt/vol] sodium dodecyl sulfate [sds], mm dithiothreitol, . mg/ml bromophenol blue) and separated by electrophoresis on sds-polyacrylamide gels. proteins were transferred to polyvinylidene fluoride membranes (amersham) using a trans-blot turbo transfer system (bio-rad). blots were blocked with % (wt/vol) elk skimmed milk powder (campina) in phosphate-buffered saline (pbs) supplemented with . % (vol/vol) tween . secondary horseradish peroxidase (hrp)-conjugated antibodies (dako) and ecl plus western blotting substrate (thermo fisher) were used to visualize protein signal. immunofluorescence microscopy. after electroporation, huh- cells were seeded on coverslips and fixed h later with % (wt/vol) paraformaldehyde in pbs. samples were permeabilized with . % (vol/vol) triton x- and incubated with antibodies, including fluorescent conjugates, diluted in % (wt/vol) bovine serum albumin (bsa) in pbs. nuclei were stained with g/ml hoechst . after embedding with prolong gold (thermo fisher), samples were analyzed with a leica tcs sp confocal laser scanning microscope, which was equipped with a ϫ objective (numerical aperture [na] . ; airy unit) and a leica hyd hybrid detector. and incubated with antibody overnight. antibody-protein complexes were then pulled down using protein a and protein g sepharose beads (ge healthcare), which were first blocked with % (wt/vol) bsa in pbs, and incubated for several hours. after repeated washing of the beads with ip buffer, proteins were eluted by heating in ϫ laemmli sample buffer. after separation on large % polyacrylamide gels and gel drying, signal was visualized using an imaging screen-k (bio-rad) and a typhoon scanner (ge healthcare). electron microscopy. transfected huh- or t cells were fixed h posttransfection in . % (wt/vol) glutaraldehyde in . m cacodylate buffer (ph . ) for h at room temperature. after washing in . m cacodylate buffer, samples were postfixed and stained at °c with % (wt/vol) osmium tetroxide in . m cacodylate buffer for h. after washing with . m cacodylate and milli-q water, cells were scraped and stained with % (wt/vol) tannic acid in milli-q water on a d rotator for h at room temperature. following washing with milli-q water, cells were spun down in heated % (wt/vol) agar in pbs, and after solidification, pellets were excised, cut into small blocks, and dehydrated in increasing concentrations of ethanol. samples were embedded in epoxy resin (lx- ; ladd research), and after polymerization, -nm sections were placed on mesh- copper em grids covered with a carboncoated pioloform layer. following poststaining with % (wt/vol) uranyl acetate and reynolds lead citrate, samples were analyzed on an fei tecnai biotwin microscope equipped with an eagle cooled slow-scan charge-coupled device (ccd) camera (fei) and operated at kv. measurements of circular profiles from d em images were done with imagej software and aperio imagescope software (leica). circular profiles were measured over their longest and shortest axes, and the geometric mean of those values was used as the diameter. one hundred circular profiles were measured for each condition. electron tomography. sections of -nm thickness were cut from the resin-embedded blocks of transfected huh- or t cells prepared as described above. prior to poststaining, colloidal gold particles of nm were applied to both sides of the em grid to serve later as fiducial markers for alignment. tomography data were recorded on an eagle ccd camera (fei) in an fei tecnai biotwin (huh- samples) or a twin ( t samples) electron microscope operated at kv, with the grids mounted on a fischione tomography holder. dual-axis tilt series of the regions of interest were collected using xplore d software (fei) at magnifications that resulted in a pixel size of . nm (biotwin data) or . nm (twin data). the angular coverage for each single-axis tilt series was °sampled in identification of a novel coronavirus in patients with severe acute respiratory syndrome a novel coronavirus associated with severe acute respiratory syndrome genomic characterization of a newly discovered coronavirus associated with acute respiratory distress syndrome in humans isolation of a novel coronavirus from a man with pneumonia in saudi arabia coronaviruses post-sars: update on replication and pathogenesis sars and mers: recent insights into emerging coronaviruses nidovirales: 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(ϩ)rna viruses rewire cellular pathways to build replication organelles the dependence of viral rna replication on co-opted host factors open reading frame a-encoded subunits of the arterivirus replicase induce endoplasmic reticulum-derived double-membrane vesicles which carry the viral replication complex formation of the arterivirus replication/ transcription complex: a key role for nonstructural protein in the remodeling of intracellular membranes antiviral innate immune response interferes with the formation of replication-associated membrane structures induced by a positive-strand rna virus two-amino acids change in the nsp of sars coronavirus abolishes viral replication do viruses subvert cholesterol homeostasis to induce host cubic membranes? ns a domain and polyprotein cleavage kinetics are critical for induction of double-membrane vesicles associated with hepatitis c virus replication alternative proteolytic processing of the arterivirus replicase orf a polyprotein: evidence that nsp acts as a cofactor for the nsp serine protease mouse hepatitis virus c-like protease cleaves a -kilodalton protein from the open reading frame a polyprotein in virus-infected cells and in vitro processing of the human coronavirus e replicase polyproteins by the virus-encoded c-like proteinase: identification of proteolytic products and cleavage sites common to pp a and pp ab nucleotide sequence of classical swine fever virus strain alfort/ and transcription of infectious rna from stably cloned full-length cdna computer visualization of three-dimensional image data using imod key: cord- - ux xg authors: josset, laurence; menachery, vineet d.; gralinski, lisa e.; agnihothram, sudhakar; sova, pavel; carter, victoria s.; yount, boyd l.; graham, rachel l.; baric, ralph s.; katze, michael g. title: cell host response to infection with novel human coronavirus emc predicts potential antivirals and important differences with sars coronavirus date: - - journal: mbio doi: . /mbio. - sha: doc_id: cord_uid: ux xg a novel human coronavirus (hcov-emc) was recently identified in the middle east as the causative agent of a severe acute respiratory syndrome (sars) resembling the illness caused by sars coronavirus (sars-cov). although derived from the cov family, the two viruses are genetically distinct and do not use the same receptor. here, we investigated whether hcov-emc and sars-cov induce similar or distinct host responses after infection of a human lung epithelial cell line. hcov-emc was able to replicate as efficiently as sars-cov in calu- cells and similarly induced minimal transcriptomic changes before h postinfection. later in infection, hcov-emc induced a massive dysregulation of the host transcriptome, to a much greater extent than sars-cov. both viruses induced a similar activation of pattern recognition receptors and the interleukin (il- ) pathway, but hcov-emc specifically down-regulated the expression of several genes within the antigen presentation pathway, including both type i and ii major histocompatibility complex (mhc) genes. this could have an important impact on the ability of the host to mount an adaptive host response. a unique set of genes was dysregulated early and permanently throughout infection with hcov-emc, and was used in a computational screen to predict potential antiviral compounds, including kinase inhibitors and glucocorticoids. overall, hcov-emc and sars-cov elicit distinct host gene expression responses, which might impact in vivo pathogenesis and could orient therapeutic strategies against that emergent virus. partially explaining the large host range of hcov-emc. this was somewhat surprising, as coronaviruses generally show strict host specificity. while recent identification of the crystal structure of hcov-emc protease suggests that a wide-spectrum cov protease inhibitor could block the catalytic site ( ) , there is currently no proven antiviral treatment for hcov-emc. viruses rely on host factors to replicate and often hijack cellular processes initiated in response to infection to ensure efficient replication ( ) . targeting cellular responses has been shown to inhibit viral replication ( , ) . furthermore, immunomodulatory drugs that reduce the excessive host inflammatory response to respiratory viruses, as seen with influenza virus infections, have therapeutic benefit (reviewed in reference ). several genome-based drug repurposing strategies successfully identified known drugs that could be reused to treat lung cancer, inflammatory bowel disease ( ) , and influenza virus infection ( ) . such an approach has the advantage of accelerating treatment availability, which could be crucial in case of an outbreak of an emerging pathogen. overall, differences in viral sequences, host cell receptor, and host range indicate that hcov-emc and sars-cov may have distinct strategies for interacting with their hosts. this fact could impact treatment strategies. to begin to assess this question, we compared the host response of human cells to hcov-emc and sars-cov infection using global transcriptomic profiling. our goal was to gain a rapid and comprehensive assessment of the host response to hcov-emc infection that could guide research on this emerging virus. importantly, we used this information to computationally predict antiviral treatment and identified a broad down-regulation of the antigen presentation pathway that may be important in vivo for the development of an adaptive immune response. to characterize hcov-emc, we sought a cell line that mimics the human airway. calu- cells, derived from epithelial cells lining the human conducting airway, can be differentiated into polarized ciliated cells and permit robust replication of several respiratory viruses, including sars-cov, influenza a virus, and respiratory syncytial virus (rsv) ( ) ( ) ( ) ( ) . therefore, calu- cells were infected with hcov-emc using a multiplicity of infection (moi) of , and results were compared with those for calu- cells infected with sars-cov urbani at the same moi. the viruses replicated to similar levels, with both peaking at Ͼ pfu at h postinfection (hpi) (fig. ) . while sars-cov has been previously shown to maintain steady replication and cell viability to hpi ( ) , hcov-emc induced substantial cytopathic effect at to hpi, with significant cell rounding and detaching. together, these data suggest that although hcov-emc and sars-cov exhibit similar replication kinetics, they elicit different host responses in lung epithelial cells. hcov-emc induces earlier and different transcriptional changes than sars-cov. to assess the global host transcriptional response following infection with hcov-emc, samples of infected calu- cells were collected throughout a -h time course postinfection. genes that were differentially expressed (de) compared to time-matched mock-infected controls were determined using the statistical cutoff of a q value of Ͻ . and an absolute log (fc) of Ͼ . these genes were compared to de genes from calu- cells infected with sars-cov across a -h time course using the same statistical cutoff (fig. ) . as previously observed ( ) , sars-cov is able to replicate actively with a surprisingly small number of significant transcriptional changes before hpi (from de genes at hpi to genes at hpi) ( fig. a) . similarly, hcov-emc induced fewer transcriptional changes before hpi than after. however, hcov-emc replication at early times postinfection induced more changes than sars-cov, with the number of de genes ranging from at hpi to genes at hpi ( fig. a) . at later times postinfection, a massive host response was observed during hcov-emc infection, with , de genes at hpi and , genes at hpi, while sars-cov induced changes of only genes at hpi with maximum changes at and hpi of , and , genes, respectively. to evaluate the similarity of transcriptional dysregulation between hcov-emc and sars-cov, we determined the percentage of overlap among de genes changing in the same direction at each time point (fig. b) . the intersection between up-or downregulated de genes for each condition was calculated separately and then averaged to determine the percentage of intersecting genes (fig. b) . the overlap between signatures at late times postinfection for a single virus was very high; for example, % of the genes de by hcov-emc at hpi were also de at hpi, and on average % of the de genes at hpi with sars-cov were also de at later times postinfection. however, the overlap between sars-cov and hcov-emc at the same time point was low; for example, only % of the de genes in response to hcov-emc at hpi were also de by sars-cov at this time point. of note, the intersection of de genes between the two infections was higher when later times postinfection for sars-cov were compared with earlier time points for hcov-emc. on average % of the fig hcov-emc replicates at a level similar to that of sars-cov in human epithelial cells. triplicate wells of calu- b cells were infected with hcov-emc (moi, ). medium from each well was collected and analyzed by plaque assay for viral growth kinetics in veroe cells. the corresponding cells were harvested for transcriptomic analysis. sars-cov titers after infection of calu- b cells at an moi of were determined using the same method ( ) . the error bars represent the standard deviations among triplicate cell samples. josset et al. hcov-emc signatures from to hpi overlapped the signatures of sars-cov from to hpi. together, these results indicate that hcov-emc induced both a more robust and a largely different host response compared to that induced by sars-cov at similar times postinfection. however, changes induced from to hpi after hcov-emc were more similar to changes induced late after sars-cov infection ( to hpi). hcov-emc massively dysregulates the host transcriptome at late times postinfection. at late times postinfection, hcov-emc induced drastic changes in the host transcriptome with , de genes at hpi and/or hpi. to characterize this late signature, we compared the log fold change (log fc) expression values of these , genes after infection with sars-cov or hcov-emc ( fig. and www.systemsvirology.org). there were , genes ( %) expressed similarly to those in sars-covinfected samples at the same or later times postinfection and , genes ( %) expressed differently than during sars-cov infection. these genes were further clustered according to their expression pattern in hcov-emc infected cells (clusters i and iii contain genes up-regulated after hcov-emc infection at to hpi; clusters ii and iv contain down-regulated genes). enrichment in canonical pathways for each of this cluster was performed and is shown fig. b . genes that were up-regulated by both hcov-emc and sars-cov (cluster i), though later by sars-cov than by hcov-emc, were primarily related to viral recognition and the activation of innate immune pathways: the il- -related pathway and activation of interferon regulatory factor (irf) by cytosolic pattern recognition receptors. genes down-regulated by both viruses (cluster ii) were related to metabolism. genes specifically up-regulated by hcov-emc were statistically enriched in only pathways: camp-mediated signaling and protein ubiquitination. because ubiquitination is involved in the innate immune antiviral response ( ) , this could represent an interesting mechanism to explore further. finally, genes specifically down-regulated after hcov-emc infection were largely related to the antigen presentation pathway. in addition, the other identified canonical pathways are strongly related to lymphocyte signaling processes. together, these data suggest that hcov-emc may quickly move to interfere with elements of the adaptive immune response, in contrast to sars-cov. the antigen presentation pathway is broadly downregulated after hcov-emc infection. as down-regulation of the antigen presentation pathway may have important implications for the development of the adaptive response, we more closely examined changes in this pathway after infection with hcov-emc (fig. ) . twenty-two genes related to major histocompatibility complex (mhc) class i, mhc class ii, or antigen presentation were found to be down-regulated after hcov-emc infection (fig. a) , while in contrast, the vast majority of these genes were up-regulated by sars-cov infection after hpi (fig. b ). interestingly, transcriptional regulators of mhc class i (nlrc ) and mhc class ii (ciita) were transiently up-regulated early after hcov-emc infection (at and hpi, respectively). other genes were down-regulated starting at hpi. importantly, both mhc class i genes (hla-a, -b, -c, -e, and -g) and class ii genes (hla-dmb, -dpa , -dpb , -dqa , -dra, -drb , -drb , -drb , and -drb ) had decreased expression values after infection. several genes from the mhc i pathway, including proteasome genes (psmb and psmb ), genes from the peptide-loading complex (pdia and tapbp), and b m, were down-regulated, as well as the gene for the invariant chain (cd ), which belongs to the mhc ii pathway. down-regulation of genes within mhc i and ii pathways was confirmed by quantitative reverse transcription-pcr (rt-pcr), with all genes tested having significantly decreased expression in hcov-emc samples (fig. c) . together, the data show that hcov-emc and sars-cov induce opposite regulation of the entire antigen presentation pathway. early and sustained transcriptional changes may be reverted by kinase inhibitors and glucocorticoids. with the goal of identifying possible drugs that will modulate the host response throughout infection and from early times postinfection, we focused on characterizing the early transcriptional changes induced by hcov-emc that remained stable throughout the infection. among the genes de early after infection (at , , , and/or hpi), ( %) remained dysregulated, with the same pattern at later times postinfection. we chose to focus on this -gene signature to avoid transient events that were specific to one time postinfection and to exclude genes with inconsistent expression patterns across the course of infection. expression values for these genes are displayed in fig. a and are available at www.systems virology.org. this early signature was enriched in genes related to the inflammatory response with il- , tumor necrosis factor (tnfr )-related pathways, and predicted activation of chemotaxis of leukocytes (see table s in the supplemental material). none of the genes were de early after sars-cov infection; however, expression of genes ( . %) was changed after hpi. this subset of genes was enriched in cell viability molecules, glucocorticoid receptor signaling, and il- -related pathways. in an effort to identify potential drugs that could block the host response to hcov-emc, we used the early -gene signature and ipa (ingenuity pathway analysis) upstream regulator analysis (see materials and methods) to identify the upstream regulators that may be responsible for gene expression changes observed early after infection. upstream regulators are defined as any molecule that can affect the expression of another molecule, including cules that are known to induce the activation of inflammatory genes that were also found up-regulated in the early signature. the top predicted inhibited regulators included four kinase inhibitors (sb , ly , u , and sp ) and one corticosteroid (triamcinolone acetonide). these molecules are known to down-regulate the same genes that were up-regulated early and across hcov-emc infection. in addition, several kinase inhibitors (including sb , ly , and u ) and a glucocorticoid (dexamethasone) were also predicted to be negative regulators of genes changed similarly after sars-cov and hcov-emc infection at late times postinfection (see table s in the supplemental material). therefore, we hypothesize that treating cells with these drugs might partially revert cov signatures and inhibit deleterious host responses and/or viral replication in the case of both sars-cov and hcov-emc. in silico and in vitro evidence for kinase inhibitors as potential anti-cov. to test these hypotheses, we first analyzed whether cells treated with identified kinase inhibitors have gene expression profiles opposite to the one induced after viral infection. to this end, we used connectivity map (cmap), which is a database of more than , drug transcriptional signatures in several cell lines ( ) . this data-driven tool allows the identification of molecules that induce similar or opposite transcriptional changes relative to the query signature, based on their connectivity scores. the connectivity score is a value between ϩ and Ϫ , where a high positive score indicates that the drug induces changes similar to those induced by viral infection, while a high negative score indicates that the drug reverses the expression of the hcov-emc signature. cmap includes transcriptional profiles of several kinase inhibitors, including two compounds that were predicted to be potential negative regulators of viral response in ipa upstream regulator analysis (sb and ly ). sb had a connectivity score of Ϫ . in mcf cells (data set s in the supplemental material), and ly had negative scores in of the cell lines tested in cmap (Ϫ . in hl , Ϫ . in mcf , Ϫ . in pc , and Ϫ . in skmel ), indicating that these two drugs reversed the expression profile of hcov-emc signature. other related kinase inhibitors present in cmap are u (a derivative of u ) and sb , which both had negative scores in pc cells (Ϫ . and Ϫ . , respectively). to further validate the potential effectiveness of identified kinase inhibitors, we evaluated the ability of the top predicted negative upstream regulator, sb , to interfere with viral replication (fig. c) . importantly, this kinase inhibitor was predicted to regulate genes that were de similarly by sars-cov and hcov-emc at late times postinfection (see table s in the supplemental material) and could therefore inhibit both viruses' replication. treating cells with m sb following sars-cov infection resulted in a significant decrease of % in the log viral titer at hpi (p Ͻ . ); these replication levels are similar to those in interferon (ifn)-treated cells. sb was less effective against emc-cov, showing no efficacy with posttreatment; however, pretreatment of infection resulted in significant decreases of % and % of the log viral titer, at and hpi, respectively (p Ͻ . ). efficacy against sars-cov can be explained by the fact that all genes that were de after sars-cov infection were predicted to be regulated by kinase inhibitors (cluster i and ii genes [ fig. ] and additional genes specific to sars-cov [data not shown]), while the , genes specific to hcov-emc (clusters iii and iv [ fig. ]) did not include any kinase inhibitor in their upstream negative regulators (see table s ). in addition, host response dynamics to sars-cov and hcov-emc were different, with a delayed and more limited response to sars-cov allowing sb posttreatment to be effective. in contrast, emc-cov rapidly induced host expression changes and thus required sb pretreatment for measurable effect. as genes specific to hcov-emc were predicted to be negatively regulated by other classes of molecules (like gemfibrozil, which targets ppar␣; z ϭ . ), it will be important to determine whether using such molecules in combination with kinase inhibitors like sb could provide a better inhibition of viral replication. together, these data demonstrate the efficacy of sb against two unique covs and validate in silico approaches to predict effective drug treatment for emergent viruses. hcov-emc was isolated from a patient who died from an acute respiratory disease similar to that caused by sars-cov. however, there are several indicators that the host responses to these two viruses may be significantly different. several cases of hcov-emc infection have resulted in renal failure, which has rarely been observed in sars-cov infection. in addition, sars-cov and hcov-emc do not use the same cell receptor, and there are important differences in their genomic sequences. this study adds strength to the assertion that "hcov-emc is not the same as sars-cov" ( ) . indeed, even though we identified specific characteristics of the sars-cov response in the hcov-emc signatures, hcov-emc induced robust and specific transcriptional responses that were distinct from those induced by sars-cov, including the broad down-regulation of mhc molecules. this study is the first global transcriptomic analysis of the cellular response to hcov-emc infection. kindler et al. performed rna-seq on human airway epithelium (hae) cells infected with hcov-emc ( ). however, their analysis was focused on viral sequences and did not include a genome-wide analysis of the host response. they did, however, use rt-qpcr (quantitative pcr) to compare expression levels of a set of genes, including ifn, rna sensor molecules, and ifn-stimulated genes (isgs), following infection with hcov-emc, sars-cov, or hcov- e (moi . ). in our study, we confirm that sars-cov and hcov-emc induce a similar up-regulation of rna sensor molecules, such as rigi, mda , and two of three genes of isgf (irf and stat ) (genes in cluster i [ fig. ] ). of note, hcov-emc titers were up to fold higher than those of sars-cov in hae cells ( ) activation of similar innate viral-sensing pathways by hcov-emc and sars-cov is not surprising given the conservation of this mechanism to detect foreign rna and familial relationships of the viruses. we also found that both viruses induced proinflammatory cytokines related to il- pathways. it has previously been shown that il- a-related gene expression exacerbates severe respiratory syncytial virus (rsv) or influenza virus infection ( , ) . il- a was predicted to be activated throughout infection with hcov-emc and may induce immune-mediated pathology that possibly contributes to a high mortality rate. il- a is known to be produced by t-helper cells, but its expression in calu cells was increased up to -fold at hpi after hcov-emc infection. interestingly, il- c and il- f, which can be produced by epithelial cells under certain inflammatory conditions and which activate pathways similar to il- a-mediated responses ( ) , were increased earlier and to a greater extent following hcov-emc infection (up to -fold at hpi for il- c and -fold at hpi for il- f). therefore, further study of the il- response may provide interesting targets to limit lung injury ( ) . a main difference between responses to hcov-emc and sars-cov was the specific down-regulation of the antigen presentation pathway after hcov-emc infection. in contrast, these genes were found to be up-regulated after sars-cov infection. several viruses have evolved mechanisms to inhibit both the mhc class i (reviewed in references and ) and class ii (reviewed in reference ) pathways. while expression of mhc class ii is usually limited to professional antigen-presenting cells, human lung epithelial cells constitutively express this complex ( ) . our data demonstrated down-regulation of the mhc class ii transactivator (ciita) after hcov-emc infection, a finding that possibly explains decreases in mhc class ii molecule expression; this is a common viral strategy used to block that pathway ( ) . mhc class ii inhibition can prevent class ii-mediated presentation of endogenous viral antigens produced within infected cells and impair the adaptive immune response. similarly, mhc class i genes were also down-regulated after hcov-emc infection; decreasing expression of mhc class i can attenuate cd t-cell-mediated recognition of infected cells and could allow immune evasion by hcov-emc. finally, psmb and psmb , parts of the immunoproteasome, were also down-regulated by hcov-emc; these components replace portions of the standard proteasome and enhance production of mhc class i binding peptides ( ) . in their absence, proteins targeted for degradation may not generate peptides that robustly bind mhc class i, thus limiting their presentation. down-regulation of psmb and psmb could counteract the host response to viral infection, including up-regulation of ubiquitins and ubiquitin ligases observed during hcov-emc infection (fig. b ) that may ineffectively target viral protein for degradation. together, the inhibition of mhc class i and ii as well as immunoproteasome construction may have an important impact on the in vivo adaptive immune response against hcov-emc. while there is no proven effective antiviral therapy against sars-cov ( ), several molecules have in vitro antiviral activity, including ribavirin, lopinavir, and type i ifn, but their benefits for patients are unclear ( ) . ifn-␣ pretreatment of cells has been shown to inhibit hcov-emc replication ( ) , but no direct antiviral therapies have been reported. targeting host factors impor-tant for the virus, instead of the virus itself, has been investigated for hiv ( ) and influenza virus ( ) . for example, inhibiting upstream regulators (such as nf-b) that control the host response to influenza virus infection has been shown to reduce virus replication in vitro and in mice ( ) . inhibition of immunophilins that interact with the viral nonstructural protein (nsp ) resulted in potent inhibition of sars-cov replication ( , ) . in this study, we characterized upstream regulators predicted to be activated (e.g., nf-b and il- , which could be targeted with specific inhibitors) and upstream regulators predicted to be inhibited. the top five inhibited regulators included one glucocorticoid and four kinase inhibitors; these drugs may be able to directly block part of the host response and impact viral replication/pathogenesis. among them, ly , a potent inhibitor of phosphatidylinositol kinase (pi k), has known antiviral activity, inhibiting the replication of influenza virus ( ) , vaccinia virus ( ) , and hcmv ( ) . sb , an inhibitor of p mapk, is also an effective antiviral against the encephalomyocarditis virus ( ), rsv ( ) , and hiv ( ) . ly and sb were also identified in connectivity map, a database of drug-associated gene expression profiles ( ) , as molecules reversing components of the hcov-emc gene expression signature. finally, sb showed promising antiviral results against both hcov-emc and sars-cov in our in vitro assay (fig. c) . further extensive studies, including dose-response tests and tests of other kinases inhibitors, are ongoing. nonetheless, these results validate our genomebased drug prediction, which allows rapid identification of effective antivirals. despite central roles of pi k and mapk pathways in regulating multiple cellular processes, many kinase inhibitors targeting these pathways have been shown to be safe and well tolerated in vivo (reviewed in references and ). it has been hypothesized that mitogenic mapk and survival pi k/akt pathways may be of major importance only during early development of an organism and may be dispensable in adult tissues ( ) . several drugs targeting jnk, pi k, and mek have shown promising therapeutic potential in humans against a variety of diseases, including cancer and inflammatory disorder ( , ) . p mapk inhibitors have also been evaluated in humans, but the first generation of molecules, including sb , has a high in vivo toxicity (liver and/or central nervous system). however, development of novel nontoxic inhibitors (e.g., ml ) ( ), more selective molecules (e.g., as ) ( ) , and administration via inhalation ( ) are promising strategies for use of this class of inhibitor for treatment of pulmonary disease. overall, these results indicate that kinase inhibitors could be used as broad anti-cov agents which might be combined with other host-targeting molecules, like peroxisome proliferator-activated receptor ␣ (ppar␣) agonists, to better inhibit hcov-emc replication. in conclusion, using global gene expression profiling, we have shown that hcov-emc induces a dramatic host transcriptional response, most of which does not overlap the response induced by sars-cov. this study highlights the advantages of highthroughput "-omics" to globally and efficiently characterize emerging pathogens. the robust host gene expression analysis of hcov-emc infection provides a plethora of data to mine for further hypotheses and understanding. host response profiles can also be used to quickly identify possible treatment strategies, and we anticipate that host transcriptional profiling will become a gen-eral strategy for the rapid characterization of future emerging viruses. calu- b cells, a clonal population of calu- cells sorted for ace expression, were grown in minimum essential media (mem; gibco) supplemented with % fetal bovine serum (hyclone) and % antibiotic antimycotic (gibco). viral titrations and propagation hcov-emc were performed in veroe cells using standard methods. human coronavirus emc (hcov-emc) was received from bart l. haagmans (erasmus medical center, rotterdam, netherlands) via mta. experiments with sars-cov were previously performed under the same conditions using the same cells ( ) . calu- cell infections. all work was performed in a biosafety level (bsl ) facility supported by redundant exhaust fans, and personnel protective equipment was worn, including tyvek suits, hoods, and hepafiltered powered air-purifying respirators as previously described ( ) . cells were washed with phosphate-buffered saline (pbs) and inoculated with virus at an moi of or mock diluted in pbs for min at °c. following inoculation, cells were washed times, and fresh medium was added to signify time zero. triplicate samples of mock-infected and virusinfected calu- b cells were harvested between and hpi. rna isolation and microarray processing. rna isolation from calu- b cells, subsequent hybridization to agilent ϫ k human hg arrays, and processing of raw data were performed as previously described ( ) . we analyzed triplicates for each time postinfection except for hpi, for which one replicate did not pass rna quality control and was therefore excluded. for direct comparison with sars-cov-infected cells, raw data from hcov-emc experiments were quantile normalized together with the sars-cov data set (geo series accession number gse ). all probes were required to meet agilent feature extraction qc criteria for all replicates at at least one infection time point ( , probes passed qc filtering). for each sample, a log fold change (log fc) value was calculated as the difference between log normalized data for this sample and the average of log normalized data for time-and data set-matched mock-infected samples. statistical analysis. differential expression was determined by comparing sars-cov-or hcov-emc-infected replicates to time-and data set-matched mock-infected controls, based on a linear model fit for each probe using the r package limma ( ). criteria for differential expression were an absolute log fc of and a q value of Ͻ . calculated using a moderated t test with subsequent benjamini-hochberg correction. differentially expressed (de) genes after hcov-emc infection at early times postinfection were defined as all genes de at , , , or hpi, and genes de at late times postinfection were defined as all genes de at or hpi. to identify genes with similar patterns of variation at early and late times postinfection, early and late signatures were intersected considering upregulated and down-regulated genes separately and then combined to define the signature of stable genes. to cluster the genes de at late times postinfection following hcov-emc infection, differential expression was also calculated directly between sars-cov-and hcov-emcinfected samples using the same criteria. a comparison of similar times postinfection was performed, except for hcov-emc-infected samples at hpi, which were compared to sars-cov samples at hpi. hcov-emc samples at hpi were compared with sars samples at hpi and later times postinfection ( , , , , , and hpi). genes similarly changed between hcov-emc and sars-cov (cluster i and ii) were defined as genes de by both viruses compared to their time-matched mocks and that were also not differentially changed between hcov-emc and sars-cov in at least one of the previously listed comparisons. genes of cluster iii and iv were genes de at late times postinfection following hcov-emc infection that were not similarly changed between hcov-emc and sars-cov. to further cluster the genes based on their expression value after hcov-emc infection, log fc values at and hpi were averaged, and clusters i and iii were genes with average log fc values of Ͼ , while cluster ii and iv genes had average log fc values of Ͻ . functional enrichment and upstream regulator analysis. functional analysis of statistically significant gene expression changes was performed using ingenuity pathways knowledge base (ipa; ingenuity systems). for all gene set enrichment analyses, a right-tailed fisher's exact test was used to calculate a p value determining the probability that each biological function assigned to that data set was due to chance alone. all enrichment scores were calculated in ipa using the probes that passed our qc filter as the background data set. upstream regulator analysis, which was used to predict regulators and infer their activation state, is based on prior knowledge of expected effects between regulators and their known target genes according to the ipa database. a z score is calculated and determines whether gene expression changes for known targets of each regulator are consistent with what is expected from the literature (z Ͼ , regulator predicted to be activated) or are anti-correlated with the literature (z Ͻ , regulator predicted to inhibited). quantitative reverse transcription pcr (rt-pcr). rna was reverse transcribed using the quantitect reverse transcription kit (qiagen). the resulting cdna samples were diluted ϫ. primer sets for sybr green quantitative rt-pcr were designed using primer ( ) . primer sequences are available in table s in the supplemental material. relative gene expression in infected samples compared to that in mock-infected cells was calculated using the Ϫ⌬⌬ct method ( ), using the myl gene as a calibrator, as the expression of myl did not significantly change in hcov-emc-infected cells in the microarray data. connectivity map. to determine whether some drugs can reverse the hcov-emc infection signature, we used the publicly available connectivity map (cmap) database (build ) ( ) . cmap is a collection of genome-wide transcriptional data from cultured human cells treated with , different compounds. we used the list of de genes that are stable after infection with hcov-emc. agilent probes were mapped to affymetrix u a probe sets using the biomart id converter tool ( ) in order to query the cmap database. results were filtered based on their negative connectivity enrichment score. antiviral in vitro assay. for in vitro evaluation of the p mapk inhibitor sb (sigma), veroe cells were incubated for h with the drug at m prior infection with hcov-emc at an moi of . 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replication of human coronaviruses sars-cov, hcov-nl and hcov- e is inhibited by the drug fk effect of the phosphatidylinositol -kinase/akt pathway on influenza a virus propagation activation of the pi k/akt pathway early during vaccinia and cowpox virus infections is required for both host survival and viral replication human cytomegalovirus up-regulates the phosphatidylinositol -kinase (pi -k) pathway: inhibition of pi -k activity inhibits viral replication and virusinduced signaling effect of p mitogen-activated protein kinase on the replication of encephalomyocarditis virus the respiratory syncytial virus small hydrophobic protein is phosphorylated via a mitogen-activated protein kinase p -dependent tyrosine kinase activity during virus infection activation of the hiv- long terminal repeat by cytokines and environmental stress requires an active csbp/p map kinase mitogen activated protein kinase inhibitors: where are we now and where are we going? development of pi k inhibitors: lessons learned from early clinical trials novel p mapk inhibitor ml has potent antiinflammatory activity in airway smooth muscle anti-inflammatory effect and selectivity profile of as , a novel and potent p mitogen-activated protein kinase inhibitor design and synthesis of inhaled p inhibitors for the treatment of chronic obstructive pulmonary disease reverse genetics with a full-length infectious cdna of severe acute respiratory syndrome coronavirus limma: linear models for microarray data primer on the www for general users and for biologist programmers analysis of relative gene expression data using real-time quantitative pcr and the (Ϫdelta delta c(t)) method biomart-biological queries made easy roles of p mapk and jnk in tgf-beta -induced human alveolar epithelial to mesenchymal transition we thank lynn law and marcus korth for valuable feedback on the manuscript.this project was funded in part by federal funds from the national institute of allergy and infectious diseases, national institutes of health, department of health and human services, under contract hhsn c and grant u ai . the findings and conclusions in this report are those of the authors and do not necessarily reflect the views of the funding agency. key: cord- -nvu tqxb authors: kim, chonsaeng; bergelson, jeffrey m. title: echovirus entry into polarized intestinal epithelial cells requires clathrin and rab date: - - journal: mbio doi: . /mbio. - sha: doc_id: cord_uid: nvu tqxb enteroviruses invade the host by crossing the intestinal mucosa, which is lined by polarized epithelium. a number of enteroviruses, including echoviruses (ev) and group b coxsackieviruses (cvb), initiate infection by attaching to decay-accelerating factor (daf), a molecule that is highly expressed on the apical surface of polarized epithelial cells. we previously observed that entry of daf-binding cvb into polarized intestinal epithelial cells occurs by an unusual endocytic mechanism that requires caveolin but does not involve clathrin or dynamin. here we examined the entry of a daf-binding echovirus, ev . we found that drugs, small interfering rnas (sirnas), and dominant negative mutants that target factors required for clathrin-mediated endocytosis, including clathrin and dynamin, inhibited both ev infection and internalization of virions from the cell surface. once virus had entered the cell, it colocalized with markers of early endosomes (eea ) and then late endosomes (lamp- ). inhibition of endosomal maturation—with sirnas or dominant negative mutants targeting rab and rab —inhibited infection and prevented release of viral rna into the cell. these results indicate that ev is internalized by clathrin-mediated endocytosis and then moves to early and late endosomes before releasing its rna. trafficking through endosomes is known to be important for viruses that depend on low ph or endosomal cathepsin proteases to complete the entry process. however, we found that ev infection required neither low ph nor cathepsins. the viral receptor induces conformational changes in the virus capsid and begins the uncoating process. such changes are observed when poliovirus interacts with its receptor, pvr ( ) , or when coxsackievirus b (cvb ) interacts with the coxsackievirus and adenovirus receptor, car ( ) ; the initial conformational changes can be detected by altered sedimentation profiles in sucrose gradients, with native virions sedimenting at s to s and altered particles sedimenting at s. rhinovirus capsids are unstable at low ph, and for some rhinoviruses uncoating occurs when the virion is delivered to an acidic endosome ( ) . however, most enteroviruses are transmitted by the fecal-oral route and must survive exposure to stomach acid; in consequence, their capsids are stable at low ph, and their uncoating appears to be independent of endosomal acidification. enteroviruses are thought to invade the host by crossing the intestinal epithelium, which is largely composed of polarized cells with distinct apical and basolateral surfaces. both cvb and many echoviruses bind to decay-accelerating factor (daf; cd ) ( ) ( ) ( ) ( ) , a complement regulatory molecule that is highly expressed on the apical surface of polarized cells ( ) . we previously ( ) observed that, to infect polarized epithelium, cvb binds daf on the cell surface, moves to the tight junction, and then enters the cell by an unusual mechanism that is independent of both dynamin and clathrin but which requires caveolin; contact with car in the tight junction leads to an essential conformational change in the cvb virion, but disruption of the capsid does not appear to occur until the virus has moved to an unidentified intracellular compartment. unlike cvb, daf-binding echoviruses do not require car for infection. to understand how these viruses gain access to polarized epithelium, we have examined the entry of a daf-binding echovirus, ev , into polarized intestinal epithelial cells. we find that ev is internalized by clathrin-mediated endocytosis and moves to early and then to late endosomes early in infection. although uncoating does not require endosomal acidification, it depends on normal endosomal maturation. these results indicate that, although cvb and ev bind to the same cell surface receptor, they enter cells by different endocytic mechanisms. further, although ev does not require low ph for uncoating, it must nonetheless move to late endosomes before uncoating can occur. we first confirmed that ev entry into caco- cells depends on daf. caco- cells were transfected with small interfering rna (sirna) targeting daf, control sirna, or sirna targeting car. transfected monolayers were exposed to ev or to cvb -rd, a virus that depends both on daf and car for infection; the number of infected cells was determined by staining for newly synthesized viral protein (fig. a) . daf sirna inhibited infection by both ev and cvb -rd; as expected, car sirna inhibited infection only by cvb . in additional experiments (not shown), anti-daf monoclonal antibody (mab) if inhibited binding of radiolabeled ev to caco- cells and inhibited ev infection of caco- cells. thus, infection of caco- cells by ev , like infection of hela cells ( ) , depends on daf. ev infection depends on dynamin and clathrin. we had previously observed that cvb infection of caco- cells occurs by a complex process that depends on caveolin but not dynamin- , appears to be independent of clathrin-mediated endocytosis, and is inhibited by depletion of membrane cholesterol ( ) . here, we examined cellular factors required for ev infection, using sir-nas, dominant negative (dn) mutants, and drugs targeting clathrin-specific endocytic mechanisms. we found that infection by ev , but not by cvb , was inhibited by sirna depletion of both clathrin and dynamin (fig. b) , by dominant negative mutants of dynamin and the clathrin adaptor protein eps (fig. c) , and by chlorpromazine (fig. d) , an inhibitor of clathrin-mediated endocytosis ( ) . dynasore, a pharmacological inhibitor of dynamin gtpase activity ( ) , also had a marked inhibitory effect on ev infection (fig. d) . (a small but statistically significant effect on cvb infection by dynasore may be nonspecific, as no inhibition of cvb was seen with dominant negative dynamin or dynamin sirna.) in contrast, depletion of caveolin- with sirna ( fig. b) , expression of dominant negative caveolin (fig. c ), or treatment with the cholesterol-depleting agent methyl-beta cyclodextrin (fig. c) , all of which inhibited cvb , had no effect on ev infection. these results indicate that infection by ev depends on both dynamin and clathrin, as would be expected if virus entry depends on clathrin-mediated endocytosis. ev entry depends on dynamin and clathrin. to determine whether clathrin and dynamin are required for events upstream of rna release, we employed the neutral red (nr) infectious center assay for virus uncoating previously used to study poliovirus entry ( ) . when virus is grown in the presence of nr, the dye is concentrated within the capsid, in close proximity to the rna genome. exposure to light activates the dye, damages the rna, and renders the virus noninfectious. however, once uncoating has occurred, the nr diffuses away from the rna, so that exposure to light no longer inhibits infection. we found that plaque formation by neutral red-labeled ev (nr-ev ) was markedly inhibited when caco- monolayers were exposed to light at and min but that the majority of nr-ev became insensitive to light by min (fig. a) . this result suggested that uncoating had largely occurred by min. to monitor requirements for uncoating, we pretreated caco- cells with inhibitors, exposed them to nr-ev , and allowed infection to proceed in the dark for min. cells were then exposed to light, harvested, replated on cell monolayers, overlaid with agar, and incubated to permit the appearance of infectious centers. to control for nonspecific effects of the inhibitors, parallel assays were performed in which the illumination step was omitted. if an inhibitor prevents or significantly delays cellular events upstream of rna release, we expect it to reduce the formation of infectious centers in illuminated, but not unilluminated, cells. in a group of preliminary experiments (not shown), we confirmed that the uncoating inhibitor r reduced infectious center formation specifically in illuminated cells, as had previously been shown for poliovirus ( ) . in contrast, brefeldin a, which acts on virus replication at a postentry step, had no effect either in illuminated or unilluminated cells. (as expected, r acted to stabilize the ev capsid, preventing the conversion of s native virions to s altered particles in infected cells, and had little or no effect on binding of radiolabeled ev to caco- monolayers.) dynasore and chlorpromazine both significantly reduced the number of infectious centers after light exposure, with little inhibitory effect seen in nonilluminated samples ( fig. b and c) . similarly, depletion of dynamin and clathrin heavy chain with sirna specifically reduced infectious center formation in illuminated samples. these results confirm that dynamin and clathrin are required for events of infection that precede release of viral rna from the capsid. to test whether clathrin and dynamin are required for internalization of virions from the virus surface rather than for another trafficking event upstream of rna release, we tested the effects of chlorpromazine and dynasore on the internalization of ev virions directly conjugated to a red fluorophore, af . cells were pretreated with drugs, exposed to ev -af at °c, and then incubated at °c for min to permit entry to occur. cells were then fixed without permeabilization and stained with anti-vp antibody and fluorescein isothiocyanate (fitc)-labeled secondary antibody (green) to detect virus still exposed on the cell surface. in control cells treated with dimethyl sulfoxide (dmso) vehicle alone, red-labeled virus was seen to accumulate in perinuclear vesicles inaccessible to the anti-vp antibody (fig. d) . similar results were seen in cells treated with the uncoating inhibitor r (fig. g ). however, in cells treated with dynasore, virus remained trapped at the cell surface, as indicated by the congruence of red and green fluorescence (fig. e, yellow) . in cells treated with chlorpromazine, virus was similarly trapped transfected with sirnas targeting daf or car or with control sirna (labeled "c") and then cultured as polarized monolayers and exposed to ev or cvb , as described in materials and methods. infected cells were counted after staining with anti-vp antibody, and nuclei were counted after staining with dapi. for each virus, results are shown as the percentages of cells infected, normalized to the result with control sirna, Ϯstandard deviations (sd) for triplicate samples. right, immunoblot to confirm depletion of car and daf by sirna. (b) ev infection is inhibited by sirnas targeting dynamin (dyne ) and clathrin heavy chain (chc) but not by sirna targeting caveolin (cav ). (c) ev infection is inhibited by dominant negative forms of dynamin and eps but not by dominant negative caveolin . polarized monolayers of cells transfected with gfp-tagged wild-type or dominant negative dynamin , eps , and caveolin were exposed to ev or cvb . infected gfp-expressing cells were counted after being stained with anti-vp antibody. not shown, wild-type gfp-dynamin had no effect on infection (compared to gfp alone). (d) ev infection is inhibited by dynasore and chlorpromazine (cpz) but not by metyl-beta-cyclodextrin (mcd). results are normalized to those obtained for control cells exposed to dmso carrier alone. in all panels, single asterisks indicate p values of Ͻ . , and double asterisks indicate p values of Ͻ . . at the cell surface (fig. f ). taken together with the data presented above, these results indicate that ev entry and infection depend on internalization of virions by clathrin-mediated endocytosis. the result with r indicates that conversion of native virions to altered particles is not required for internalization of virions into the cell. rab and rab are important for infection and entry. cargoes internalized by clathrin-mediated endocytosis move rapidly to early endosomes in the cell periphery and then to more central late endosomes and lysosomes. endosomal maturation is accompanied by changes in lipid and protein content, as well as by progressive acidification ( ) . two small gtpases, rab and rab , are critical for vesicular traffic through the endosomal system: rab controls the delivery of cargoes to the early endosome, whereas rab regulates the maturation of late endosomes and endosome-lysosome fusion ( ) . to begin to characterize the in- was bound to caco- monolayers at °c, unbound virus was washed off, and then monolayers were overlaid with agar and incubated at °c. at intervals, monolayers were exposed to light and then incubated further to permit plaques to develop. (b) rna release depends on dynamin. neutral red infectious center assays were performed with cells pretreated with dynasore in dmso (labeled "dyna") or with dynamin (dyn ) sirna, as described in materials and methods. results are normalized to those obtained with nonilluminated controls exposed to dmso or negative-control sirna (labeled "c"). (c) rna release depends on clathrin-mediated endocytosis. neutral red infectious center assay performed with cells pretreated with chlorpromazine (cpz; compared to untreated control cells) with clathrin heavy chain sirna (chc; compared to cells treated with control sirna). (d) cells exposed to red-labeled ev for minutes were stained, without permeabilization, with anti-vp antibody and with fitc-labeled secondary antibody, to detect virus on the cell surface. virus (red) is internalized and localized to perinuclear and other cytoplasmic vesicles. (e) cells treated with dynasore; treated as described for panel c. virus remains largely on the cell surface, as indicated by green and yellow signals. (f) cells treated with chlorpromazine. virus remains largely on the cell surface, as indicated by green and yellow signals. (g) cells treated with r . tracellular trafficking events required for ev entry, we tested whether rab and rab were required for virus infection. sirnas targeting both rab and rab (fig. a) , as well as dominant negative forms of rab and rab (fig. b) , inhibited ev infection of caco- cells. as previously reported ( ) , infection by cvb -rd was inhibited by reagents targeting rab but not rab . these results suggested that ev might be delivered to late endosomes during the entry process. to test this, we examined the colocalization of ev with the early endosomal marker eea ( fig. a and b ) and the late endosomal marker lamp- ( fig. a and b) at intervals after infection. colocalization with eea increased steadily until min after infection (fig. b) and then decreased; colocalization with lamp- increased more slowly (fig. b) , reaching its maximum at min after infection. these results suggest that ev is delivered first to early endosomes and then to late endosomes. in the neutral red uncoating assay, sirnas targeting either rab or rab inhibited infectious center formation after light exposure, with little inhibitory effect seen in nonilluminated samples (fig. c) . thus, both rab and rab are required for events that occur upstream of rna release. taken together, these results indicate that ev is delivered to late endosomes before uncoating occurs. ev uncoating does not require endosomal acidification or cathepsin activity. for a number of viruses, acidification of the endosome provides an important trigger for structural changes that permit fusion of viral and endosomal membranes or release of nucleic acid from the protein capsid. for some, the relatively mild acidity of early endosomes is insufficient, and delivery to more acidic late endosomes is required before entry can occur ( , ) . enteroviruses such as echovirus , which enter the body by way of the intestine, must survive exposure to gastric acid and are generally quite resistant to low ph ( ) . to test whether ev entry depends on the low ph of the late endosome, we used two agents that prevent endosomal acidification by different mechanisms, ammonium chloride ( ) and the endosomal proton pump inhibitor bafilomycin a ( ) . neither ammonium chloride nor bafilomycin inhibited ev infection of caco- cells (fig. a) , although both agents effectively blocked infection by vesicular stomatitis virus, a virus that is known to require endosomal acidification for fusion and entry ( ) . in addition to providing an acidic environment, endosomes are rich in proteases; endosomal cysteine proteases (primarily cathepsins l and b) have been shown to be important for a number of viruses, including reovirus ( ), ebolavirus ( , ) , murine hepatitis virus ( ), and severe acute respiratory syndrome (sars) coronavirus ( ) . to examine the possible role of these cathepsins in entry by ev , we treated caco- cells with inhibitors of cathepsins b and l at concentrations known to inhibit entry by reovirus ( ) . no effect on ev infection was detected (fig. b) , despite significant inhibition of cathepsin b and l activity in treated cells (fig. c and d) . similarly, the protease inhibitor . not shown, wild-type gfp-rab and -rab had no effect on infection (compared to gfp alone). (c) rna release. neutral red infectious center assays performed in cells pretreated with sirnas targeting rab a, - b, - c, and - , compared to results for cells pretreated with negativecontrol sirna. e- , which inhibits a variety of cysteine proteases, including cathepsins b and l, had no effect on ev infection (data not shown). thus, ev entry requires virus trafficking to late endosomes but does not depend on endosomal acidification or the activity of the major endosomal cathepsins. the results we report here indicate that ev , after binding to daf on the apical surface of polarized epithelial cells, is internalized by clathrin-mediated endocytosis and then moves to early and late endosomes before releasing its rna into the cell. drugs, sirnas, and dominant negative mutants that perturb clathrinmediated endocytosis were found to inhibit both ev infection and internalization of virions from the cell surface. internalized virions were seen to colocalize with early and late endosomal markers, and sirnas and dominant negative mutants targeting rab gtpases essential for endosomal maturation inhibited infection, acting upstream of rna release. like ev , cvb also initiates infection of caco- cells by attaching to daf. however, the two viruses use different mechanisms for entry. cvb entry requires virus interaction with car, a protein localized specifically to tight junctions, and occurs by an unusual dynamin-independent mechanism that depends on membrane cholesterol, phosphorylation of caveolin ( ) , and the internalization of the tight junction protein occludin ( ) , among other factors. in contrast, ev internalization depends on host factors associated with clathrin-mediated endocytosis. introduction of isolated enteroviral rna into the cell cytoplasm is sufficient to initiate infection. thus, the critical event in entry is the release of viral rna from the capsid to the cytoplasm. how this occurs is not well understood. for poliovirus, the first step in uncoating is triggered when the virion interacts with its receptor, pvr: the virion undergoes a conformational change, leading to exposure of previously buried hydrophobic peptides and release from the capsid of a small internal protein, vp . but the resulting altered virion, referred to as the a particle, still retains the rna genome. a second conformational change, and possibly an intracellular cue, is required for the genome to exit, leaving behind a second identifiable altered virion, the s empty capsid ( ) . in contrast to poliovirus interactions with pvr, some viruses are not converted to a particles when they contact their receptors. the receptor for minor-group rhinoviruses, the ldl receptor, does not induce conformational changes but instead mediates the delivery of these acid-susceptible viruses to endosomes, where uncoating is initiated by acidification ( , ) . cvb is converted to a particles during infection, but a particles are not formed when virus binds to daf; conversion to a particles and productive infection both depend on interaction with another receptor, car ( , ) . although ev binds to human daf on transfected rodent cells, infection does not ensue ( , ) ; introduction of naked viral rna into the same cells results in infection (our unpublished data), suggesting that the block to infection occurs upstream of rna release. like cvb , ev has been noted to form a particles during infection, but it is not converted to a particles when it is exposed to soluble forms of daf; it has thus been suggested that a second cellular factor may be essential for infection ( ) . whether this factor is a second receptor protein or an intracellular cue of another sort is uncertain. the intracellular site where uncoating occurs also remains in question. direct visualization of rna release has established that poliovirus uncoating in hela cells occurs rapidly and quite close to the plasma membrane ( ) . in contrast, indirect evidence suggests that, in endothelial cells, poliovirus uncoating is completed slowly, after virus has moved to a perinuclear compartment ( ); cvb uncoating in caco- cells, as determined by appearance of s particles, also occurs deep within the cell ( ) . we found that ev uncoating, as defined by the neutral red assay, was completed at approximately min, when virus colocalization with lamp- was maximal. the requirement for rab indicates that ev uncoating depends on endosomal maturation, and it suggests that the late endosomal environment may provide a signal important for rna release. acidification itself does not seem to be important, as bafilomycin and ammonium chloride had no effect on infection; further, although endosomal cathepsins are important for entry of several viruses, infection was not affected by inhibition of cathepsin activity. ebolavirus entry was recently shown to depend on the niemann-pick c protein ( , ) , a cholesterol transport protein resident in late endosomes ( ) . we observed no effect on ev infection when caco- cells were treated with u a (data not shown), an inhibitor of cholesterol synthesis, which induces a cellular phenotype similar to that seen in niemann-pick c deficiency and which has been shown to block ebola virus entry ( ) . thus, if a specific endosomal factor is required for ev uncoating, it remains to be identified. the observation that ev must reach late endosomes before uncoating is unexpected for an enterovirus that does not depend on endosomal acidification. our data do not show directly that uncoating occurs in the late endosome itself, and we cannot exclude the possibility that trafficking through the endosome is an essential step in a pathway that leads to another intracellular organelle where uncoating is completed; such is the case with sv , which moves through endosomes on its way to the endoplasmic reticulum (er), where destabilization of the capsid is catalyzed by factors present in the er lumen ( , ) . we are currently exploring whether ev entry depends on additional host factors associated with specific aspects of endosomal maturation or with trafficking between late endosomes and other intracellular compartments. caco- cells (atcc htb- ) were cultured in high glucose minimal essential medium containing % fetal bovine serum, march/april volume issue e - ® % nonessential amino acids, and penicillin-streptomycin. chinese hamster ovary (cho) cells stably transfected with cdna encoding human car, human daf, or empty vector have been described previously ( ) . echovirus (wallace; atcc vr- ) and cvb -rd ( ) were prepared in hela cells, concentrated by ultracentrifugation through a sucrose cushion, and titered by plaque assay on hela cells. vesicular stomatitis virus, provided by ron harty (university of pennsylvania), was prepared and the titer was determined in bhk- cells as described ( ) . antibodies and chemicals. car-specific rabbit polyclonal antibody (h- ) and horseradish peroxidase (hrp)-conjugated gapdh antibody (catalog no. sc- ) were obtained from santa cruz biotechnology. murine monoclonal antibodies specific for daf (catalog no. ), clathrin heavy chain (catalog no. ), caveolin (catalog no. ), and anti-eea antibody (catalog no. ) were obtained from bd. rabbit polyclonal antibody specific for dynamin was from abcam (catalog no. ab ), rabbit polyclonal anti-rab was from stressgen (catalog no. kap-gp ), and rabbit polyclonal anti-rab was from sigma (catalog no. r ). murine monoclonal antibody specific for lamp- (clone h b ) was from the developmental studies hybridoma bank at the university of iowa, and monoclonal antibody specific for vesicular stomatitis virus (vsv) m protein (clone h ) was obtained from douglas lyles (wake forest university). dynasore, bafilomycin a , cathepsin b inhibitor (catalog no. ), and cathepsin l inhibitor (catalog no. ) were obtained from calbiochem. chlorpromazine (cpz), methyl-beta-cyclodextrin (m␤cd), and nh cl were from sigma. compound r was provided by janssen pharmaceuticals. plasmids and sirnas. plasmids encoding green fluorescent protein (gfp)-tagged wild-type (wt) and k a dominant negative (dn) dy-namin ( ) were provided by mark mcniven (mayo institute, rochester, ms); gfp-tagged wt and y f dn caveolin ( ) were from ari helenius (swiss federal institute of technology, zurich); eps -gfp wt and dn ( ) were provided by alice dautry-varsat (institut pasteur, paris). gfp-tagged wt and dn (s n) rab were provided by george bloom (university of virginia), and wt and dn (t n) rab were provided by craig roy (yale university). transfection with plasmids was performed using the amaxa nucleofector system (lonza). transfected cells were plated at ϫ cells/well in collagen-coated -well chamber slides (bd) and used for virus infection after days. control and human car sirnas have been described previously ( ) . the sequence of daf sirna is = gua cca ggu ggc aua uua utt =. sirnas targeting dynamin (m- - ), clathrin heavy chain (m- - ), and caveolin- (m- - ) were purchased from dharmacon. rab sirnas (rab a [ = agg aau cag ugu ugu agu att =], rab b [ = gaa agu caa gcc ugg uau utt =], and rab c [ = caa uga acg uga acg aaa utt =]) were synthesized based on sequences in a previous report ( ) , and rab sirna (sc- ) was purchased from santa cruz. sirnas targeting rab and rab were used at a nm concentration; other sirnas were used at nm. transfection was performed with lipofectamine (invitrogen). cells were transfected in -well plates; to optimize the knockdown efficiency, a second transfection was performed h after the first. after an additional h, the monolayers were trypsinized and cells were replated at ϫ cells/well in collagen-coated -well chamber slides (bd biosciences), and infections were performed days later. depletion of targeted proteins was confirmed by immunoblot analysis of parallel samples. virus infection and internalization. caco- cells were plated on collagen-coated -well chamber slides (bd biosciences) at ϫ cells per well and infected days later. to measure infection, virus (ev , cvb -rd, or vsv; pfu/cell) in minimum essential medium (mem) with mm hepes was permitted to bind to polarized monolayers for h at °c. monolayers were then washed with phosphate-buffered saline (pbs), complete medium was added, and cells were incubated for h at °c. infection was terminated by fixation and permeabilization for min with a : mixture of ice-cold methanol-acetone. monolayers were stained with anti-vp antibody (ncl-entero; novocastra) to detect infected cells and with ', -diamidino- -phenylindole (dapi) to detect cell nuclei, and images were captured with an olympus bx immunofluorescence microscope, using a ϫ objective. three or four fields ( to , cells) were captured for each monolayer. the numbers of infected cells and the total number of dapi-stained nuclei were quantified using imagej software (http://rsbweb.nih.gov/ij/). for entry staining, ev , purified on sucrose gradients, was labeled with a -fold molar excess of alexafluor (af- ) succinamidyl ester (molecular probes), using a protocol previously described for labeling ev . af -ev ( pfu/cell) was allowed to bind to cells for h at °c at pfu/cell. after a pbs wash, complete medium was added and cells were incubated for min at °c. virus internalization was stopped by fixation with % paraformaldehyde (pfa) for min, and excess pfa was removed with pbs containing mm nh cl. surface-associated virus was detected by staining unpermeabilized monolayers with anti-vp antibody and anti-mouse secondary antibody conjugated to fitc. slides were mounted with vectashield (vector laboratories). images were captured with a zeiss lsm confocal microscope using a ϫ oil objective. z-stack images were obtained at . -m intervals and viewed with the lsm reader plugin for imagej. inhibitory drugs were used at the following concentrations: dynasore, m; bafilomycin a , nm; cathepsin b inhibitor, m; cathepsin l inhibitor, m; chlorpromazine, g/ml; m␤cd, mm; nh cl, mm; brefeldin a, g/ml; r , g/ml. in general (see exceptions below), cells were pretreated for min at °c in complete medium containing the drug, and then virus was permitted to bind in drugcontaining medium at °c for h. unbound virus was removed, and then drug-containing medium was replaced and infection was allowed to proceed at °c for h. for experiments with dynasore and m␤cd, medium with % nuserum (bd biosciences, bedford, ma) in place of fetal bovine serum (fbs) was used, because serum components can decrease the efficacy of those drugs ( ) . in experiments with dynasore, drug-containing medium was replaced with normal medium after h. because m␤cd acts quickly to deplete cholesterol from the plasma membrane, cells were pretreated with m␤cd for min and drug was not used again during the course of infection. neutral red infectious center assay (uncoating assay). to prepare neutral red (nr)-labeled virus (nr-ev ), hela cells were infected with ev ( pfu/cell) in the presence of g/ml nr. after overnight incubation, virus was harvested and purified in the dark. neutral red infectious center assays using inhibitor drugs or sirnas were performed as described by brandenburg et al. ( ) with slight modifications. in experiments with sirnas, caco- cells were transfected and allowed to form polarized monolayers in -well plates before infection. in experiments with drugs, monolayers in -well plates were pretreated with a drug for h at °c. in the dark, nr-ev , in binding buffer containing the drug, was allowed to attach to cells at °c for h; binding buffer was then removed and infection was initiated by adding warmed culture medium, containing the drug if appropriate. after min at °c, virus-infected cells were exposed to white light for min at room temperature; preliminary experiments indicated that a -min incubation was required for the majority of input virus to escape light-induced nr damage. duplicate monolayers were maintained in the dark and used as unilluminated controls. cells were detached with trypsin-edta and then counted, diluted, and replated onto fresh caco- monolayers in -well plates, in the absence of inhibitors. agarose overlay was added after h. after days, plaques were developed and counted. in each of four independent experiments, the numbers of plaques in the drug-treated or illuminated wells were normalized to the number of plaques in the unilluminated control well (expressed as a percentage). therefore, no error estimates are provided for the controls, which were defined as %. capsid conformational changes. conversion of native virions to s a particles was detected essentially as described in reference . monolayers were incubated with radiolabeled ev at °c, unbound virus was removed, and cells were either maintained in the cold or incubated at °c, either in the presence of r or in dmso alone. at min, cells were lysed, debris was removed, and recovered virus was subjected to centrifugation in to % sucrose gradients. colocalization with endosomal markers. caco- monolayers were incubated with af -ev at °c. at -min intervals, monolayers were fixed with a : mixture of ice-cold methanol-acetone and stained with antibodies specific for eea- or lamp- followed by fitc-conjugated secondary antibody and examined by confocal microscopy. images with the best focus in the red (virus) channel were captured and analyzed. to exclude noninternalized virus, the cytoplasmic region of each cell was outlined with the freehand selection tool of imagej. colocalization of red and green signal was determined, using the colocalization test plugin in the wcif imagej bundle to calculate pearson's correlation coefficient for each cell. three independent experiments were performed. in each experiment, at least five images were analyzed for each time point. measurement of cathepsin b and l activity. cathepsin activity was measured as described by ebert et al. ( ) with slight modifications. caco- cells were preincubated with cathepsin b or l inhibitor ( m) for h and washed with pbs and lysed with mm sodium acetate (ph ), mm edta, and . % triton x- . insoluble debris was removed, and cell lysate was mixed with cathepsin b substrate ( m; calbiochem catalog no. ) and incubated for min at room temperature or with cathepsin l substrate ( m; invitrogen catalog no. r ) and incubated for . h at °c. for cathepsin b activity, fluorescence was measured using a synergy multimode microplate reader (bio-tek) with excitation at nm and emission at nm. for cathepsin l activity, fluorescence was measured using a fluostar fluorometer (bmg labtechnologies) with excitation at nm and emission at nm. analysis of experimental variation. in all figures, error bars indicate the means Ϯ standard deviations for or more independent samples. single asterisks indicate p values of Ͻ . , as determined by student's t test; double asterisks indicate p values of Ͻ . . enteroviruses: poliovirus, coxsackieviruses, echoviruses, and newer enteroviruses overview of taxonomy receptors cell entry: a biological and structural perspective mechanisms of endocytosis caveolin, a protein component of caveolae membrane coats induction of mutant dynamin specifically blocks endocytic coated vesicle formation dynaminmediated internalization of caveolae dynamin at the neck of caveolae mediates their budding to form transport vesicles by gtp-driven fission from the plasma membrane of endothelium virus entry by endocytosis the poliovirus receptor: a hook, or an unzipper? interaction with coxsackievirus and adenovirus receptor (car), but not with decay accelerating factor (daf), induces a particle formation in a daf-binding coxsackievirus b isolate uncoating of human rhinovirus serotype from late endosomes decay-accelerating factor, a glycosylphosphatidylinositol-anchored complement regulatory protein, is a receptor for several echoviruses coxsackievirus b adapted to growth in rd cells binds to decay-accelerating factor (cd ) coxsackieviruses b , b , and b use decay accelerating factor as a receptor for cell attachment decay accelerating factor (cd ) identified as a receptor for echovirus using celics, a rapid immuno-focal cloning method interaction with decay-accelerating factor facilitates coxsackievirus b infection of polarized epithelial cells virus-induced abl and fyn kinase signals permit coxsackievirus entry through epithelial tight junctions mis-assembly of clathrin lattices on endosomes reveals a regulatory switch for coated pit formation dynasore, a cell-permeable inhibitor of dynamin imaging poliovirus entry in live cells endocytosis and molecular sorting rab gtpases coordinate endocytosis coxsackievirus entry from epithelial tight junctions requires occludin and the small gtpases rab and rab entry of bunyaviruses into mammalian cells picornaviridae: the viruses and their replication fluorescence probe measurement of the intralysosomal ph in living cells and the perturbation of ph by various agents the cytotoxic action of diphtheria toxin and its degradation in intact vero cells are inhibited by bafilomycin a , a specific inhibitor of vacuolar-type h(ϩ)-atpase pathway of vesicular stomatitis virus entry leading to infection cathepsin l and cathepsin b mediate reovirus disassembly in murine fibroblast cells endosomal proteolysis of the ebola virus glycoprotein is necessary for infection role of endosomal cathepsins in entry mediated by the ebola virus glycoprotein endosomal proteolysis by cathepsins is necessary for murine coronavirus mouse hepatitis virus type spike-mediated entry inhibitors of cathepsin l prevent severe acute respiratory syndrome coronavirus entry conformational changes, plasma membrane penetration, and infection by human rhinovirus type : role of receptors and low ph inhibition of coxsackie b virus infection by soluble forms of its receptors: binding affinities, altered particle formation, and competition with cellular receptors interaction between echovirus and its receptor, decay-accelerating factor (cd ): evidence for a secondary cellular factor in a-particle formation poliovirus entry into human brain microvascular cells requires receptor-induced activation of shp- ebola virus entry requires the cholesterol transporter niemann-pick c small molecule inhibitors reveal niemann-pick c is essential for ebola virus infection niemann-pick c is a late endosome-resident protein that transiently associates with lysosomes and the trans-golgi network bap and bip are essential for dislocation of sv from the endoplasmic reticulum to the cytosol simian virus depends on er protein folding and quality control factors for entry into host cells single amino acid changes in the virus capsid permit coxsackievirus b to bind decay-accelerating factor dynamin-and lipid raftdependent entry of decay-accelerating factor (daf)-binding and non-daf-binding coxsackieviruses into nonpolarized cells disruption of golgi structure and function in mammalian cells expressing a mutant dynamin caveolar endocytosis of simian virus reveals a new two-step vesicular-transport pathway to the er ap- /eps interaction is required for receptormediated endocytosis the coxsackievirus and adenovirus receptor interacts with the multi-pdz domain protein- (mupp- ) within the tight junction analysis of clathrinmediated endocytosis of epidermal growth factor receptor by rna interference we thank marc mcniven, ari helenius, george bloom, craig roy, and alice dautry-varsat for plasmids, ron harty for vsv, douglas lyles for anti-vsv antibody, and janssen pharmaceuticals for r . we are grateful to kunal patel for teaching us a number of the techniques involved in this work. we thank andrea stout and jasmine zhao of the university of pennsylvania cell and developmental microscopy core for assistance with confocal imaging, and michael sebert for help in performing cathepsin assays. carolyn coyne and michael sebert provided valuable comments on the manuscript. this work was supported by nih r ai and the plotkin endowed chair in infectious diseases at children's hospital of philadelphia. key: cord- -zyzgk z authors: chang, stewart t.; sova, pavel; peng, xinxia; weiss, jeffrey; law, g. lynn; palermo, robert e.; katze, michael g. title: next-generation sequencing reveals hiv- -mediated suppression of t cell activation and rna processing and regulation of noncoding rna expression in a cd (+) t cell line date: - - journal: mbio doi: . /mbio. - sha: doc_id: cord_uid: zyzgk z next-generation sequencing (ngs) enables the highly sensitive measurement of whole transcriptomes. we report the first application to our knowledge of this technology to the analysis of rna from a cd (+) t cell line infected with intact hiv. we sequenced the total mrna from infected cells and detected differences in the expression of both host and viral mrna. viral reads represented a large portion of the total mapped sequencing reads: approximately % at h postinfection (hpi) and % at hpi. we also detected a small but significant suppression of t cell activation-related genes at hpi. this suppression persisted and expanded by hpi, providing new possible markers of virus-induced t cell cytopathology. by hpi, the expression of over % of detectable host loci was also altered, indicating widespread alteration of host processes, including rna processing, splicing, and transport to an extent not previously reported. in addition, next-generation sequencing provided insights into alternative viral rna splice events and the expression of noncoding rnas, including microrna host genes. t he hallmark of aids is the loss of cd ϩ t cells, the primary target of human immunodeficiency virus type (hiv- ) infection. infected cd ϩ t cells undergo fundamental changes that eventually result in cell death and the release of new virus particles (reviewed in references and ). following the uptake of virus, the viral genome is reverse transcribed and integrated into the host genome. the host machinery is then used to produce viral transcripts that are either spliced into smaller transcripts that serve as the template for viral proteins or left unspliced to be incorporated into new virus particles. microarray analyses have shown that infected cells respond to these assaults with gene expression changes in a number of pathways, including apoptosis, cell cycle, cholesterol biosynthesis, and inflammation ( ) ( ) ( ) ; reference and references therein). many of these cellular responses are also reflected at the level of the host organism. for example, gene expression in the lymph nodes of simian immunodeficiency virus (siv)-infected asian pig-tailed macaques (a model of pathogenic hiv infection) reflects strong and sustained type i interferon responses ( ) . a sim-ilar initial interferon response is seen in the natural host, african green monkeys, but it eventually subsides and the infection resolves ( ) . despite intense investigation into the molecular events following infection in these and other studies, fundamental gaps in knowledge still remain. for instance, the extent to which host and viral gene expression respond to each other is still poorly understood. new technologies enabling the measurement of both host and viral rna may help to address such shortcomings. in this study, we used next-generation sequencing (ngs) to examine changes in the transcriptome of t cells infected with replication-competent hiv. ngs offers a number of benefits for the study of host-pathogen interactions. increased sensitivity and accuracy over microarrays allow important events such as the initial host response to viral replication to be studied in greater detail ( ) . also, host and viral rna transcripts can be assayed simultaneously, rather than on separate technical platforms, allowing greater reproducibility and increased confidence in the results. finally, because ngs allows total rna to be assayed, insights are not limited to annotated transcripts or even protein-coding genes. indeed, previous work in our group has shown that many noncoding rna species are differentially expressed in virus-infected cells ( ) . we report here the effects of hiv- infection on the expression of polyadenylated rnas in a cd ϩ t cell line. using ngs, we detected small but significant changes in host gene expression affecting t cell function that coincided with the initiation of viral rna production at h postinfection (hpi). these changes intensified near peak viral replication at hpi when a multitude of other host processes were disrupted as well. in addition, by using ngs, we observed the dramatic expansion of viral mrna expression and detected new viral splice events occurring during viral replication and differential expression of noncoding rna species, including microrna host genes. these findings provide an unprecedented and comprehensive view into the transcriptomelevel changes that occur within t cells infected with replicating hiv. in this study, we investigated changes in host and viral transcription occurring in a t lymphoblast-based model of hiv infection. sup-t cells ( ϫ ) were infected in triplicate with hiv- strain lai at a multiplicity of infection (moi) of which resulted in near-complete infection at hours postinfection (hpi). the phenotype of infected cells (including appearance, viability, and the amount of viral mrna and protein) was used to guide the selection of samples for ngs. at hpi, viral mrna could be detected in infected cells at appreciable levels (see fig. s in the supplemental material). few cells expressed the viral antigen p gag, and visually, the cells appeared to be intact. by hpi, high copy numbers of viral mrna were present (fig. s ). nearly all cells showed the presence of p gag antigen, syncytia were observed, and nonsyncytial cells had increased in volume. by hpi, the effects of viral infection were more pronounced. the amount of viral rna peaked (fig. s ), and cell viability decreased. the bulk of cell death occurred between and hpi. on the basis of these observations, we selected two time points to analyze by ngs: and hpi concurrent with the beginning of viral rna accumulation and the occurrence of near-peak rna levels prior to cell death, respectively. we isolated rna from infected and mock-infected replicate cell samples at these two time points, created cdna libraries from polyadenylated rna, and subjected the libraries to analysis by ngs. for each sample, we obtained on average over million -nucleotide (nt) reads mapping to either hiv or human genomes. a large number of reads mapped to the viral genome ( . to million depending on the time point, see table s in the supplemental material). by hpi, viral reads constituted~ % of the total mapped reads, and by hpi, this proportion increased to~ %. reads mapping to the viral genome indicated the presence of rna splicing events. in addition to splicing events involving known splice sites, we also observed evidence of five splicing events involving one or more previously unobserved splice sites (see fig. s in the supplemental material). we tested the presence of one of these sites (located = of the annotated splice acceptor site ) by quantitative pcr (qpcr) and observed an amplicon size consistent with the usage of the site (fig. s a to s c) . hiv- infection results in a small set of differentially expressed host genes at hpi. to characterize the host response to hiv infection, we mapped the remaining nonviral reads to refseq-annotated human gene loci and quantified them as fpkm (fragments per kilobase of exon per million mapped fragments). we selected a set of host genes whose expression spanned a range of values and measured the expression levels directly by qpcr (see fig. s in the supplemental material). measurements by ngs showed a high degree of correlation with qpcr results at both time points (r ϭ . and r ϭ . at and hpi, respectively), indicating a close match between the two methods. we then identified specific host genes that were differentially expressed (de) during infection. comparing fpkm for each gene in infected cells to time-matched mock-infected cells, we found that a total of genes were de at hpi (representing all fold changes with benjamini-hochberg-adjusted p values of less than . ; table ). by hpi, the number of de genes had increased to , representing over % of all , gene loci detected under stringent criteria (table ) . closer examination of these values showed that a large proportion of de genes occurred with smallmagnitude changes. in particular, % ( of ) and % ( , of , ) of the expressed genes exhibited -to . -fold changes at and hpi, respectively ( table ). the observed precision of the sequencing-based measurements (fig. s ) supported the use of low-fold change thresholds. other values for the statistical threshold were also used, and for all but the most stringent thresholds (when fewer than genes were observed to be up-or downregulated at hpi), a similar predominance of small-magnitude changes was observed (see table s in the supplemental material). comparing the directionality of change at both time points, we found that nearly all of the de genes at hpi continued to be regulated in the same manner (i.e., up-or downregulated) at hpi. specifically, of the de genes at hpi were also de at hpi (with benjamini-hochberg-adjusted p Ͻ . ), and the majority of these ( of ) exhibited the same direction of change relative to mock infection, often with increased degree of change (as indicated by more-intense red or green coloration at hpi than at hpi in a heat map of fpkm fold changes, fig. ). this indicated that regulation coincident with the beginning of viral replication at hpi largely persisted and intensified near peak viral replication at hpi, even for initially low fold changes. hiv- infection impacts core t cell functionality by hpi. we determined the possible impact of these de genes by analyzing the corresponding annotations (biological processes, molecular functions, and pathways) using the nih david resource. we found that t cell activation and differentiation were the most overrepresented annotations among de host genes at hpi overall ( fig. a) . other annotations associated with de genes included protein kinase regulation, dna recombination, and caspase activity regulation ( fig. a ). in addition, several of the de genes at hpi encoded transcriptional regulators; these genes include egr , klf , and myc. interestingly, four of the nine genes de at hpi but not at hpi also encoded transcriptional regulators (creb l , hexim , znf , and zkscan ), indicating regulation specific to early viral replication. seven genes contributed to the overrepresentation of t cell activation among de genes at hpi (bcl b, cd d, egr , ikzf , irf , rag , and sox ), six of which were downregulated, indicating a net suppression of t cell activation (as t cell differentiation in fig. ). five t cell activation-related de genes (bcl b, egr , irf , ikzf , and sox ) encoded transcription factors, some of which had known relevance to hiv infection. for example, the bcl b gene product binds directly to the hiv- sample relative to averaged time-matched mock-infected cell samples. genes were segregated by the direction of change relative to mock infection at hpi, and hierarchical clustering was done within each directional group. genes that were not also de at hpi (purple type) and genes that were also de at hpi but with changed directionality (gold type) are indicated. annotations indicate overrepresented categories in david (fig. ). matches to top-scoring categories in each david annotation cluster (matching numbers in fig. ) (solid black squares) and matches to related categories in the same david cluster as the top-scoring category (solid gray squares) are indicated. reg, regulation. long terminal repeat (ltr) and inhibits ltr expression ( ) . bcl b downregulation may therefore allow more efficient hiv- replication. other de genes related to t cell activation not encoding transcription factors also had known relevance to hiv infection. for example, the cd d product presents lipid antigens on the surface and is directed by hiv- nef for internalization and degradation ( ) . downregulation of cd d at the mrna level would further reduce surface expression and facilitate immune evasion. we complemented functional analysis in david by gene set enrichment analysis (gsea). this method does not rely on the prior determination of de genes (e.g., by applying a p value threshold) but instead uses a ranked gene list as input and is therefore well suited for identifying annotations among genes with small-magnitude changes in expression ( ) . gsea identified additional genes that were downregulated and contributed to the suppression of t cell activation; these genes include the cd , cd , cd , cd , and sit genes encoding t cell-specific surface molecules (see fig. s in the supplemental material). many of the genes associated with t cell activation in david and gsea also had roles in other pathways, indicating possible pleiotropic effects (e.g., adora a and rag which also contribute to caspase activity [ fig. ]) . hiv- induces large-scale changes in transcription by hpi. by hpi, large-scale changes in the transcriptomes of hivinfected cells were evident (table ; see the heatmap in fig. s a in the supplemental material). consistent with the large number of de genes at hpi, we found that a wide range of biological processes was affected as determined by further analysis using david ( fig. b ; complete listing in table s in the supplemental material). t cell activation and differentiation continued to be overrepresented and were associated with an increased number of de genes (as lymphocyte differentiation [ fig. b] ). other fundamental cellular functions associated with de genes at hpi included dna metabolism, transcription, mrna processing and splicing, translation, protein degradation, and cell cycle control (fig. b ). this breadth of functional regulation suggests that hiv- infection resulted in the effective transcriptional reprogramming of sup-t cells by hpi. suppression of t cell functionality persists and expands at hpi. as was the case at hpi, genes related to t cell activation and differentiation were predominantly downregulated at hpi. all of the t cell activation-related de genes at hpi continued to exhibit the same direction of change at hpi (fig. ). other t cell activation-related genes de at hpi included cd d, cd q, and rhoh, all of which encode membrane proteins crucial for t cell receptor function. a network depicting known interactions among proteins encoded by t cell activation-related de genes at hpi showed two highly connected nodes: lck (lymphocytespecific protein kinase) and tp (p ), both of which were strongly downregulated at hpi (fig. a) . our observed downregulation of tp differed from the upregulation observed in previous microarray studies and may indicate a cell type-specific effect ( ) . genes involved in other core t cell functions, such as proliferation, survival, and antigen presentation, were also downregulated at hpi. these genes included cd d, cd , cxcr , tnfrsf , and treml . tob (transducer of erbb ), a negative regulator of t cell activation, proliferation, and interleukin (il- ) production, showed increased expression and may contribute to the downregulation observed in other genes (data not shown). a connection between tob expression and hiv- infection has not previously been mentioned. overall, the increased number of de genes related to t cell activation indicated increased suppression of this pathway by hpi. ribonucleoprotein complex biogenesis and rna processing. gene sets related to ribonucleoprotein complex biogenesis were the most overrepresented annotations among de genes at hpi (fig. b) . ribonucleoproteins contribute to diverse functions within the cell, including microrna synthesis, ribosomal assembly, and translation ( ) . this overlap was reflected at the gene level as well. in particular, ribonucleoprotein complex biogenesis shared many de genes at hpi with another top annotation cluster, rna processing. this set of genes encoding the ribonucleoprotein component of the rna processing machinery was almost entirely downregulated and included many members of the heterogeneous nuclear ribonucleoprotein (hnrnp) and small nuclear ribonucleoprotein (snrnp) families. in a network identifying interactions among the proteins encoded by these genes, a number were found to be highly connected (fig. b ). one of these was hnrnpa whose gene product regulates the splicing of eukaryotic and viral mrna ( ) . hnrnpa and other hnrnp gene products are known to affect the localization of hiv- gag/pol mrna, and their overexpression has previously been shown to reduce hiv- replication (fig. b) ( ) . other de genes at hpi related to ribonucleoprotein biogenesis and rna processing are also known to affect hiv- replication directly; these genes include tardbp (tar dna-binding protein ) whose protein binds the transactivation response (tar) element of integrated hiv- dna and represses hiv- transcription ( ) ( ) ( ) . downregulation of hnrnpa , tardbp, and other related genes may therefore allow more efficient hiv- replication in sup-t cells. rna transport. the importance of host regulation of rna fate was underscored by another cluster of gene sets related to rna transport (fig. b) . host factors for rna transport are known to be coopted by hiv- to allow the export of unspliced, partially spliced, or fully spliced viral mrnas out of the nucleus ( ) . in particular, the host factors crm (exportin ) and ran gtpase are used for the export of unspliced viral mrna, while the host factor nxf (nuclear rna export factor ) facilitates the export of fully spliced viral mrna in a ran-independent manner ( ) . in our data set, we observed no change in the expression of the crm -encoding gene xpo , but several members of the ran signaling pathway were downregulated, suggesting that the overall export of unspliced viral rna was suppressed (see fig. s b in the supplemental material; data not shown for xpo ). however, in contrast to other rna transport-related genes, nxf and nxf were expressed more highly in infected cells, suggesting that hiv infection may have selectively enhanced the export of fully spliced viral rna (fig. s b) . limited upregulation of host processes at hpi. despite the presence of an almost equal number of up-and downregulated genes at hpi, relatively few gene sets (functions, processes, or pathways) were associated with upregulated genes. when up-and downregulated de genes at hpi were analyzed separately, more annotation clusters were observed among downregulated genes ( versus among upregulated genes with significance defined by modified fisher's exact test as p Ͻ . ). a similar result was obtained by gsea (see fig. s a and s b in the supplemental material). these results suggested that upregulated genes were distributed across many gene sets with few occurring in particular functions or pathways. among the few upregulated pathways were stress-activated/jnk cascade signaling and ion transport (fig. s c) . consistent with these observations, hiv- nef has been shown to activate jnk signaling, ultimately activating the caspase cascade and triggering cell death ( ) . the triggering of apoptosis at hpi is consistent with our observation that infected cells began to die following the -hpi time point. hiv- infection does not trigger innate sensing at hpi. notably, innate immunity was absent from the processes associated with de genes at hpi, suggesting that hiv- infection impaired t cell activation while evading virus-sensing mechanisms ( fig. a) . with the exception of irf , which was downregulated at hpi (fig. ) , interferon response factors (irfs) (irf , irf , irf , and irf ) showed no significant change in expression at hpi (data not shown). furthermore, specific irf targets, including irf target genes ifi , ifi , isg , and isg , were also not differentially expressed at this time point ( , ) . irf targets were of particular interest, as intact cytoplasmic hiv- dna has been shown to activate irf in cd ϩ t cells ( ) . our observed lack of irf target gene expression is consistent with previous observations that replicating hiv- suppressed irf activity ( ) . we also examined the levels of inflammationrelated genes previously observed to be differentially regulated in hiv infection studies ( ): anxa , b m, and cd (generally upregulated) and cd , cd , il- , and il- (generally downregulated). these genes were also either not expressed at detectable levels or unchanged in expression at hpi (data not shown). innate immunity and the inflammatory response continued to be absent from the significantly upregulated gene sets at hpi (by gsea [see fig. s a in the supplemental material]). irf was more strongly downregulated at hpi than at hpi, but other irf genes (irf , irf , irf , and irf ) continued to show no significant change in expression ( fig. ; data not shown). in a separate experiment, we confirmed that irf was downregulated at hpi by qpcr (by an average of %; data not shown). other interferon (ifn)-inducible genes were differentially expressed at this time point but in different directions, such as b m and ifi (both downregulated) and ifi and isg (both upregulated). this lack of concerted change may have offset the detection of interferon response gene sets as up-or downregulated at hpi by gsea. instead, at hpi, the related gene sets of cellular defense and immune response were found to be primarily downregulated (by gsea [fig. s b] ). several of the downregulated genes associated with these gene sets were also associated with t cell activation; these genes include cd d, cd , cd , rag , and tnfrsf (fig. s d) . additional immune response-specific genes downregulated at hpi included il and il r, suggesting that hiv- may have impaired the ability of infected cells to signal other cells or respond to extracellular cues. by mapping sequencing reads to refseq transcript annotation, we also observed changes in the expression of several non-protein-coding rna species. specifically, reads mapping to refseq transcripts that began with nr were considered noncoding. as before, expression of these transcripts was compared between infected and mock-infected samples. at hpi, only one annotated noncoding rna (nr_ encoding c orf ) was found to be differentially expressed (benjamini-hochberg-adjusted p Ͻ . ). however, by hpi, annotated noncoding rnas were found to be differentially expressed by the same criterion, with changed by an average of -fold or greater (fig. a) . several classes of noncoding rnas were represented, including microrna host genes, hypothetical genes and open reading frames (orfs), small nucleolar rnas (snornas), and pseudogenes (fig. a) . de pseudogenes often matched their protein-coding counterparts in directionality and degree of regulation, which suggests that regulatory regions were maintained and polyadenylated transcripts were produced (data not shown). we also observed differential expression of four annotated mi-crorna host genes in infected cells, three of which were downregulated (mir hg, mir , and mir ), while the remaining one was upregulated (mir f), all with -fold or greater changes. the downregulation of mir hg (by . -fold) was of particular interest, as mir hg encodes a cluster of micrornas, including mir- , - a, - a, - b, - a, and - (fig. b) ; in contrast, the other microrna host genes encode only single micrornas. reads mapped to mir hg = of the mature microrna sequences, indicating that the postexcised polyadenylated product may have been detected (fig. b) . we also observed a concurrent upregulation of known targets of mir hgencoded micrornas, including pcaf, a host factor required for tat-induced hiv- gene expression ( ) . hiv infection and host noncoding rna. in this study, we used ngs to measure all of the polyadenylated rnas in cd ϩ t lymphoblasts infected with intact, replication-competent hiv. ngs offers a number of potential benefits to the study of viral infections; among them is the ability to detect non-protein-coding rnas expressed during infection. in a previous study, we used ngs to detect long noncoding rnas in the lungs of severe acute respiratory syndrome (sars) coronavirus (cov)-infected mice ( ) . many of these rnas were found to be differentially expressed, and unique signatures of infection could be identified ( ) . in this study, we also detected several noncoding rna species in hiv-infected cells that were differentially expressed (de). in most cases, the functions of these noncoding rnas and their relevance to infection remains unknown. one noncoding rna of interest, the microrna host gene mir hg, encoded a cluster of micrornas and was strongly downregulated during infection. while our method of rna library preparation precluded direct detection of mature micrornas, the ngs data set allowed the expression of both regulators and targets of micrornas to also be observed. for example, we observed a concurrent upregulation of the known target host gene pcaf, a cellular factor required for hiv replication ( ) . in contrast to triboulet et al. ( ) who observed that pcaf was upregulated at the protein level but not the mrna levels in infected peripheral blood mononuclear cells (pb-mcs), we found that pcaf was upregulated at the mrna level in infected sup-t cells. this may indicate alternative modes of pcaf regulation in different cell types. in addition, mir hgencoded micrornas have hundreds of other candidate targets in the targetscan database ( ) . several of these candidates were found to be coexpressed with pcaf in our data set, including klf (unpublished data). klf encodes a zinc finger transcription factor whose precise function is unknown but whose sequence is located proximally to a single-nucleotide polymorphism strongly associated with hiv- plasma levels ( ) . we also observed differential expression of factors that may have mediated mir hg downregulation. for example, o'donnell et al. ( ) found that c-myc regulates expression of mir hg. consistent with that study, we observed that myc and mir hg were concordantly expressed, as myc was downregulated at and hpi (fig. ) . myc suppression by hiv- may therefore underlie a number of subsequent of transcriptional events. early regulation of host transcription factors. we also observed discordance between the large amount of viral rna present (~ % of total mappable rna) and the limited amount of altered host gene expression at hpi (~ % of detectable gene loci). that is, despite the use of host machinery and the presence of foreign mrna, host transcription remained relatively unchanged. this result suggests that at hpi, viral transcription occurred on top of largely undisturbed transcription of host genes. the host genes that we detected as differentially expressed at hpi showed that t cell activation and differentiation were suppressed in in- fected cells, likely via the downregulation of t cell activationspecific transcription factors (bcl b, irf , ikzf , and sox ) . the regulation of these and transcription factors specific for other functions (e.g., egr , klf , and myc) may explain how hiv initiated replication with minimal disruption to host gene expression at hpi but elicited larger-scale changes later in infection. the suppression of t cell activation that we observed was consistent with previous studies on the response of t cells to infection by chemokine (c-x-c motif) receptor (cxcr )-tropic versus chemokine (c-c motif) receptor (ccr )-tropic hiv- strains ( ) ( ) ( ) . for example, sirois et al. ( ) found that key t cell activation-related genes, including lck, were downregulated at hpi by a cxcr -tropic strain, whereas these same genes were upregulated by a ccr -tropic strain. our study utilized the cxcr -tropic strain lai, and it would be interesting to generate ngs data for a ccr -tropic virus for comparison. interestingly, our observations of small-magnitude differences in expression were consistent with those of sirois et al. ( ) , who found the expression of t cell activation-related genes to be changed by . fold or less using reverse transcription-pcr (rt-pcr). large-scale disruptions to host transcription near peak viral replication. by hpi, extensive reprogramming of the host transcriptome affecting a multitude of pathways had occurred. remarkably, these large-scale changes occurred without concerted upregulation of innate immunity at the gene set level, suggesting that hiv- evaded viral sensing mechanisms. by this time point, the most affected pathways related to rna fate determination, including rna processing. while hiv-related rna processing has been studied intensively, the contribution of most host factors remains incompletely defined ( ) . modulation of this pathway by the virus may allow the proportion of unspliced, partially spliced, and fully spliced hiv rna to be adjusted depending on the stage of the hiv life cycle. our finding that over genes related to rna processing were differentially expressed suggests that hiv- infection results in a more complete modulation of rna processing than previously identified ( , ) . however, our observed downregulation of rna processing was consistent with results from an earlier study using ngs to investigate changes in cd ϩ t lymphoblasts infected with an hiv-based, nonreplication-competent vector at hpi ( ) . altered regulation of this and other pathways has been observed using microarrays as well, although in general, we observed changes in greater numbers of genes affecting these pathways using ngs ( , , , , ) . finally, we compared our results from ngs to other types of data available regarding hiv-infected cells, including proteinprotein interaction (ppi) data and small interfering rna (sirna)-based screens. in an analysis of hiv-related ppi data, van dijk et al. ( ) identified sets of human proteins highly connected to host and viral proteins ( ) . several proteins related to t cell activation were identified as highly connected; these proteins included cd , cxcr , and lck (see table s in the supplemental material). downregulation of these members, as we observed, would therefore potentially affect the functions of many other proteins as well. together, these data emphasize the high degree to which hiv- suppressed this pathway. in addition, many of the pathways associated with de genes were identified as essential for hiv- replication in a recent meta-analysis of sirna-based screen data ( ) . these pathways included dna binding, rna splicing, rna export, nuclear transport, and protein complex assembly (table s ) . while the sirna data suggest that hiv- ini-tially requires these pathways to be active to establish an infection, our data suggest that hiv- also later suppresses and inactivates these pathways during active replication. in conclusion, using ngs, we have reported a number of unique findings in hiv-infected cells: the direct measurement of viral and host rna, the detection of small changes in the abundance of mrna transcripts, the identification of novel viral rna splice events, and the assay of multiple forms of noncoding rnas, including insights into the regulation of micrornas. these observations give additional insights into the panoply of changes that occur in host cells infected with hiv and provide the groundwork for using new sequencing technologies in future studies investigating the host response to viral infection. sup-t cells were obtained from american type culture collection (crl- ) and propagated in rpmi medium (gibco) supplemented with % fetal bovine serum (hyclone), penicillin ( u/ml), streptomycin ( g/ml), and glutamax-i. hiv- lai strain (catalog no. ) was obtained from the nih aids research and reference reagent program (germantown, md) and propagated in sup-t cells. u -magi-cxcr cem cells were obtained from m. emerman through the aids research and reference reagent program, and the virus titer in these cells was measured by the protocol of deminie and emerman ( ) . typical titers reached infectious units per ml. infections were carried out at a multiplicity of infection (moi) of and performed in triplicate. mock-infected samples received sup-t cell conditioned medium and were also performed in triplicate. the infectious dose was optimized to achieve % infected cells at hpi with % cell viability as measured by trypan blue exclusion assay. infected cells were visualized by immunofluorescence assay with rabbit hiv- sf p antiserum kindly provided by biomolecular technologies through the aids research and reference reagent program. rna preparation and next-generation sequencing. total rna was extracted from ϫ cells per sample using a mirvana kit (applied biosystems/ambion, austin, tx), and the quality and concentration of the rna were determined by an agilent bioanalyzer. samples were submitted for sequencing to illuminafasttrack sequencing services (hayward, ca). cdna libraries were generated using illumina mrna-seq kit. the quality and concentration of these libraries were determined by an agilent bioanalyzer. the libraries were clonally amplified on a cluster generation station using illumina version cluster generation reagents to achieve a target density of approximately , ( k)/mm in a single channel of a flow cell. the resulting libraries were then sequenced on a genome analyzer iix using illumina version . sequencing reagents which generated single reads of nucleotides (nt). image analysis, base calling, and error estimation were performed using illumina analysis pipeline (version . ). read mapping and transcript quantification. we mapped the -nt reads to human ribosomal sequences using the short-read aligner software bowtie to remove potential rrna sequences ( ) . we then mapped the remaining unmapped reads to the hiv genome (genbank accession no. k ) using the gapped aligner software tophat, which predicts hiv splicing junctions and maps intron-spanning reads to known splicing junctions ( ) . to quantify transcript expression, we mapped all reads that remained unmapped to the human reference genome (hg , build grch , downloaded from ucsc genome browser, http://genome.ucsc .edu) using the gapped aligner software tophat. refseq transcript annotations were supplied to facilitate the mapping of reads spanning known splicing junctions. on the basis of these human genome mapping results, we then estimated the levels of expression at both the transcript and locus levels for refseq-annotated genes using the transcript assembly software cufflinks ( ) . read sequences that mapped to more than one genomic location were excluded from expression quantification. for visualization, bam files were generated using tophat and samtools ( ) and displayed using the ucsc genome browser. splice site variant detection and testing. the positions of known viral splice sites were found in the literature ( ) , and the corresponding sequences were identified in strain pnl - (genbank accession no. af ). based on sequence identity to strain lai, a list of known lai-specific splice sites was generated and compared to the ngs tophat output. splice patterns involving splice sites sd and sa / * were tested by quantitative pcr (qpcr) using primers psk f ( = cagggacttg aaagcgaaag =; locations to in hivbrucg accession no. k ) and psk r ( = tggggcttgttccatctatc =; locations to ). quantitative reverse transcription (rt-pcr). rna was reverse transcribed using the quantitect reverse transcription kit (qiagen, valencia, ca). the resulting cdna samples were diluted ϫ. abi taqman assays were run for each sample in triplicate (see fig. s c in the supplemental material). relative expression was calculated using the ⌬⌬c t method with averaged ⌬c t values (where c t stands for threshold cycle) for the oaz gene as a calibrator, as the expression of oaz did not significantly change between and hpi in the ngs data. intracellular viral rna load was quantified as previously described ( ) . relative change of irf was determined by qpcr using primers psk (forward primer [ = tctg gctttttcctctgagc =] and reverse primer [ = atgcttttctgg ggtcactg =]). differential expression analysis. to compare transcript expression across different conditions, we first normalized transcript abundances by the following methodology. transcript abundance was quantified as fpkm (fragments per kilobase of exon per million mapped fragments) values estimated by cufflinks. we chose one sample arbitrarily as a reference. distributions of log -transformed fpkm values between the reference and remaining samples were compared by quantile-quantile plots. we determined the scaling factor for each sample as the median difference of the corresponding quantile values of the sample and reference. only genes/transcripts with raw fpkm values of Ն in all samples were considered in the estimation of scaling factors. we retained those genes/transcripts with nonzero fpkm values in % of the samples of at least one biological condition (our detection criterion). an offset of was added to all normalized values to facilitate the comparisons involving one or more fpkm values of zero and to reduce the variability of the log ratios for low expression values. transcripts were mapped to refseq gene loci, resulting in , loci with detectable reads. the data are available at http://www .viromics.washington.edu or upon request. the normalized expression data were analyzed for differential expression by using linear model methods as implemented in the r package limma ( ) . p values were derived from linear model-based t tests between infected and time-matched mock-infected samples. unless otherwise noted, we defined differential expression by benjamini-hochberg-adjusted p values of less than . based on the assumption that a false discovery rate of % provided an acceptable balance of false-positive control and statistical power. fold changes (fc) were derived from comparing the means of these groups, and multiple groupings of fold changes were used ( . to . fc, . to . fc, and . ϩ fc) based on previous observed fold change ranges observed in high-throughput and in particular ngs data in virus-infected systems ( ) . david and gsea analyses. to identify annotations among de genes, we used the nih david resource ( , ) . default settings were used in david with go_bp_fat, go_cc_fat, go_mp_fat, bio-carta, and kegg_pathway gene set annotations. complementary analysis was performed using gene set enrichment analysis (gsea) ( ) . default settings were used in gsea with gene ontology (go) categories (c .all.v . gene sets) and biocarta and kegg pathways (c .all.v . gene sets) and , gene set-based permutations. leading-edge analysis was performed within gsea to derive genes for hierarchically clustering upand downregulated gene sets. interactions between de genes were identified using ingenuity pathways analysis (ipa) software (ingenuity systems, redwood city, ca). networks were generated within ipa based on direct, literature-curated interactions. subsets of genes were selected for input into ipa based on de gene overlaps with david annotation clusters. heat maps were generated using the r package gplots. comparisons to published networks of protein-protein interaction (ppi) data were made using cytoscape ( ) . this work was funded by public health service grants p da and p rr from the national institutes of health. we thank matthew thomas and richard green for technical assistance and marcus korth for editorial assistance. we also thank peter sloot and david van dijk for generously sharing expertise related to proteinprotein interaction networks. supplemental material for this article may be found at http://mbio.asm.org /lookup/suppl/doi: . /mbio. - /-/dcsupplemental. microarray data on gene modulation by hiv- in immune cells: - molecular biology of human immunodeficiency virus type large-scale monitoring of host cell gene expression during hiv- infection using cdna microarrays functional genomics analyses of differential macaque peripheral blood mononuclear cell infections by human immunodeficiency virus- and simian immunodeficiency virus cellular gene expression upon human immunodeficiency virus type infection of cd (ϩ)-t-cell lines transcriptional profiling in pathogenic and nonpathogenic siv infections reveals significant distinctions in kinetics and tissue compartmentalization a comparison of massively parallel nucleotide sequencing with oligonucleotide microarrays for global transcription profiling unique signatures of long noncoding rna expression in response to virus infection and altered innate immune signaling bcl b is a general transcriptional repressor of the hiv- long terminal repeat in t lymphocytes through recruitment of the nurd complex hiv- down-regulates the expression of cd d via nef gene set enrichment analysis: a knowledgebased approach for interpreting genome-wide expression profiles rna and disease hnrnp a controls hiv- mrna splicing through cooperative binding to intron and exon splicing silencers in the context of a conserved secondary structure role of cellular rna processing factors in human immunodeficiency virus type mrna metabolism, replication, and infectivity the multiple roles of tdp- in pre-mrna processing and gene expression regulation cloning and characterization of a novel cellular protein, tdp- , that binds to human immunodeficiency virus type tar dna sequence motifs tdp- : multiple targets, multiple disease mechanisms? retroviral mrna nuclear export elements regulate protein function and virion assembly nuclear mrna export: insights from virology nef induces apoptosis by activating jnk signaling pathway and inhibits nf-kappab-dependent immune responses in drosophila irf- -dependent and augmented target genes during viral infection transcriptional profiling of interferon regulatory factor target genes: direct involvement in the regulation of interferon-stimulated genes the cytosolic exonuclease trex inhibits the innate immune response to human immunodeficiency virus type human immunodeficiency virus type mediates global disruption of innate antiviral signaling and immune defenses within infected cells suppression of microrna-silencing pathway by hiv- during virus replication most mammalian mrnas are conserved targets of micrornas distinct genetic loci control plasma hiv-rna and cellular hiv-dna levels in hiv- infection: the anrs genome wide association study c-myc-regulated micrornas modulate e f expression r and x hiv envelopes induce distinct gene expression profiles in primary peripheral blood mononuclear cells differential effects of r and x human immunodeficiency virus type infection on cd ϩ cell proliferation and activation r and x hiv viruses differentially modulate host gene expression in resting cd ϩ t cells regulation of hiv- alternative rna splicing and its role in virus replication microarray study reveals that hiv- induces rapid type-i interferon-dependent p mrna upregulation in human primary cd ϩ t cells analysis of hiv- expression level and sense of transcription by high-throughput sequencing of the infected cell temporal gene regulation during hiv- infection of human cd ϩ t cells impact of viral infection on the gene expression profiles of proliferating normal human peripheral blood mononuclear cells infected with hiv type rf identifying potential survival strategies of hiv- through virus-host protein interaction networks host cell factors in hiv replication: metaanalysis of genome-wide studies quantitation of virus stocks produced from cloned human immunodeficiency virus dna ultrafast and memory-efficient alignment of short dna sequences to the human genome tophat: discovering splice junctions with rna-seq transcript assembly and quantification by rna-seq reveals unannotated transcripts and isoform switching during cell differentiation the sequence alignment/map format and samtools alternative splicing of human immunodeficiency virus type mrna modulates viral protein expression, replication, and infectivity detection and quantification of human immunodeficiency virus type p antigen in dried whole blood and plasma on filter paper stored under various conditions linear models and empirical bayes methods for assessing differential expression in microarray experiments bioinformatics enrichment tools: paths toward the comprehensive functional analysis of large gene lists systematic and integrative analysis of large gene lists using david bioinformatics resources cytoscape: a software environment for integrated models of biomolecular interaction networks key: cord- -chvvtr d authors: fidel, paul l.; noverr, mairi c. title: reply to Özdemir, “measles-mumps-rubella vaccine and covid- relationship” date: - - journal: mbio doi: . /mbio. - sha: doc_id: cord_uid: chvvtr d nan the question as to whether there may be a cumulative effect of live attenuated vaccines for the nonspecific beneficial immune effects is interesting. while the current clinical trials are not investigating this issue directly, we have focused on the mmr vaccine as it is widely available and has the potential for any or all of the three components to induce the mdscs. however, based on our data in the animal model of fungal/bacterial sepsis, very strong long-lasting protection is afforded from one administration of the attenuated fungal isolate ( ) . hence, cumulative effects may not be important with the exception of extending the time for optimal protection with timely boosters. finally, while it is true that we do not know how long the trained innate immunity persists, the randomized clinical trial of mmr versus placebo in health care workers and the nonhuman primate study that will test mmr or bcg in a model of covid- infection will go far to answer these questions. to date, the trained innate response with bcg suggests the immunity is functional for approximately year based on infant vaccinations ( ) . the studies in the animal model of fungal/bacterial sepsis show that the mdscs are induced quickly (within days of vaccination) and are relatively longlived (up to days) ( , ) . data also show that the protection afforded by the trained innate response can be achieved with multiple lethal challenges over time ( ) . since these are mouse studies with relatively short lifespans, we can predict a fairly long half-life for the immune-tolerant trained response in humans. indeed, the sequelae and hospitalization associated with covid- infection are much reduced in children, teenagers, and even young adults in their early s ( , ) . children often get the mmr vaccine up to three times by age . thus, it is possible that the nonspecific protective effects provided by the live attenuated vaccines are quite long-lived. until more is known, however, we are suggesting that the mmr vaccination be given to eligible adults who have not had the vaccination within a year, as an immune preventive against the worst sequelae of any subsequent covid- infection. could an unrelated live attenuated vaccine serve as a preventive measure to dampen septic inflammation associated with covid- infection? significantly improved covid- outcomes in countries with higher bcg vaccination coverage: a multivariable analysis exploratory analysis of immunization records highlights decreased sars-cov- rates in individuals with recent non-covid- vaccinations candida/staphylococcal polymicrobial intra-abdominal infection: pathogenesis and perspectives for a novel form of trained innate immunity homologous protein domains in sars-cov- and measles, mumps and rubella viruses: preliminary evidence that mmr vaccine might provide protection against covid- mmr vaccine appears to confer strong protection from covid- : few deaths from sars-cov- in highly vaccinated populations immune protection against lethal fungal-bacterial intra-abdominal infections can a century-old tb vaccine steel the immune system against the new coronavirus spectrum of trained innate immunity induced by low-virulence candida species against lethal polymicrobial intra-abdominal infection epidemiology of covid- among children in china detection of covid- in children in early key: cord- -grrd jmt authors: tournier, jean-nicolas title: pandemic legion history more complex than previously thought date: - - journal: mbio doi: . /mbio. - sha: doc_id: cord_uid: grrd jmt nan morens and colleagues describe in the "early pandemic history" section the story of pathogens emerging around , years ago at the time of neolithic agricultural revolution. as such, diseases such as "measles, smallpox, tuberculosis (tb), [and] gastric cancer (caused by helicobacter pylori)" are cited as consequences of "conditions of intense human-animal proximity and environmental alterations." this assertion of the dating of the origin of these aforementioned pathogens is partially misleading. both viruses (i.e., those causing measles and smallpox) emerged probably much later, while mycobacterium tuberculosis and helicobacter pylori started their association with humans before the agricultural revolution. historical records of viruses are scarce, and the reconstruction of their evolutionary history might be difficult ( ) ( ) ( ) . however, a recent phylogenetic study of a strain has placed measles virus (mv) divergence from rinderpest virus during the sixth century before the common era (bce), possibly coinciding with the rise of large cities allowing measles epidemic sustainability ( ) . for smallpox, the exact date of divergence of variola virus (varv) from a zoonotic strain is more disputed, as molecular data gave an estimation of emergence for the most recent common ancestor between the th and the th century, while skin lesions seen in the mummy of ramses v, who died in bce, suggested earlier interactions ( ) . camelpox virus and taterapox virus infecting gerbils very likely shared a common ancestor with varv that might have evolved from a common rodent orthopoxvirus ( , ) . a recent study of ancient varv samples from viking remains discovered a sister clade and predated varv emergence as early as ce, confirming written accounts of likely smallpox infections in europe from the late th century ( ) . in contrast, the interactions of m. tuberculosis and h. pylori with humans represent much more sophisticated and longer stories. a thorough phylogeographic analysis of modern h. pylori diversity has shown that the homo sapiens became its specific host before he started his migration out of africa , years ago and no later than , years ago ( ) . for tb, recent studies have clearly demonstrated that in opposition to a frequently reported idea, human tb is not a zoonosis derived from bovine tb arisen as a consequence of cattle domestication ( ) . m. tuberculosis and mycobacterium bovis share more than . % nucleotide identity, and while they probably do have a common ancestor, m. bovis is definitely not the parent of m. tuberculosis ( , ) . concerning m. tuberculosis emergence dating, several studies are conflicting ( ) , but at least one study estimated that the most recent common ancestor of m. tuberculosis existed , years ago ( ) . moreover, on yersinia pestis, the agent of plague, morens and colleagues mentioned the "plague of athens" ( to bce) as "perhaps the first recorded pandemic," although there is a wealth of data proving a much earlier presence of y. pestis starting at least , years ago ( , ) . eventually, the conception of the agricultural revolution affecting health through direct emerging infections from domesticated livestock needs to become more balanced. the mv example has shown that emergence by accident is not the rule and that a pathogen needs more conditions than a close contact with human for sustainable success. pandemic covid- joins history's pandemic legion evolutionary analysis of the dynamics of viral infectious disease dating the emergence of human pathogens ancient pathogen genomics as an emerging tool for infectious disease research measles virus and rinderpest virus divergence dated to the sixth century bce diverse variola virus (smallpox) strains were widespread in northern europe in the viking age poxviruses and the evolution of host range and virulence genome sequence diversity and clues to the evolution of variola (smallpox) virus myths and misconceptions: the origin and evolution of mycobacterium tuberculosis the complete genome sequence of mycobacterium bovis ecology and evolution of mycobacterium tuberculosis out-of-africa migration and neolithic coexpansion of mycobacterium tuberculosis with modern humans emergence and spread of basal lineages of yersinia pestis during the neolithic decline the history of helicobacter pylori: from phylogeography to paleomicrobiology key: cord- -bnqmwst authors: hyser, joseph m.; collinson-pautz, matthew r.; utama, budi; estes, mary k. title: rotavirus disrupts calcium homeostasis by nsp viroporin activity date: - - journal: mbio doi: . /mbio. - sha: doc_id: cord_uid: bnqmwst many viruses alter intracellular calcium homeostasis. the rotavirus nonstructural protein (nsp ), an endoplasmic reticulum (er) transmembrane glycoprotein, increases intracellular levels of cytoplasmic ca( +) ([ca( +)]cyto) through a phospholipase c-independent pathway, which is required for virus replication and morphogenesis. however, the nsp domain and mechanism that increases [ca( +)]cyto are unknown. we identified an nsp domain (amino acids [aa] to ) that inserts into membranes and has structural characteristics of viroporins, a class of small hydrophobic viral proteins that disrupt membrane integrity and ion homeostasis to facilitate virus entry, assembly, or release. mutational analysis showed that nsp viroporin activity was mediated by an amphipathic α-helical domain downstream of a conserved lysine cluster. the lysine cluster directed integral membrane insertion of the viroporin domain and was critical for viroporin activity. in epithelial cells, expression of wild-type nsp increased the levels of free cytoplasmic ca( +) by . -fold, but nsp viroporin mutants maintained low levels of [ca( +)]cyto, were retained in the er, and failed to form cytoplasmic vesicular structures, called puncta, which surround viral replication and assembly sites in rotavirus-infected cells. when [ca( +)]cyto was increased pharmacologically with thapsigargin, viroporin mutants formed puncta, showing that elevation of calcium levels and puncta formation are distinct functions of nsp and indicating that nsp directly or indirectly responds to elevated cytoplasmic calcium levels. nsp viroporin activity establishes the mechanism for nsp -mediated elevation of [ca( +)]cyto, a critical event that regulates rotavirus replication and virion assembly. disrupt calcium homeostasis in ways that favor virus replication, assembly, and/or release ( , ) . the best-characterized viroporins that disrupt calcium homeostasis are the enterovirus b proteins ( ) . biochemical analysis indicates that b forms a transmembrane hairpin structure that inserts into the endoplasmic reticulum (er), golgi, and plasma membranes ( ) . the ability of b to increase levels of cytoplasmic ca ϩ ([ca ϩ ]cyto) depends on the cationic and amphipathic nature of the pore-forming domain and is essential for virus release ( ) . rotaviruses (rv) are triple-layered nonenveloped viruses, have genomes composed of segments of double-stranded rna, and cause life-threatening viral gastroenteritis in children worldwide ( , ) . rv infection alters cellular calcium homeostasis by increasing [ca ϩ ]cyto by -to -fold, increasing cellular uptake of ca ϩ by -fold, depleting agonist-releasable er calcium stores, and substantially increasing plasma membrane cation permeability ( ) ( ) ( ) . elevated cytoplasmic and er luminal calcium levels are crucial for virus replication and morphogenesis, since virus yields are decreased if [ca ϩ ]cyto is reduced by chelating extracellular calcium with edta, buffering [ca ϩ ]cyto with , -bis(oaminophenoxy)ethane-n,n,n=,n=-tetraacetic acid tetra(acetoxymethyl) ester (bapta-am), blocking plasma membrane l-type calcium channels with methoxyverapamil, or blocking sarco/endoplasmic reticulum calcium atpase (serca) pumps with thapsigargin (tg) ( , , , ) . all of these changes in calcium homeostasis are recapitulated by expressing the rotavirus nonstructural protein (nsp ) alone in insect and mammalian cells ( ) ( ) ( ) . nsp is an er transmembrane glycoprotein that acts through a phospholipase c (plc)independent pathway to elevate [ca ϩ ]cyto, suggesting that it may disrupt calcium homeostasis independent of the er-associated inositol , , -trisphosphate receptor (ip r) calcium release channel. the current studies sought to identify the domain of nsp that elicits the plc-independent elevation of cytoplasmic calcium levels, to identify the mechanism by which that domain functions, and to understand whether calcium release by nsp regulates later steps in the rv replication cycle. the nsp membrane-destabilizing domain has structural similarities to known viroporins. one goal of these studies was to identify the nsp domain that induces er calcium permeability and the plc-independent elevation of [ca ϩ ]cyto. previously, an er-proximal polybasic domain (amino acids [aa] to ) was shown to permeabilize both mammalian and escherichia coli membranes ( , ) . in bl (de )plyss e. coli, nsp expression disrupts inner membrane integrity, leading to t lysozymemediated cell lysis ( ) . since other viroporins cause lysis in this assay ( ) ( ) ( ) , we hypothesized that this domain of nsp functioned as a viroporin. we analyzed nsp for the following structural motifs common to viroporins: oligomerization domains, lysine-or arginine-rich basic regions, and amphipathic ␣-helices ( , ) . previous studies showed that nsp oligomerizes through a coiled-coil domain (ccd; aa to ) ( ) . next, based on the helical propensity of nsp aa to (nsp ), helical wheel models of the simian agent (sa ) nsp putative viroporin domain were generated. the n-terminal helix is predicted to be amphipathic and contains a cluster of lysine residues on one face of the helix. the c-terminal helix is also predicted to be amphipathic, with distinct polar and nonpolar surfaces (fig. a) . these subdomains were named the pentalysine domain (pd) and amphipathic domain (ad). to determine if the pd, ad, and ccd were necessary for membrane permeabilization, sa nsp truncation and deletion constructs were tested for membrane-destabilizing activity (mda) using the bacterial lysis assay (fig. b) . in the absence of iptg (isopropyl-␤-d-thiogalactopyranoside), bacteria bearing an nsp - expression vector continued to grow (fig. c , black line), but induction of nsp expression led to rapid cell lysis (red line). cell lysis was not directly caused by nsp because the optical density of bl (de ) e. coli (lacking lysozyme) expressing nsp - remained stable (see fig. s in the supplemental material). expression of nsp , which includes only the pd and ad, slowed the kinetics but not the extent of cell lysis (fig. c , orange line). deletion of the pd had no effect on mda (fig. c , blue line), but deletion of the ad abrogated lysis (green line). immunoblot analysis showed that all four constructs were expressed, though detection of nsp - required that -fold more cell lysate had to be analyzed, reflecting a lower level of expression (fig. d ). the mda of nsp suggested that the putative viroporin domain could oligomerize independently of the ccd. to test this, partially purified nsp - was analyzed by immunoblotting following reducing or nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds-page) (fig. e ). in the absence of ␤-mercaptoethanol (bme), the major species detected had the fastest-migrating band of~ kda, and slower-migrating species were also detected, with their molecular masses increasing in~ -kda increments. in the presence of bme, the fastest-migrating band was~ kda, and slower-migrating species increased in molecular masses of~ -kda increments. these data indicate that nsp - forms disulfide-stabilized dimers that further oligomerize into higher-molecular-weight species that are stabilized by the lipidmimetic sds in the running buffer. the putative viroporin domain contains two conserved cysteine residues (fig. a , arrowheads) that could support disulfide bond formation, and sds-stabilized oligomers have been observed for other viroporins ( ) ( ) ( ) . together, these data show that the viroporin domain oligomerizes independently of the ccd and is sufficient for nsp mda and that this activity is mediated by the ad amphipathic ␣-helix. pd lysine residues , , and promote nsp mda. the above-described results suggested that the pd is dispensable for mda when this domain is deleted from nsp , leaving the ad with a free amino terminus. however, clustered lysine residues are important for the activity of other viroporins ( , ) , so we next sought to determine whether the five clustered lysine residues were important for nsp mda in the context of the full viroporin domain. first, using site-directed mutagenesis, all five positively charged lysine residues (aa , , , , and ) in the sa nsp - truncation were replaced by glutamic acid, a negatively charged residue that would not disrupt the helical propensity. this mutation blocked mda, indicating that one or more lysine residues were crucial for nsp -mediated membrane disruption (data not shown). to determine which lysine residues were important, each lysine was replaced by glutamic acid individually or in combinations of two or three within the context of sa nsp . this slightly shorter construct was used to make the generation of multiple mutations easier, and no lysine-to-glutamic acid mutants were tested in the e. coli lysis assay. the od of uninduced (Ϫiptg, black line) or nsp -expressing cultures was determined at -min intervals for minutes and presented as the percent optical density relative to the optical density at the time of induction. (bottom) immunoblot of nsp expression using monoclonal antibody b - / . (b, top) lysine-to-alanine and lysine-to-histidine mutants were tested in the e. coli lysis assay, as described above. (bottom) immunoblot of nsp expression using monoclonal antibody b - / . difference in the rate or extent of bacterial cell lysis was observed between nsp - and nsp (compare red lines of fig. c and a). the mda of wild-type (wt) nsp - (red line) was equivalent to that of the single lysine-to-glutamic acid mutant k e ( fig. a , orange line) and the other single mutants (data not shown). double or triple mutations of any combination of k , k , and k to glutamic acid also showed wild-type mda (data not shown). double or triple mutations of any combination of k , k , and k to glutamic acid (k , , e) reduced nsp - mda ( fig. a) , with the triple k , , e mutation (gray line) showing continued growth similar to that of the uninduced negative control. immunoblot analysis of total cell lysates showed expression of each mutant ( fig. a, bottom) . these data indicate that the c-terminal lysine res-idues (aa , , and ) within the pd were important for nsp mda. lysine is uniquely suited for membrane interaction because the positively charged -amine can form an electrostatic interaction with the negatively charged phosphate head group, bringing a protein in close proximity to the membrane ( ) . positively charged residues (lysine or arginine) are highly conserved within the pd among all serogroup a rotavirus nsp sequences ( ) , suggesting that the charge may be functionally relevant. to determine whether the charges of residues , , and were crucial for nsp mda, we constructed lysine-toalanine (a nonpolar residue) and lysineto-histidine (an aromatic basic residue) mutants and tested them in the bacterial lysis assay (fig. b) . while both the double (k , h) (fig. b , maroon line) and triple (k , , h) (blue line) lysine-tohistidine mutants showed wild-type mda, the double lysine-to-alanine mutant (k , a) (fig. b , gray line) showed impaired activity, and the triple lysine-to-alanine mutant (k , , a) (purple line) had no mda, similar to that of the k , , e mutant (fig. b ). immunoblot analysis confirmed similar expression of the constructs (fig. b , bottom). these data indicate that mutations that disrupt the positive charge of the pd also disrupt nsp mda. amphipathicity of the viroporin domain is necessary for mda. in viroporins, the amphipathic ␣-helix forms the pore lumen upon oligomerization of the protein. thus, disruption of nsp ad amphipathicity would be predicted to abolish mda. to test this prediction, a six-residue mutant was constructed that changed aa to from ifntll to asdasa and substantially decreased the amphipathic moment of the ad (see fig. s in the supplemental material). as expected, the asdasa mutant had no mda (fig. a , gray line). to identify residues on the polar and nonpolar sides of the ad crucial for mda, we made point mutations in the context of sa nsp . residues on the nonpolar side were replaced by either serine or asparagine (small polar residues), whereas residues on the polar side were replaced by either alanine or leucine (small and large nonpolar residues). the mda of the ad mutants clustered into the following three groups: wild-type mda (c s, f s, n a, t a, l s, k l, and n a t a) ( the importance of ␣-helical secondary structure was investigated by inserting a proline-glycine dipeptide (pg) at various places within the pd or ad (see fig. s in the supplemental material) within the nsp - construct. since pg disrupts helices, if the structure at the site of insertion is important, then the mda would be reduced. pg insertion after lysine , which is not important for mda, had wild-type mda (see fig. s , orange line, in the supplemental material). pg insertion after residues or reduced the rate, but not the extent, of mda (see fig. s , green and blue lines). pg insertion after residues and (within the ad) significantly impaired and abolished mda, respectively (see fig. s , dark blue and gray lines, respectively). thus, the propensity of the viroporin domain to form a helical structure is important, though to differing degrees depending on the location. nsp bacterial cytotoxicity is mediated by the viroporin domain. while bacterial lysis in the above-described assays was mediated by t lysozyme, it remained unclear whether nsp was cytotoxic to cells. since the optical density of cells lacking lysozyme upon induction of nsp - expression remained unchanged (see fig. s in the supplemental material), nsp expression may cause cell death in the absence of cell lysis. to determine if wild-type or mutant nsp proteins decreased cell viability, the number of cfu/milliliter plated in the absence or presence of mm iptg was determined (fig. ) . wild-type nsp - , which lacks the viroporin domain, was used as the negative control and showed a decrease of only ϫ cfu/ml. in contrast, the viability of wild-type nsp - -expressing cells dropped by ϫ cfu/ml, a Ͼ % decrease. the ntk mutant had a similar drop in cell viability, but expression of the k , , e, f s l s, and asdasa mutants showed a significantly attenuated loss of viability. thus, these data show that nsp itself is cytotoxic to bacteria without causing cell lysis, and the putative viroporin domain is the primary mediator of this cytotoxicity. the nsp viroporin domain is inserted into membranes. based upon the structure of the b viroporin, the nsp viroporin domain would be predicted to form a two-helix transmembrane hairpin. however, this predicted structure conflicts with the accepted topology of full-length nsp , wherein aa to is an er signal sequence that directs transmembrane insertion, and the viroporin domain is peripherally associated with the cytoplasmic surface of the membrane ( ) . thus, we sought to determine if, in the absence of the transmembrane domain encompassing aa to , the viroporin domain is peripherally associated with membranes, as predicted by the current topology model, or inserted through membranes, as would be expected if nsp is a viroporin. a series of truncations (fig. a ) that contain both the viroporin domain and the soluble ccd (nsp ), only the viroporin domain (nsp ), or only the ccd (nsp - ), a soluble tetrameric coiled-coil domain ( ) , were expressed in bacteria. the cells were separated into soluble, peripheral, or integral membrane fractions and analyzed by immunoblotting (fig. b ). both nsp and nsp were found solely in the integral membrane protein fraction, but nsp - was found predominantly in the soluble fraction, indicating the viroporin domain directed membrane insertion of the nsp - and nsp truncations. to determine if the pd or ad mutants altered membrane insertion of the viroporin domain, we compared the membrane fractionation profiles of k , , , , e and asdasa with that of wild-type nsp (fig. c ). as before, wild-type nsp was found entirely in the integral membrane fraction, as was the asdasa mutant. the k , , , , e mutant migrated slightly faster due to a greater net negative charge and was found primarily in the peripheral membrane protein fraction, with a minor amount detected in the integral membrane fraction. thus, while disruption of both the positive charge of the pd and the amphipathic organization of the ad blocked nsp mda, only disruption of the positive charge prevented transmembrane insertion of the viroporin domain. together, these data point to a role for the pd in membrane insertion of nsp . to assess whether mutations of the viroporin domain affected the expression or folding of full-length nsp expressed in mammalian monkey kidney cells, nsp -enhanced green fluorescent protein (egfp) fusion proteins for wt or k , , e,ntk and asdasa viroporin mutants were tested by immunoblotting. wt nsp -egfp was expressed, and endo-␤-nacetylglucosaminidase h (endo h) treatment, to remove n-linked carbohydrates, increased nsp migration, indicating that it was glycosylated (fig. d ). both the ntk and asdasa mutants were expressed and glycosylated similarly to wt nsp (ntk data not shown). k , , e was barely detectable by immunoblotting, migrated similarly to unglycosylated nsp , and was not susceptible to endo h treatment; however, treatment with the proteosome inhibitor mg ( m) increased levels of nonglycosylated k , , e (fig. d) , suggesting that it might be targeted for endoplasmic reticulum-associated degradation (erad) ( ) . these data were confirmed by flow cytometry, as the mean fluorescence intensity (mfi) for k , , e was significantly lower than that of the wt and increased % with mg treatment (fig. e ). to ensure that k , , e was still membrane associated, we sepa- rated cells into soluble, peripheral, and integral membrane fractions, marked by immunoblot detection of gfp, gm , and stim- , respectively (see fig. s in the supplemental material). in the absence of mg , the wt and asdasa were detected exclusively in the integral membrane fraction. in the presence of mg , k , , e was found primarily in the integral membrane fraction, but some protein was detected in the soluble and peripheral membrane protein fractions (fig. f ). together, these data suggest that nsp is initially targeted to the er membrane via the noncleaved signal sequence (aa to ), but the lysine cluster directed translocation of the viroporin domain (aa to ) into the membrane. altering the charge of the lysine cluster blocked membrane insertion of the viroporin domain in e. coli and targeted nsp for erad-mediated degradation in mammalian cells. thus, the lysines facilitate proper nsp folding by directing viroporin domain insertion into the membrane. viroporin mutants fail to elevate intracellular calcium levels in mammalian cells. intracellular expression of fulllength nsp in eukaryotic cells leads to increased er permeability and a significant increase in [ca ϩ ]cyto ( , ) . we tested the mutants characterized using the e. coli lysis assay to determine if the viroporin domain is responsible for the increased [ca ϩ ]cyto using the fluorescent calcium indicator indo- . using flow cytometry, we measured the mean [ca ϩ ]cyto of cells expressing egfp, the wt, or viroporin mutant nsp -egfp (fig. ) . expression of wt nsp -egfp increased the ratio of calcium-bound to calcium-free indo- fluorescence, shifting the egfp-positive population to the right (fig. a, blue) , but histograms for cells expressing nsp -egfp k , , e or asdasa remained clustered to the left (fig. a , red and green, respectively). cytoplasmic calcium levels (fig. b) ]cyto causes rapid formation of the puncta, and in rotavirus-infected cells, nsp colocalizes with lc in these puncta that surround viroplasms, cytoplasmic inclusions where genome replication and progeny virus assembly occur ( ) . thus, assessment of nsp puncta formation acts as a surrogate for testing the ability of viroporin mutants to form the viroplasm-associated puncta. the subcellular localization and distribution of wt or mutant nsp -egfp fusion proteins were analyzed in normal medium ( . mm cacl ), which supports spontaneous puncta formation for wildtype nsp . the extent of er localization for egfp, wt nsp - egfp, and the three nsp -egfp viroporin mutants (k , , e, ntk, and asdasa) was determined by colocalization with an er-targeted dsred fluorescent protein using confocal microscopy (fig. a) . egfp was found throughout the cell and did not localize to the er or form puncta (fig. a, first row) . as seen previously, wt nsp -egfp localized partially to the er compartment and to distinct puncta that did not contain the dsred-er marker (fig. a, second row) . in contrast, nsp -egfp viroporin mutants were localized primarily in the er, with cells expressing k , , e and asdasa lacking puncta (fig. a , third and fifth rows, respectively), with a few small discrete puncta that did not contain dsred-er being observed in cells expressing ntk. next, we tested if changes in [ca ϩ ]cyto directly regulated puncta formation by quantitating the number of nsp -egfpexpressing cells containing puncta (i) in normal dmem, (ii) after treatment with the intracellular calcium chelator bapta-am, or (iii) after treatment with tg, a serca pump inhibitor that elevates cytoplasmic calcium levels. this experiment was designed to determine if bapta treatment to buffer the elevated [ca ϩ ]cyto caused by the viroporin activity of wt nsp would decrease puncta formation and if tg treatment to pharmacologically elevate [ca ϩ ]cyto would induce puncta formation of nsp viroporin-deficient mutants. in normal dmem, wt nsp -egfp formed discrete puncta in . % of cells, but the viroporin mutants formed significantly fewer puncta, with rates of . % for k , , e, . % for ntk, and . % for asdasa (fig. b , black bars) (p Ͻ . ). bapta-am treatment of wt nsp -egfp-expressing cells reduced puncta formation to . % (fig. b) (p Ͻ . ). an overall decrease in the number of punctacontaining cells was also observed in bapta-treated viroporin mutant-expressing cells (fig. b ) (p of Ͻ . for ntk and as-dasa). finally, cytoplasmic calcium levels were elevated pharmacologically with tg to determine if exogenous calcium stimulation would induce puncta formation of the viroporin nsp -egfp mutants. tg treatment did not significantly increase the number of puncta-containing wt nsp -egfp-expressing cells (fig. b) . in contrast, tg treatment increased puncta formation by . % for k , , e, . % for ntk, and . % for asdasa (fig. b, light gray) . thus, mutation of the viroporin domain decreased spontaneous puncta formation; however, pharmacological elevation of [ca ϩ ]cyto induced puncta formation of the mutant proteins. together, these data show (i) that the viroporin mutants were specifically deficient in the elevation of [ca ϩ ]cyto but not in the ability to form puncta, (ii) that elevated [ca ϩ ]cyto triggers the trafficking of nsp out of the er and into cytoplasmic puncta, and (iii) that elevation of [ca ϩ ]cyto and formation of puncta are separable steps within this process. seeking to define the mechanism for the plc-independent increase in [ca ϩ ]cyto, we investigated a previously described nsp domain (aa to ) with membrane-destabilizing activity that mediates cytotoxic effects in both e. coli and mammalian cells ( , ) . this study reports a comprehensive biochemical and mechanistic characterization of a viroporin domain from a viral nonstructural protein that alters cellular calcium homeostasis to regulate the progression of virus replication and assembly. the major new findings of this study are as follows: (i) nsp aa to were structurally similar to those of the enterovirus b protein and functionally consistent with the defining characteristics of viroporins, (ii) the nsp pd mediated integral membrane insertion of the viroporin domain, (iii) nsp viroporin mutants that failed to induce e. coli lysis and cytotoxicity also failed to elevate [ca ϩ ]cyto in mammalian cells, and (iv) elevation of [ca ϩ ]cyto regulates the subcellular distribution of nsp by triggering the movement of nsp out of the er and into cytoplasmic puncta. using the e. coli lysis assay, we were able to show that the pd and ad are functionally distinct motifs within the viroporin domain. the pd functioned as a membrane insertion motif, but the pd alone did not support viroporin activity. in contrast, viroporin activity was mediated by the ad (aa to ), and mutation of this motif blocked viroporin activity but not membrane insertion. the ad also plays a role in nsp oligomerization by promoting the formation of high-molecular-weight nsp multimers that were lost by disrupting this domain ( ) . we confirmed this observation, since expression of the viroporin domain alone (aa to ) had viroporin activity and formed disulfide-bonded dimers and sds-stable oligomers (fig. e) , which have been seen with other viroporins ( , ) . therefore, oligomerization of the viroporin domain can occur in the absence of the ccd. though direct evidence that this domain forms a pore is needed, this can be demonstrated only by an atomic structure of nsp , which is hampered by the necessity to use detergent to extract nsp during purification. while full-length nsp is targeted to the er membrane by an uncleaved signal sequence (aa to ) ( ), membrane insertion of the viroporin domain was mediated by the clustered lysine residues. these data support a new topology model for nsp , where transmembrane insertion of the viroporin domain leads to a -pass trans-membrane topology (fig. ) . the nsp viroporin domain likely forms a two-helix hairpin, similar to that of hcv p , because the c terminus is known to be exposed to the cytoplasmic side of the er membrane and functions as an intracellular receptor for immature virions ( , ) . oligomerization of nsp around the amphipathic ␣-helix would then create an aqueous channel through which ca ϩ could pass. this new model incorporates and is consistent with two early studies of nsp topology that identified either aa to or aa to as being the single transmembrane segment ( , ) . while transmembrane translocation of the highly charged pentalysine motif seems energetically unfavorable, this phenomenon has been seen for both cationic antimicrobial peptides and other viroporins ( , ) . this was recently demonstrated through molecular modeling of an hcv p nmr structure, which shows that the conserved dibasic motif is embedded in the membrane, with the lysine and arginine side chains directly interacting with phosphate moieties of the lipids ( ) . further, lysine and arginine have long acyl chains that can invade and displace membrane lipids by snorkeling through the lipid environment to facilitate the side chain nitrogen and head group oxygen interaction ( ) . such polybasic clusters in viral proteins may constitute membrane insertion motifs. we previously showed that nsp forms a novel vesicular compartment concomitantly with increased [ca ϩ ]cyto ( ) and that these structures associate with the autophagy protein lc and surround viroplasms, cytoplasmic inclusions in rotavirus-infected cells that support virus replication. the formation of these vesicular puncta by exogenous expression of nsp serves as a surrogate for the formation of viroplasm-associated puncta. in these studies, mutation of the viroporin domain prevented the elevation of cytoplasmic ca ϩ levels, which correlated with the disruption of viroporin activity and loss of e. coli cytotoxicity. while the ntk mutant did not induce lysozyme-mediated cell lysis, it showed e. coli cytotoxicity and elevation of calcium levels similar to those of wt nsp , indicating that mda can be retained in the absence of cell lysis. the use of flow cytometry and indo- as a ca ϩ indicator allowed singlecell analysis of a much larger population of nsp -expressing cells than in previous studies that used single-cell microscopy and fura- ( , ) . additionally, indo- is less sensitive to compartmentalization into ca ϩ -storage organelles, allowing more accurate measurements of [ca ϩ ]cyto ( ) . nsp viroporin activity could trigger the elevation of cytoplasmic ca ϩ levels in several ways that are not necessarily mutually exclusive. in the er, progressive depletion of the er ca ϩ stores by er-associated nsp could activate store-operated ca ϩ entry (soce) and indirectly increase plasma membrane permeability by opening cellular ca ϩ entry channels ( , ) . additionally, expression of nsp increases plasma membrane permeability to mono-and divalent cations ( ) . since nsp traffics to the plasma membrane in rv-infected cells, it is possible that nsp viroporin activity could directly increase plasma membrane permeability to ca ϩ and possibly other ions ( , ) . both mechanisms rely on nsp viroporin activity; however, a detailed analysis of how nsp affects ca ϩ at both the er and plasma membrane will be necessary to determine the relative importance that either direct permeabilization or soce activation, or both, has on the elevation of intracellular ca ϩ levels during a rotavirus infection. previous studies suggested that the movement of nsp from the er into the punctate nsp /lc vesicles was regulated by [ca ϩ ]cyto, but since the mechanism of nsp -mediated er calcium release was unknown, the dependence on calcium for this process could not be characterized further ( ) . we demonstrated that failure of the viroporin mutants to spontaneously form puncta was a direct consequence of their inability to elevate cytoplasmic ca ϩ levels by measuring puncta formation after buffering (bapta-am) or elevating (tg) [ca ϩ ]cyto. under normal conditions, puncta formation by nsp occurs spontaneously and rapidly; however, mutants of either the pentalysine domain (k , , e) or amphipathic domain (asdasa) were unable to form puncta. tg stimulation of viroporin mutant nsp puncta formation demonstrated that the mutations specifically blocked the elevation of [ca ϩ ]cyto but not the ability to form puncta. thus, disruption of cellular ca ϩ homeostasis and puncta formation are separable events, and nsp not only increases [ca ϩ ]cyto by viroporin activity but also appears to be a sensor for changes in [ca ϩ ]cyto. the nsp viroporin mutants developed in these studies will be useful tools to determine the precise [ca ϩ ]cyto that triggers nsp puncta formation. these studies demonstrate that nsp viroporin activity is responsible for the elevation of [ca ϩ ]cyto in rotavirus-infected cells, which was first reported nearly years ago ( ) , and appears to regulate several changes in the subcellular distribution of other rv proteins. first, elevation of cytoplasmic ca ϩ levels regulates the formation of viroplasms, the rv replication complex. nonstructural protein (nsp ), a component of viroplasms, has two pseudo-ef-hand ca ϩ binding sites and elevated levels of cytoplasmic ca ϩ , and ca ϩ binding triggers the aggregation of soluble nsp into a viroplasm-like structure ( ) . second, in response to elevated levels of ca ϩ , nsp traffics out of the er and into puncta that surround viroplasms. third, the assembly of the rv outer capsid protein vp onto virions requires high ca ϩ levels inside the er ( ) . since rna interference (rnai)-mediated knockdown of nsp prevents the proper assembly of viroplasms and causes the mislocalization of several other rv proteins ( , ) , it appears that nsp viroporin activity functionally regulates the progression of rv infection and assembly by altering the cytoplasmic ca ϩ levels. the regulatory function fulfilled by nsp viroporin activity is unique among viroporins, which function primarily in virus entry (influenza m ) ( ), virus release (hcv p , hiv vpu, coronavirus e, and polyomavirus vp /agnoprotein) ( , , , ) , or apoptosis (rsv sh) ( ) . while picornavirus b elevates ca ϩ levels in infected cells, the role that elevated ca ϩ levels plays in the replication cycle for these viruses is not well characterized ( ) . thus, as is shown here for rotavirus nsp , it is possible that the use of viroporins to modulate processes important for replication complex assembly, genome replication, and virus assembly is a mechanism utilized by more viruses than is currently appreciated. expression vectors. e. coli expression constructs were generated by ligationindependent cloning (lic) using the pet ek/lic system (emd biosciences, san diego, ca). wild-type nsp -egfp was constructed by inserting the sa nsp (genbank accession no. af . ) coding region into pegfp-n (clontech). internal deletions and mutations were generated by using the quikchange mutagenesis kit (stratagene, la jolla, ca) or encoding the desired mutation in the forward primer, and all constructs were sequenced (lone star laboratories, houston, tx). e. coli lysis assay. assessment of nsp viroporin activity in e. coli was performed essentially as described previously ( ) . overnight cultures were diluted : into fresh lb and grown until the optical density at nm (od ) was . to . , and mm iptg was added to induce protein expression. od measurements of each culture were taken before iptg induction and at -min intervals postinduction for min using a multiwell plate spectrophotometer. immunoblot analysis. samples were mixed with sample buffer, boiled for minutes, run on to % tris-glycine or to % tris-tricine polyacryamide gradient gels (bio-rad, hercules, ca), and transferred onto a nitrocellulose membrane (ge healthcare bio-sciences corporation, piscataway, nj) as previously described ( ) . bacterially expressed nsp - -his was partially purified using ni ϩ -nitrilotriacetic acid (nta) beads (ge healthcare bio-sciences) as previously described ( ) and separated by sds-page as described above, except sample buffer lacking bme was used under nonreducing conditions. antibodies used were nsp mab b - / ascites, anti-penta-his antibody (qiagen, valencia, ca), anti-egfp monoclonal antibody (clontech), anti-gm monoclonal antibody (bd transduction laboratories, san jose, ca), and anti-stim- antibody (sigma-aldrich, st. louis, mo). e. coli viability assay. stationary-phase cultures of e. coli bl (de ) for the indicated nsp constructs were serially diluted in lb (without ampicillin), and l of each dilution was plated on lb-ampicillin plates in the absence or presence of mm iptg. the plates were incubated overnight at °c, and the number of cfu per milliliter was calculated. membrane protein fractionation. bl (de )plyss broth cultures were grown to an od of . to . , and protein expression was induced with mm iptg and cultured for h. the cells were pelleted by centrifugation ( , ϫ g, h) and resuspended in ml ice-cold phosphatebuffered saline (pbs) (total protein lysate fraction [t]). a -ml aliquot was sonicated in pbs using a probe sonicator (soluble protein fraction [s]). the membranes were pelleted by centrifugation ( , ϫ g, h) and resuspended in ml mm sodium carbonate for min on ice (peripheral membrane protein fraction [p]). the membranes were again pelleted and resuspended in ml % sds-pbs (integral membrane protein fraction [i]). equal buffer volumes were used to maintain the same relative protein concentration as that of the starting material. equivalent amounts of each fraction were analyzed by sds-page. cells and transfection. african green monkey ma kidney cells and human embryonic kidney (hek) t cells were maintained and transfected as previously described ( ) . in experiments using n-(benzyloxycarbonyl) leucinylleucinylleucinal-z-leu-leu-leu-al (mg ), at h posttransfection, the medium was replaced with fresh opti-mem containing m mg . in all cases, cells were incubated overnight at °c. confocal microscopy. ma cells were fixed with % paraformaldehyde and permeabilized with . % triton x- for min. cells were stained with to-pro- (invitrogen), and coverslips were mounted onto slides using prolong gold antifade reagents (molecular probes, eugene, or). mounted slides were observed using a carl zeiss lsm meta confocal microscope with a ϫ immersion oil objective (carl zeiss, germany). the pinhole was set to , and pixel time was set at . s for scanning averages per track on each slice, and z-stack slices were set to m. the collected images were processed using lsm image software (carl zeiss, inc., thornwood, ny). indo- calcium measurements. indo- ( g; molecular probes) was resuspended in l % f- and l fetal bovine serum (fbs) at °c. the indo- loading buffer used was hanks' balanced salt solution (hbss; invitrogen) supplemented with % bovine serum albumin (bsa) (hbss-bsa) and . m indo- . at approximately h posttransfection, the cells were gently washed with hbss-bsa, and indo- loading buffer was added for min at °c. cells were pelleted, resuspended in alpha-mem (no phenol red) plus % fbs plus mm hepes, and maintained at °c until analyzed. flow cytometry analysis was performed using an lsrii system running facsdiva software (bd biosciences, franklin lakes, nj). indo- fluorescence was excited by a uv laser ( nm), and ca ϩ -free and ca ϩ -bound emissions were split using a lp dichroic filter. ca ϩ -free emission was collected with a / -nm band-pass filter, and the ca ϩ -bound emission was collected with a / -nm band-pass filter. egfp fluorescence was excited by the argon laser ( nm), and emission was collected with a / -nm band-pass filter. the mean bound ca ϩ -to-free ca ϩ fluorescence ratio (r) was determined for each sample. the concentration of calcium was calculated by using the following equation: ca ϩ (nm) ϭk d (r Ϫ r min )s f /(r max Ϫ r)s b . treatment of cells with mm edta and m ionomycin was used to determine the fluorescence ratios at zero (r min ) and saturated (r max ) calcium, respectively. s f and s b are the fluorescence intensities of the calcium-free and -bound dyes, respectively. the dissociation constant of indo- is nm. experiments were performed in triplicate, and results are presented at the mean calculated calcium level. puncta formation assay. ma cells were either loaded with m bapta-am for h at °c at h posttransfection or maintained in normal medium. at approximately h posttransfection, a subset of the transfected cells were treated with m thapsigargin (tg) for h, and then, all the cells were fixed in % paraformaldehyde. the number of nsp -egfp-expressing cells containing diffuse rather than punctate nsp -egfp was counted in random fields per well using the ϫ lens objective on an olympus ix inverted epifluorescence microscope. cells with a completely uniform reticular nsp -egfp distribution were scored as having no puncta; however, cells with the presence of even one punctate structure were scored as having puncta. statistical analysis. statistical differences between groups were determined using a two-tailed student's t test. p values of Ͻ . were considered significant. supplemental material for this article may be found at http://mbio.asm.org /lookup/suppl/doi: . /mbio. - /-/dcsupplemental. figure s , tif file, . mb. figure s , tif file, . mb. figure s , tif file, . mb. figure s , tif file, . mb. table s , pdf file, . mb. ion homeostasis, channels, and transporters: 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formation of viroplasmlike structures by the rotavirus nsp protein is calcium regulated and directed by a c-terminal helical domain rotavirus glycoprotein nsp is a modulator of viral transcription in the infected cell silencing the morphogenesis of rotavirus role of the coronavirus e viroporin protein transmembrane domain in virus assembly epitope mapping and use of epitope-specific antisera to characterize the vp * binding site in rotavirus sa nsp key: cord- -h wvx gw authors: imperiale, michael j.; casadevall, arturo title: the importance of virology at a time of great need and great jeopardy date: - - journal: mbio doi: . /mbio. - sha: doc_id: cord_uid: h wvx gw nan a s we enter , one needs to look no farther than the daily news reports to appreciate the ongoing burden of viral diseases. last year, ebola reemerged in west africa, claiming thousands of lives and affecting many thousands more. cases of middle east respiratory syndrome (mers) continue to be reported, with the possibility of a severe acute respiratory syndrome (sars)-like epidemic ever present. chikungunya virus has spread to the western hemisphere, and the infection is now epidemic in the caribbean and southern united states. we are also living in a world in which hundreds of millions of people are chronically infected with hepatitis b and c viruses (hbv and hcv, respectively). the rate of new hiv infections has declined, but millions remain infected, and it, too, has already cut short far too many lives. viruses account for up to % of all human cancers, and although a large percentage of new human papillomavirus (hpv) and hbv infections can now be prevented by vaccination, many are already infected, and the vaccines are not being used to their full potential. we are in the middle of our annual encounter with influenza virus, never knowing when the next strain to which there is little or no preexisting immunity will arise. in - , a mismatch between the h n strain in the influenza vaccine and the circulating virus has led to a poorly protective vaccine, which highlights the need for new vaccines. in recent months, there has been a recurrence of measles in the united states associated with a refusal by some parents to vaccinate their children, and the outbreak continues at the time of this writing ( ). this accounting is of only some viruses and is limited to those that infect humans! there can be no argument that humankind's best hope of preventing and treating these diseases comes from a vigorous research enterprise. vannevar bush recognized this after world war ii in the landmark report "science, the endless frontier," in which he convinced the u.s. government that investment in basic research at universities would yield tremendous dividends (https:// www.nsf.gov/od/lpa/nsf /vbush .htm). indeed, we have now almost eliminated polio due to the development of vaccines and have converted hiv infection from a certain death sentence to a largely manageable state, and, as mentioned above, we have our first anticancer vaccines by preventing some viral infections. the tremendous reduction in mortality from such diseases as variola, measles, and rubella came about only because the causative viruses were identified, cultivated, attenuated, and made into effective vaccines by biomedical research. in addition to having applied these practical findings, we have gained important fundamental insights into the biology not only of viruses but also of the cells that they infect, and that information is being applied to find cures against other diseases, such as cancer. all these advances, and more, have come about because of public trust in science and investment in scientific research. despite all this good news, much remains to be done. it was recently estimated that there are , mammalian viruses ( ), many of which may have the potential for human transmission. even if only a small fraction of these viruses can jump into humans and cause disease, humanity is living under a tremendous threat from viral zoonoses. less expensive drugs are needed for treating those with viral infections such as hepatitis c. while the hpv vaccine can prevent many infections, there are viral types that are not covered by the vaccine, and many millions of people were infected before the vaccine came on the market. infections with numerous other viruses are not treatable due to the lack of effective antivirals. clearly, vaccines against hiv, hepatitis c, and ebola, to name a few, would save countless lives. new pathogens continue to emerge, and existing nonviral pathogens become resistant to common antibiotics. hence, we are living at a time of great need for the discipline of virology. we think that the field of virology and, by extension, the field of microbiology are at a critical crossroads. funding for research in the united states and elsewhere is stagnant, if not losing pace with inflation. working with the most pathogenic organisms requires even higher costs and is heavily regulated. some senior scientists are rethinking their career choices, and there is growing concern that young scientists will be discouraged from entering the field, especially in areas of controversial research, such as studies of the transmissibility of highly pathogenic influenza viruses ( ). while some have argued that virology is a dying field, that assertion has been elegantly refuted by dan dimaio ( ). adding to these stresses, scientists and society are struggling with a new antiintellectual movement that challenges scientific conclusions, from anthropomorphically induced climate change to the absence of any link between vaccines and autism. the rise of antivaccine movements is of particular concern to society, for reduced vaccination rates threaten to undermine some of the greatest accomplishments of virology and public health in the th century. the combination of reduced funding, increased regulation, experimental controversies, and the emerging antiscience intellectual milieu is a toxic blend that makes this time one of great jeopardy for virology. while we scientists cannot directly control funding or regulations, we can take charge of some aspects of the research enterprise in a way to ensure that it continues to benefit society. first, we can continue to advocate for better funding by the federal government. this requires engaging our elected officials both directly and indirectly by continuing to educate them and the public at large about the importance of fundamental research in infectious diseases. the advocacy group research!america has a number of helpful tips on its website (http://www.researchamerica.org). second, we need to demonstrate to the public that we are being good stewards of their investment by working safely in the laboratory. there have been a number of high-profile biosafety lapses over the past year, and the negative publicity surrounding these events may lead to more regulation and less funding support for exactly the types of research that we most critically need. we therefore argue that each of us needs to pay special attention to biosafety in and the longer term. third, in controversial areas, such as studies of transmissibility involving pathogens with pandemic potential, it needs to be clearly articulated why some types of experiments need to be done by vigorously engaging in scientific debate using the tools of science, all the while acknowledging that there are risks and taking every step to mitigate those risks. third, every scientist needs to become a foot soldier in confronting the pervasive spread of antiscientific attitudes, such as the antivaccination movement, which threaten to undermine the great advances society has made in so many aspects of everyday life, including reducing mortality from many infectious diseases. although virology is currently at the epicenter of these converging storms, the issues that it faces are relevant to all of microbiology and, by extension, to all of science and the society that it serves. a little added effort on our parts will go a long way to ensuring continued public confidence in what we do to make their lives healthier. measles cases exceed in us outbreak a strategy to estimate unknown viral diversity in mammals is the debate and "pause" on experiments that alter pathogens with pandemic potential influencing future plans of graduate students and postdoctoral fellows? mbio is virology dead? mbio the views expressed in this article do not necessarily reflect the views of the journal or of asm key: cord- -rdlinzrn authors: gralinski, lisa e.; sheahan, timothy p.; morrison, thomas e.; menachery, vineet d.; jensen, kara; leist, sarah r.; whitmore, alan; heise, mark t.; baric, ralph s. title: complement activation contributes to severe acute respiratory syndrome coronavirus pathogenesis date: - - journal: mbio doi: . /mbio. - sha: doc_id: cord_uid: rdlinzrn acute respiratory distress syndrome (ards) is immune-driven pathologies that are observed in severe cases of severe acute respiratory syndrome coronavirus (sars-cov) infection. sars-cov emerged in to and led to a global outbreak of sars. as with the outcome of human infection, intranasal infection of c bl/ j mice with mouse-adapted sars-cov results in high-titer virus replication within the lung, induction of inflammatory cytokines and chemokines, and immune cell infiltration within the lung. using this model, we investigated the role of the complement system during sars-cov infection. we observed activation of the complement cascade in the lung as early as day following sars-cov infection. to test whether this activation contributed to protective or pathologic outcomes, we utilized mice deficient in c (c (–/–)), the central component of the complement system. relative to c bl/ j control mice, sars-cov-infected c (–/–) mice exhibited significantly less weight loss and less respiratory dysfunction despite equivalent viral loads in the lung. significantly fewer neutrophils and inflammatory monocytes were present in the lungs of c (–/–) mice than in c bl/ j controls, and subsequent studies revealed reduced lung pathology and lower cytokine and chemokine levels in both the lungs and the sera of c (–/–) mice than in controls. these studies identify the complement system as an important host mediator of sars-cov-induced disease and suggest that complement activation regulates a systemic proinflammatory response to sars-cov infection. furthermore, these data suggest that sars-cov-mediated disease is largely immune driven and that inhibiting complement signaling after sars-cov infection might function as an effective immune therapeutic. from coronaviruses circulating in animal markets in china ( ) . emergence of this novel virus led to a global outbreak of respiratory disease, with over , human cases and % mortality ( , ) . in , a new, related zoonotic coronavirus was identified in the middle east, designated middle east respiratory syndrome coronavirus (mers-cov), causing severe respiratory disease with greater than % mortality (www .who.int/emergencies/mers-cov/en) ( ) . both sars-cov and mers-cov cause a range of disease from asymptomatic cases to severe acute respiratory distress syndrome (ards) and respiratory failure ( ) . notably, metagenomics and synthetic virus recovery strategies have since revealed the existence of large pools of preepidemic sars-like bat coronaviruses which replicate in primary human airway epithelial cells. these viruses are poised for emergence because they both efficiently use human ace entry receptors and resist existing vaccines and immunotherapeutics ( , ) . due to the ongoing threat and continued emergence of new, highly pathogenic coronaviruses from animal reservoirs, a thorough understanding of the host-virus interactions that drive sars-cov pathogenesis will aid the public health response to current and future coronavirus outbreaks ( ) . the importance of complement in sars-cov pathogenesis is controversial. previous studies have investigated the role of known polymorphisms in the mannose-binding lectin (mbl) and mbl-associated serine protese- (masp ) genes in sars-cov infection outcome following the outbreak but with conflicting results. one retrospective analysis showed that people with low or deficient serum mbl levels were more likely to become infected with sars-cov ( ) than those with high mbl levels, suggesting that mbl and complement activation play a role in protecting the host from infection. however, a second study found no association between mbl haplotype and sars-cov infection status ( ) . additionally, it was shown mbl can bind to the sars-cov spike protein in vitro by some groups ( ) but not by others ( ) . examination of the role of the downstream complement gene masp found no association between genotype and sars susceptibility ( ) . together, the results leave a general uncertainty about the role of complement in response to sars-cov infection. despite the existing body of literature, the role of complement in sars-cov pathogenesis has never been directly assessed in vivo. the complement system is an ancient arm of the innate immune response comprised of multiple proteins whose reactive cascade of cleavage products can coordinate the inflammatory response at the sites of infection and can be directly antimicrobial. consisting of more than soluble and cell surface-associated proteins, complement is a major component of innate immunity that functions to recognize and eliminate invading pathogens ( ) . activation of the complement system occurs through multiple mechanisms that include three welldescribed pathways, the classical, lectin, and alternative complement activation pathways ( ) , and results in proteolytic processing of various components of the complement system, including c , c , and c . proteolytic processing of c generates an array of cleavage products that are involved in amplification of complement activity through formation of c and c convertases, opsonization of pathogens, and attraction and activation of leukocytes of both the innate and adaptive arms of the immune response. several studies, including a recent study showing that complement blockade results in reduced disease in a mers-cov human dpp transgenic (hdpp -tg) mouse model ( ) , have elucidated protective and pathogenic roles for the complement system following infection by a variety of viral pathogens ( ) . furthermore, the complement system has well-described roles in other pulmonary diseases ( ) , especially after influenza virus and respiratory syncytial virus infection ( ) ( ) ( ) . in this study, we assessed the role of the complement system in the pathogenesis of sars-cov infection. building from a systems biology analysis that suggested that complement was modulated during sars-cov infection, we confirmed that complement was activated upon sars-cov challenge. mice deficient in c (c -/-), the central protein of the complement signaling pathway, were protected from sars-cov-induced weight loss and had reduced pathology, improved respiratory function, and lower levels of inflammatory cytokines/chemokines in the lung and periphery. importantly, the kinetics and magnitude of virus replication in c -/and wild-type mice were the same, showing that complement does not play a role in controlling virus replication. we observed complement deposition in the lungs of sars-cov-infected mice, suggesting that complement activation results in immune-mediated damage to the lung. additionally, serum activation indicates that complement-mediated systemic inflammation may drive the pathogenic response to sars-cov infection. together, the results indicate that complement plays a critical role in sars-cov pathogenesis and that inhibition of the complement pathway might be an effective therapeutic to coronavirus-mediated disease. complement is activated in sars-cov ma -infected mice. while work by other laboratories has shown that c -deficient mice are extremely susceptible to both h n and h n influenza virus infection ( ) , the role of complement in sars-cov infection has not yet been evaluated in vivo. using a systems biology-based approach, we identified the complement pathway as a high-priority target for control of sars-cov ma (the mouse-adapted sars-cov) pathogenesis based on weighted gene correlation network analysis (wgcna) of rna transcripts in the lungs of mice infected with a lethal versus a sublethal dose of sars-cov ma ( ) . because the complement signaling cascade is activated through proteolytic cleavage events, we also assessed lung proteomics samples for complement protein abundance. c b, cfb, and c all had significantly higher abundances in the lungs of mice infected with a lethal dose of sars-cov ma than in those of mice infected with a sublethal dose (fig. a) . complement activation is measured by detection of pathway component cleavage products. c , the master regulator of complement signaling, is cleaved into c a and c b following creation of c convertase. c activation products (c fragments c a, c b, ic b, c dg, and c c) were detected by western blotting in lung tissue of sars-cov ma -infected mice, but not in control mice, as early as day postinfection (dpi) (fig. b) , confirming that sars-cov ma infection activates the complement pathway. to test the importance of the complement signaling pathway in sars-cov pathogenesis, we infected c -/mice and c bl/ j controls with sars-cov ma . control mice exhibited approximately % transient weight loss, with peak weight loss at day postinfection (fig. a) . in contrast, the c -/mice were significantly protected from infection, with no significant weight loss evident at any time point. surprisingly, viral titers in the lung were similar in c -/and c bl/ j controls (fig. b) , indicating that the lack of disease in c -/mice is uncoupled from viral replication efficiency and that complement signaling is not necessary for sars-cov ma clearance from the lung. we further measured sars-cov ma -induced disease by assessing respiratory function using whole-body plethysmography following infection of c bl/ j and c -/mice. enhanced pause (penh) is a calculated measure of airway resistance that we have associated with airway debris following sars-cov ma infection ( ) . the % exhalation force (ef ) measures the exhalation force midbreath, which increases as breathing becomes more difficult. finally, the ratio of peak expiratory flow (rpef) is the time to peak expiratory flow and has been associated with wheezing following infection. all three metrics have been shown to change significantly following sars-cov ma infection, with penh and ef increasing following infection and rpef decreasing. combined, these measurements show that sars-cov ma -infected animals have altered exhalation patterns in their breathing. c -/mice exhibit a decreased change in penh and ef levels following sars-cov ma infection relative to those of infected c bl/ j control mice; however, rpef values were similar between infection conditions ( fig. c to e). together, these data indicate that despite the lack of weight loss in c -/mice, the absence of the complement pathway did not alter host control of viral the respiratory function of sars-cov ma -infected c bl/ j and c -/mice and mock-infected mice was measured using a buxco whole-body plethysmography system for penh, a measure of calculated airway resistance (c), ef , midbreath expiratory flow (d), and rpef, the rate of peak expiratory flow (e). *, p Ͻ . between mock-infected mice and a given condition. (c to e) three mice were used for each infection group, and two mock-infected mice were used. replication or completely abolish respiratory disease following sars-cov ma infection. in order to determine which arm of the complement pathway contributes to sars-cov ma pathogenesis, we infected knockout mice lacking components upstream of c . c -deficient mice lack the ability to signal through both the classical and lectin pathways, while fb-deficient mice lack the ability to signal through the alternative pathway. both mouse strains showed reduced weight loss relative to that of infected control mice (see fig. s in the supplemental material) at days postinfection; however, neither c -/nor fb -/mice reproduced complete protection from weight loss observed in c -/controls. together, the results suggest that multiple arms of the complement pathway may be activated and contribute to sars-cov-mediated disease through c activation. reduced lung pathology in c -/mice. analysis of sars-cov ma -infected lung sections showed that the absence of c resulted in reduced, but still significant, lung pathology. at day postinfection, only minor effects on lung disease were observed with airway denudation and debris, the main histopathological phenotypes at this early time point; levels of pathology were similar between wild-type and knockout mice ( fig. ; table s ). c -/mice exhibited more airspace inflammation, including eosino- phils, at dpi than their wild-type controls, although this relationship was reversed later in infection. at dpi, c bl/ j mice displayed pronounced lung pathology, including inflammatory cells in the large airway and parenchyma, perivascular cuffing, thickening of the interstitial membrane, and low levels of intra-alveolar edema. in contrast, c -/mice showed reduced scores in these areas, consistent with the improved respiratory function observed in fig. . notably, the lung pathology results were not as pronounced as the complete absence of weight loss, suggesting a possible distinction between lung disease and overall pathogenesis. we also considered whether the decrease in sars-cov ma pathogenesis in c -/mice was due to reduced lung damage in the absence of complement pathway signaling. to investigate this possibility, we looked for signs of complement deposition on sars-cov ma lung tissue. at both and dpi, we observed scattered positive staining for complement in the lungs of sars-cov ma -infected mice, suggesting that local tissue damage might contribute to sars-cov pathogenesis (fig. ) . interestingly, staining was consistently found in the parenchyma of the lung and not in the large airways, which are the other main site of sars-cov ma replication. no positive staining was observed in the lungs of c -/mice. diminished infiltration of the lungs of a select immune population of infected c -/mice. in order to identify and quantitate inflammatory cells in the lung, we performed flow cytometry at dpi. in parallel with humans exhibiting lung pathology, c -/mice exhibited significant pulmonary infiltration following sars-cov ma infection, but this inflammation was reduced relative to that observed in wild-type mice. sars-cov ma -infected c bl/ j and c -/mice had similar total cell counts as well as similar percentages of cd -positive cells in their lungs (data not shown). consistently with what was observed in human sars-cov patients ( ) , lymphopenia was observed in the lungs of both sars-cov ma -infected c bl/ j and c -/mice with reduced percentages of b cells (fig. a ) and cd t cells relative to those in mockinfected mice following infection. despite similar overall lymphocyte levels, small but significant differences were observed in levels of t cell activation between infected c bl/ j and c -/mice; both cd and cd t cells in c -/mice expressed more ki- ( fig. c and d) , an intracellular marker of proliferation, than those in c bl/ j controls. analysis of myeloid cells in the lung showed that infected c bl/ j mice had significantly higher levels of neutrophils, particularly nonactivated neutrophils, in the lung than infected c -/mice ( fig. b and e) . furthermore, inflammatory monocytes, which have previously been associated with increased sars-cov ma pathogenesis ( ), were significantly increased in the lungs of wild-type mice but not c -/mice (fig. b) . finally, we observed significantly more dendritic cells and alveolar macrophages in the lungs of sars-cov ma -infected c -/mice than in the lungs of infected c bl/ j mice. together, although c -/mice produced a robust immune cell infiltration following sars-cov infection, they had significant reductions in both inflammatory monocytes and neutrophils relative to controls; both cell types that are associated with sars-cov pathogenesis ( ) . conversely, the presence of activated t cells is associated with recovery following infection ( ) . in addition to examining inflammatory cells, we evaluated the vascular integrity of the lung following sars-cov ma infection in the presence and absence of c . we observed no differences in the numbers of platelets present in the bronchoalveolar lavage (bal) fluid between c bl/ j and c -/mice at either or days postinfection, indicating that the absence of c does not appear to significantly alter vascular permeability following infection with sars-cov (fig. s b) . cytokine and chemokine levels are significantly decreased in the lungs of c -/mice. to further investigate the inflammatory response to sars-cov ma infection, we measured cytokine and chemokine protein levels in the lung in the presence and absence of complement signaling. multiple protein expression patterns were observed in response to infection. mip a, mip b, and mcp are all highly expressed in the lung following sars-cov ma infection of both c bl/ j and c -/mice (fig. a) , indicating that some inflammatory signaling remains intact in the absence of c . granulocyte colony-stimulating factor (g-csf), interleukin (il- ), tumor necrosis factor alpha (tnf-␣), and il- a comprised a group of cytokines and chemokines that were more highly produced in the lungs of c bl/ j mice than in c -/mice (fig. b) , all peaking at days postinfection. notably, these cytokines all have a role in the production, recruitment, or differentiation of neutrophils, consistent with the flow cytometry results in fig. b . with the exception of rantes, all inflammatory cytokines and chemokines were measured at the highest levels at dpi, indicating that the host immune response is triggered quickly following infection with sars-cov ma . together, these results indicate that the absence of complement has an impact on the magnitude of some cytokines and chemokines in the lung; however, robust production can occur in either the presence or the absence of c . sars-cov ma induces systemic complement activation. the absence of complement signaling resulted in reduced sars-cov ma pathogenesis, as measured by weight loss and a partial reduction of respiratory dysfunction, pathology, immune infiltration, and cytokine responses in the lung. we hypothesized that systemic disease coupled with no change in viral titer might also drive important elements of complement-mediated disease. therefore, we examined sera from wild-type and c -/mice for signs of systemic disease following infection. western blot analysis showed increased levels of c a-derived fragments in the serum, indicating systemic complement activation in sars-cov ma -infected mice at dpi (fig. a) . given this result, we next examined cytokine and chemokine protein levels for markers of inflammation in the sera of sars-cov ma -infected mice. both mcp- and rantes levels were elevated in the serum following infection, regardless of mouse genetic background (fig. b) . however, numerous cytokines and chemokines, such as il- , g-csf, and kc (keratinocyte chemoattractant or cxcl ) were present in significantly higher abundance in the lungs of c bl/ j mice than in those of c knockout mice (fig. c) . we further examined the possibility that sars-cov ma infection leads to complement deposition outside the lung and found no signs of increased complement staining in the kidney (fig. d) . although no complement deposition was seen, the presence of both activated complement and inflammatory cytokines in the sera likely contributes to a systemic inflammatory response that drives sars-cov ma -mediated weight loss following infection. the complement system is a critical part of the host immune response to bacterial and viral infection. originally identified in the s as a heat-sensitive, nonspecific complement to the more specific adaptive immune pathways ( ), the complement system is one way that the innate immune system detects and responds to foreign antigens. because of its potential to damage host tissues, the complement system is also tightly regulated through a number of inhibiting proteins that are constitutively present in the serum ( ). it has previously been shown that complement pathway signaling is critical for the protective host immune response to various bacterial infections ( ) as well as some influenza virus and flavivirus infections ( , , ) . furthermore, viruses, including herpesviruses, poxviruses, astroviruses, flaviviruses, and retroviruses, encode genes to help them evade detection by the complement system ( ) , strong evidence that complement is important in the host antiviral response. the host factors that drive protective ( ) or pathogenic ( ) complement-associated responses in viral infection are not well understood. of particular concern, the anaphylatoxins c a, c a, and c a are produced during activation of the complement signaling cascade; they have potent proinflammatory properties and can trigger inflammatory cell recruitment and neutrophil activation ( ) . c a and c a blockade has been proposed as a treatment for acute lung injury ( ) , and anti-c a antibody has been shown to protect mice from infection with influenza virus ( ) and, more recently, mers-cov ( ) . complement recognition is important for the control of paramyxoviruses ( ), dengue virus ( ) , and human t lymphotrophic virus type (htlv- ) ( ), and many more viruses have developed means of evading detection by the complement system ( ) . in contrast, the data presented here, in conjunction with recent findings for ross river virus ( , ) , influenza virus ( ) , and well-established autoimmune disease ( ) , demonstrate that complement system activation can also lead to exacerbated disease. previous reports clearly established the ability of mannose-binding lectin (mbl) to bind to the sars-cov spike protein ( ), dependent on an n-linked glycosylation site; however, the role of complement signaling in sars-cov pathogenesis was unclear ( , , , ) . in this study, we demonstrate that the complement system is activated following sars-cov ma infection (fig. b) . however, we did not observe any change in viral titer in c -/mice (fig. b) , indicating an important difference between in vivo and in vitro studies and the use of viral pseudoparticles. the absence of complement signaling resulted in protection from sars-cov ma -induced weight loss, as shown through the use of both c -deficient mice ( fig. a) and activation pathway-specific knockout mice (fig. s ). respiratory function in c knockout mice was improved relative to that of control mice, although significant changes in penh and rpef were still observed, indicating that the elimination of complement signaling did not completely remove the effects of sars-cov ma infection. while analysis of the cellular inflammatory response to sars-cov ma infection revealed modest changes in histopathology and overall inflammatory cell recruitment to the lungs, significant differences were observed in pathogenic inflammatory monocyte and neutrophil populations, indicating that complement signaling contributes to the broader immune response to infection. immunohistochemical staining revealed that sars-cov ma infection induced complement deposition in the lung (fig. ) , similar to that associated with pathogenesis in ross river virus-infected mice ( ) and some influenza virus infections ( ) , and it is likely that complement deposition contributes to pulmonary disease and inflammatory cell recruitment. the cytokines and chemokines il- , il- , kc (cxcl ), and g-csf have higher abundances in the lungs of sars-cov ma -infected wild-type mice than in c -/mice and are all known to promote neutrophil recruitment. indeed, significantly more neutrophils were observed in the lungs of sars-cov ma -infected c bl/ j mice than in the c -/mice (fig. b) . interestingly, while there were fewer neutrophils present, the neutrophils found in the lungs of c -/mice infected with sars-cov ma had significantly more staining of major histocompatibility complex class ii (mhc ii) and the costimulatory molecules cd and cd (fig. e) , indicating a state of activation ( ) . unpublished data from our laboratory have consistently demonstrated higher neutrophil counts in the lungs of mice with severe disease than in those of mice with only mild pathogenesis. additionally, neutrophilia in human sars-cov patients was associated with a poor outcome of infection ( ) , and studies of native rat coronavirus ( ) found both a protective and a pathogenic role for neutrophils following infection. combined, these data demonstrate that the absence of complement provides significant improvements in pulmonary disease following sars-cov ma infection and suggest that a nonpulmonary cause might also contribute to the lack of weight loss in c -/mice. importantly, our data demonstrate that sars-cov ma infection activates the complement system systematically as well as in the lung (fig. a and b) . wild-type c bl/ j mice exhibited an increased abundance of serum cytokines and chemokines in response to sars-cov ma infection (fig. c ) in comparison to c -/mice. in particular, the pyrogenic cytokine il- ( ) is present at higher abundance in the lungs and sera of c bl/ j mice than in those of c -/mice. il- ␣ and tnf-␣ are also more abundant in the lungs of c bl/ j mice, suggesting that wild-type but not c -/mice develop a fever response to infection that contributes to weight loss and respiratory dysfunction phenotypes. while the precise mechanism of complement activation following sars-cov ma infection is still unclear, it is likely through recognition of the viral spike glycoprotein and partially mediated by mbl (see fig. s in the supplemental material). the anaphylatoxins produced by the activated complement pathway, c a, c a, and c a, have important immunostimulatory roles in vascular permeability and inflammatory cell recruitment ( , ) . c a and c a in particular are noted for their roles in causing mast cell degranulation, initiating a cytokine storm, promoting vascular permeability, and contributing to acute lung injury ( , , ) . furthermore, a c a antibody blockade was recently shown to protect in a model of highly susceptible mers-cov mice ( ) . although there are no reports of mast cell activation following sars-cov or mers-cov infection, activation has been observed both in vitro and in vivo following infection with influenza virus ( ) and may occur following other severe respiratory infections, including coronaviruses. mast cells release cytokines, including il- , il- , and tnf-␣, consistent with the inflammatory profile observed following sars-cov ma infection. while this work cannot definitively conclude that the complement anaphylatoxins and mast cell activation contribute to sars-cov ma pathogenesis, the data are consistent with this possibility, and the concept warrants further investigation. complement pathway activation is a hallmark of bacterial infection, and genetic deficiencies in the complement pathway result in enhanced susceptibility to streptococcus pneumoniae, neisseria meningitidis, and haemophilus influenzae infections ( ), as well as to sepsis. interestingly, it has also been shown that sars-cov ma infection stimulates tlr ( , ) , which is classically known as the lipopolysaccharide (lps) receptor ( , ) and important for recognition of many bacterial infections. combined, these data suggest that host recognition of sars-cov ma infection may activate similar pathways recognizing a bacterial infection, leading to immune signaling cascades that cause systemic disease and enhance viral pathogenesis. while systemic activation of the complement pathway may be useful during a bacterial infection, it is less so during a localized acute viral infection, such as sars-cov. furthermore, complement activation, in conjunction with the presence of neutrophils, is known to cause increased vascular permeability, a condition that is also observed following sars-cov infection ( ) ( ) ( ) and was associated with poor outcome. baseline complement activation also increases with age ( , ), consistent with increased sars-cov morbidity and mortality in aged populations. finally, it has previously been reported that serum c a levels are predictive of ards development ( ) and that, in the absence of complement, animals are protected from bacterially induced "shock lung" ( ) , data consistent with the pathogenic role that we have found for complement following sars-cov ma infection. in this work, we demonstrate that sars-cov ma infection activates the complement pathway and that complement signaling contributes to disease following infection. this disease is likely mediated by complement protein deposition in the lung as well as systemic complement activation and inflammation. notably, the absence of c has no impact on viral titer, unlike what has been observed following influenza virus infection. despite these differences, it is notable that mers-cov and h n influenza virus-induced acute lung injury and pulmonary inflammation are reduced in mice that are treated with either a c a receptor (c ar) antagonist or antibodies to c a ( , ) . a similar treatment might be effective in mitigating sars-cov ma -induced disease, as sars, mers, and influenza have common disease manifestations, including development of acute lung injury. given the large array of zoonotic strains poised for cross-species transmission, broad-based inhibitors of emerging coronavirus infections are a high priority ( ) . pinpointing the precise arms of the complement pathway that contribute to sars-cov will help further identify therapeutic targets while minimizing unnecessary inhibition of the immune response. this work suggests that investigation of anticomplement drugs for treatment of coronavirus infections is warranted and would pair well with direct antiviral therapeutics. stocks of recombinant mouse-adapted sars-cov (ma ) ( ) were propagated and their titers were determined in vero e cells and stored as single-use aliquots at Ϫ °c as previously described ( ) . tissue titers of ma were determined by plaque assay on vero e cells as previously described ( , ) , with a limit of detection of pfu. all experiments using live virus were performed in a class ii biological safety cabinet in a certified biosafety level laboratory with negative air pressure and redundant exhaust fans; personnel wore personal protective equipment, including tyvek suits, hoods, and powered air-purifying respirators. mouse experiments. c bl/ j (stock number ) and c -/-(stock number ) mice were purchased from jackson laboratories. fb Ϫ/Ϫ mice were generously provided by charles jennette (unc), and c -/mice were provided by mark heise (unc). all animal husbandry and experiments were performed in accordance with all university of north carolina at chapel hill institutional animal care and use committee guidelines. age-matched ( -to -week-old) female mice were anesthetized with a mixture of ketamine-xylazine and intranasally inoculated with l of phosphate-buffered saline (pbs) or pfu of sars-cov ma diluted in pbs. mice were monitored for disease signs and weighed at -h intervals. microarray and proteomics analysis. microarray and proteomics analyses were performed on a time course and according to a dose-response study published by gralinski et al. by following the same methods ( ) . the proteomics data (experiment sm ) are publically available through the pnnl (http://omics.pnl.gov) web portal. briefly the mean intensity (abundance) for each protein was then graphed as an average ( mice for each infection, mice for mock infection) for each group at each time point. missing or absent values were not scored; however, if no value was observed in any of the samples at a time point, the sample was registered with a single , representing "not detected." histological analysis. at the times indicated in the figures, mice were euthanized using an overdose of isoflurane, and lung tissue was fixed in % formalin. tissues were embedded in paraffin, and -m sections were prepared. to determine the extent of inflammation and tissue pathology, tissues were stained with hematoxylin and eosin and scored in a blind manner from (no sign of phenotype) to (widespread and severe phenotype). platelet counts. for bronchoalveolar lavage (bal), immediately following euthanasia, ml of pbs was injected into the lung through the trachea by using a -gauge exel safelet catheter tip (fisher). this fluid was then drawn back out and used for subsequent analysis. two hundred fifty microliters of bal fluid was used for absolute counting of gross cell types using an abaxis vetscan hm analyzer. complement deposition staining. lung sections from sars-cov ma -, ross river virus-, or mock-infected mice were stained for the presence of c by the animal histopathology and laboratory medicine core at the university of north carolina. sars-cov ma lung samples were tested from -week-old mice at , , , and days after infection with pfu of virus. staining was performed using a goat anti-mouse c primary antibody (mp biomedicals). immunoblot analysis. mice were perfused with pbs, and then lung tissue was dissected and homogenized in lysis buffer ( mm tris [ph . ], mm nacl, % nonidet p- , . % deoxycholate, and . % sodium dodecyl sulfate [sds] supplemented with complete protease inhibitor cocktail [roche]). total protein concentrations were determined by using the coomassie plus assay kit (pierce). dilutions of serum or -to -g aliquots of protein were diluted in an equal volume of ϫ sds sample buffer, and sds-polyacrylamide gel electrophoresis was performed. proteins were transferred onto polyvinylidene fluoride membranes (bio-rad). membranes were blocked in ϫ pbs- % milk- . % tween and incubated with goat anti-mouse c antibody ( : , ; cappel) overnight at °c. membranes were washed in pbs- . % tween and incubated with rabbit anti-goat-horseradish peroxidase ( : , ; sigma) for h at room temperature. after a washing step, proteins were visualized by enhanced chemiluminescence (amersham) according to the manufacturer's instructions. flow cytometry. following euthanasia at days postinfection, mice were perfused with ml of pbs via cardiac puncture. lungs were dissected, minced, and incubated for min with vigorous shaking at , and cd -percp-efluor (clone e ; ebioscience). after being stained, cells were fixed in % paraformaldehyde overnight and then stored in pbs until acquisition within h. a minimum of , events were collected using an lsrii cytometer (becton, dickinson), and analysis was completed using flowjo software version (treestar). all samples were first evaluated through subsequent gates for (i) mononuclear cells, (ii) doublet exclusion, (iii) dead-cell exclusion based on uptake of a fixable live/dead cell discriminator (invitrogen), and cd -lca expression before downstream analyses. reported cell frequencies were normalized to the percentage of total cd -lca ϩ events, where appropriate. whole-body plethysmography. respiratory function was measured using whole-body plethysmography as described by menachery et al. ( ) . briefly, mice were loaded into individual chambers and allowed to acclimate for min before a -min measurement window. measurements were recorded every s for a total of measurements per time point per mouse. cytokine and chemokine protein analysis. the small center lung lobe of each mouse was homogenized in ml of pbs and briefly centrifuged to remove debris. fifty microliters of homogenate was used to measure cytokine and chemokine protein abundance using a bio-plex pro mouse cytokine -plex assay (bio-rad) according to the manufacturer's instructions. statistical analyses. percent starting body weights, viral titers, and inflammatory cell numbers were evaluated for statistically significant differences by the mann-whitney test or student's t test using graphpad prism software. accession number(s). the microarray data were previously deposited in the geo database under accession number gse ( ). supplemental material for this article may be found at https://doi.org/ . /mbio . - . research was supported by grants from the niaid of the nih (ai to r.s.b. and m.t.h., ai to r.s.b., ai to r.s.b., and k ag to v.d.m.). animal histopathology was performed by the animal histopathology and laboratory medicine core at the university of north carolina, which is supported in part by an nci center core support grant ( p ca - ) to the unc lineberger comprehensive cancer center. the unc flow cytometry core facility is supported in part by cancer center core support grant p ca to the unc lineberger comprehensive cancer center. the funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication. the content is solely the responsibility of the authors and does not necessarily represent the official views of the nih. molecular evolution of the sars coronavirus during the course of the sars epidemic in china characterization of a novel coronavirus associated with severe acute respiratory syndrome severe acute respiratory 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respiratory syndrome coronavirus host range expansion in human airway epithelium flow cytometric analysis of macrophages and dendritic cell subsets in the mouse lung key: cord- -mizygp j authors: beall, anne; yount, boyd; lin, chun-ming; hou, yixuan; wang, qiuhong; saif, linda; baric, ralph title: characterization of a pathogenic full-length cdna clone and transmission model for porcine epidemic diarrhea virus strain pc a date: - - journal: mbio doi: . /mbio. - sha: doc_id: cord_uid: mizygp j porcine epidemic diarrhea virus (pedv) is a highly pathogenic alphacoronavirus. in the united states, highly virulent pedv strains cause between and % mortality in suckling piglets and are rapidly transmitted between animals and farms. to study the genetic factors that regulate pathogenesis and transmission, we developed a molecular clone of pedv strain pc a. the infectious-clone-derived pedv (icpedv) replicated as efficiently as the parental virus in cell culture and in pigs, resulting in lethal disease in vivo. importantly, recombinant pedv was rapidly transmitted to uninoculated pigs via indirect contact, demonstrating virulence and efficient transmission while replicating phenotypes seen in the wild-type virus. using reverse genetics, we removed open reading frame (orf ) and replaced this region with a red fluorescent protein (rfp) gene to generate icpedv-Δorf -rfp. icpedv-Δorf -rfp replicated efficiently in vitro and in vivo, was efficiently transmitted among pigs, and produced lethal disease outcomes. however, the diarrheic scores in icpedv-Δorf -rfp-infected pigs were lower than those in wild-type-virus- or icpedv-infected pigs, and the virus formed smaller plaques than those of pc a. together, these data describe the development of a robust reverse-genetics platform for identifying genetic factors that regulate pathogenic outcomes and transmission efficiency in vivo, providing key infrastructural developments for developing and evaluating the efficacy of live attenuated vaccines and therapeutics in a clinical setting. dustry in asia ( , ) . during this ongoing outbreak, new strategies are desperately needed to understand pathogenic mechanisms and the functions of viral genes and to provide new technologies to combat this disease. pedv appears to recognize cd , an aminopeptidase n protein, as a receptor for entry into pig cells, as well as the sugar coreceptors heparan sulfate and n-acetylneuraminic acid ( , ) . pedv can infect multiple cell types in vitro, including swine, human, primate, and bat cells, suggesting the possibility of adaptation and spread to other species ( ) . the pedv genome is composed of , nucleotides (nt) encoding seven known open reading frames (orfs) expressed from both genomic and subgenomic mrnas ( ) . subgenomic mrnas are arranged in a nested fashion from the = end of the genome. pedv encodes the traditional coronavirus structural proteins: a receptor-binding spike glycoprotein (s), the envelope protein (e), the membrane glycoprotein (m), and the nucleocapsid protein (n). the spike glycoprotein is a type i membrane glycoprotein composed of s and s external domains, a transmembrane domain, a c-terminal cytoplasmic domain, and a signal peptide. the s protein plays a role in virulence, growth adaptation, receptor binding, and viruscell membrane fusion ( , ) . e of pedv (pedv-e) upregulates stress pathways in the host cell, induces antiapoptotic factors, and is important for viral budding ( ) . pedv-n induces cell stress and prevents apoptosis through similar pathways and prolongs the host cell's s phase ( ) . additionally, pedv contains at least three additional orfs: orf a, orf b, and orf . orf a and -b encode two viral proteases that process these large precursor polyproteins into nonstructural proteins, including the viral replicase and associated rna-modifying enzymes that are critical for full-length and subgenomic positive-and negative-strand rna synthesis ( ) . orf regulates virus production and encodes an ion channel important for viral fitness but is not required for viral replication in vitro ( , ) . in this study, we generated the first infectious cdna clone of a virulent north american pedv strain, pc a ( ) . parental genomic and orf deletion recombinant viruses were generated using the infectious cdna clone system; the latter was also engineered to express red fluorescent protein (rfp). both recombinant viruses are replication competent in vitro and pathogenic in neonatal gnotobiotic (gn) piglets. parental and recombinant viruses were efficiently transmitted to uninoculated pigs via indirect contact, allowing for genetic studies into the molecular mechanisms regulating virus transmission and pathogenesis. the availability of an infectious clone for pedv will allow us further opportunities to understand gene function and genetic variants in pedv pathogenesis and transmission, leading to better-informed design of vaccines and therapeutics. we have developed molecular clones for several highly pathogenic swine and human coronaviruses, using class ii restriction endonucleases, to directionally assemble a full-length cdna viral genome from a set of sequentially designed smaller cdnas ( ) ( ) ( ) ( ) ( ) ( ) . to develop a molecular clone for pedv, the highly virulent pc a strain (fig. a ) was sequenced and synthesized as six contiguous pedv subclones designated a to f (fig. b) . subclones a/b, b/c, c/d, and d/e are joined by unique sapi restriction endonuclease cleavage sites (at nucleotide positions , , , and , respectively) that allow for directional assembly into a full-length cdna without alteration of the viral amino acid sequence. subclones e and f are joined at a unique bsai site at nucleotide position . in subclone f, a single bsai restriction site in pedv-pc a was removed by introducing a silent mutation at position , effectively marking the recombinant genome (fig. c) . thus, each fragment contains a unique set of class ii restriction enzyme sites flanking the genomic sequence that allow for unique -nt over- hangs between each fragment. this specificity allows for systematic, efficient, and directional assembly of the complete pedv genome by in vitro ligation. the pedv a fragment contains a t start site, whereas the f fragment terminates in a residues, allowing for in vitro transcription and capping of a polyadenylated full-length transcript. pedv-orf is an accessory orf encoding a putative ion channel protein that is oftentimes deleted from some natural isolates or following in vitro passage, suggesting that it encodes nonessential functions in vitro and/or in vivo ( ) . to generate a fluorescently marked pedv genome mutant, orf in the pedv f fragment was replaced with red fluorescent protein (rfp), named tomato-red (fig. d) . the mutant was created using native restriction enzyme recognition sequences that allowed for the preservation of the orf transcription regulatory sequence (trs), which regulates subgenomic rna expression (fig. d) . recovery of recombinant viruses. to isolate recombinant wild-type and rfp-expressing recombinant pedvs, each plasmid fragment was digested with noted restriction enzymes, purified, and ligated to create a full-length pedv cdna genome. using the t rna polymerase, full-length transcripts were synthesized in vitro as previously described by our group ( , ) . as previous swine and human coronavirus infectious clones displayed improved recovery rates and replication efficiency in the presence of the supplemented n gene transcript ( , ) , capped pedv-n gene transcripts were mixed with the full-length genomic transcripts prior to their electroporation into vero cells. within to h postelectroporation, subgenomic recombinant-virus mrna could be detected via reverse transcription-pcr (rt-pcr). after we isolated recombinant virus from pig intestinal contents after inoculation, wild-type and recombinant viruses replicated to titers that approached or exceeded ϫ pfu/ml in vero cells, equivalent to titers commonly reported in the literature ( fig. a) . a recombinant infectious-clone-derived pedv (icpedv) produced a plaque morphology (fig. b ) similar to that of the parental strain and formed syncytia characteristic of pedv in culture (fig. c) . notably, icpedv-⌬orf -rfp displayed a reduced plaque size compared to that of either pc a or icpedv (fig. b) , indicative that orf may be important for in vitro growth of the virus and suggestive of possible attenuation of the orf deletion mutant. at and h postelectroporation with icpedv-⌬orf -rfp, fluorescent red cells were seen in cell culture, both within individual cells and within larger syncytia (fig. d) . characterization of recombinant viruses. to evaluate protein expression in our recombinant pedv, we cloned and expressed pedv-s and pedv-n in venezuelan equine encephalitis virus strain virus replicon particles (vrp) and isolated vrp-pedv-s and vrp-pedv-n. the vrp were inoculated into the footpads of mice, and polyclonal pedv-n and pedv-s antisera were collected after a day boost. using western blot techniques, we confirmed the presence of the -/ -and~ -kda pedv-s and pedv-n proteins, respectively, in cells infected with icpedv, icpedv-⌬orf -rfp, and parental pedv in vitro (fig. a) . thus, molecularly derived viruses have protein expression phenotypes similar to those of the parental virus. to further confirm the presence of the recombinant virus postelectroporation, we reverse transcribed genomic rna from virions in the culture media and then sequenced them to demonstrate the presence of the distinguishing bsai cloning site in infectiousclone viruses (fig. b) . additionally, we sequenced the leadercontaining subgenomic mrna transcripts to ensure that both our recombinant viruses and their parental strain shared the wild-type transcription regulatory sequence (trs) required for normal coronavirus replication and growth kinetics (fig. c ). together, these data definitively demonstrate that both recombinant clones generated replicating recombinant virus in vitro. icpedv replication and pathogenesis in gn piglets. pedv pc a is highly pathogenic in newborn piglets and is rapidly transmitted to littermates. to determine if icpedv replicated parental pedv pc a in vivo pathology and transmission phenotypes, gnotobiotic (gn) pigs were orally inoculated with icpedv (passage [p ]), icpedv-⌬orf -rfp (p ), or pc a (p ) and housed with uninfected indirect-contact pigs ( table ) . challenged animals demonstrated fecal viral rna shedding, and diarrhea started to days postinfection (dpi) in all three virus groups ( fig. a ; table ). importantly, uninoculated indirect-contact pigs within each group demonstrated both robust virus shedding and diarrhea, confirming the transmissibility of both pc a and the recombinant virus (fig. b ). all three viruses replicated to similar peak titers ( to log genomic equivalents [ge]/ml). however, pc a and icpedv group pigs had more-severe diarrhea (highest fecal score of ) than icpedv-⌬orf -rfp group pigs (highest score of ). the day of harvest for each piglet was dependent on clinical fecal scores or occurred upon the death of the piglet. notably, days of harvest varied both between virus types and within virus groups. because of the animal numbers in each group and because of the variation within each pedv group, the day of harvest cannot be used as a reliable indicator of the relative degree of viral virulence or attenuation. the pathogenesis of icpedv in gn pigs also replicated the pathogenic phenotype of pedv strain pc a, which had been collected from the same swine farm on the same day as pc a ( ) . histopathological examination showed severe villous atrophy in pedv pc a-and icpedv-inoculated pigs and moderate-tosevere villous atrophy in icpedv-⌬orf -rfp-infected pigs (fig. a ). the villous height/crypt depth (vh/cd) ratio of the jejunum of a mock-inoculated pig was significantly higher than those of pc a-, icpedv-, and icpedv-⌬orf -rfp-infected pigs (p Ͻ . ) ( table ) . immunohistochemistry (ihc) for pedv-n confirmed the presence of recombinant virus throughout the small intestine (duodenum, jejunum, and ilium) in both icpedv-and icpedv-⌬orf -rfp-infected pigs ( fig. b ; table ). in addition, icpedv antigens were detected in the large intestine (colon). these results indicate that the cell culture supernatants of icpedv and icpedv-⌬orf -rfp contained infectious recombinant virus particles that replicated well in gnotobiotic pigs. while the recombinant icpedv replicated the clinical phenotypes of parental pc a in vivo, icpedv-⌬orf -rfp infection resulted in a partial attenuation in pigs based on lower diarrhea scores. the rapid infection of contact pigs suggests efficient transmission of icpedv and icpedv-⌬orf -rfp, replicating both parental pc a and circulating u.s. strain transmission phenotypes. emerging viruses pose a considerable threat to humans and society by causing morbidity and mortality in human populations or causing significant losses in important food sources and trade, leading to economic instability and loss of critical protein sources, especially in poor rural populations. porcine epidemic diarrhea virus is a serious livestock pathogen that is causing significant economic losses in the swine industry internationally. to date, over a billion piglets have died globally. live vaccine has been used historically to combat pedv outbreaks in asia; however, live vaccines available today are ineffective in preventing outbreaks of circulating pandemic strains, including u.s. outbreak strains, and have not significantly reduced the global disease burden ( ) . other important nidovirus infections of swine include transmissible gastroenteritis virus (tgev), its related respiratory variant designated porcine respiratory coronavirus (prcv), and porcine reproductive and respiratory disease virus (prrsv), which have caused major economic losses to the swine industry since the late s ( ) ( ) ( ) . in addition to pedv, an emerging coronavirus, porcine deltacoronavirus, has recently been reported in swine, demonstrating the possibility of continued emergent threats to this important food industry ( , ) . given the apparent increase in the number of new swine viruses identified over the past years, it seems clear that management practices and/or other changes in the ecosystem are providing an environmental setting that promotes the emergence of new viral pathogens for the swine industry. if so, these data document the need for the development of new, rapid-response intervention platforms for disease control in critical livestock populations that are centrally linked to human health. in this article, we describe the first molecular clone for a highly pathogenic u.s. strain of pedv, pc a, isolated from an outbreak in ohio in june ( ) . both the parental pedv pc a strain and its derivative recombinant cloned virus were genetically stable and fully pathogenic in neonatal gnotobiotic pigs, demonstrating that icpedv provides not only a strategy that allows for the systematic evaluation of the role of viral genes in pathogenesis, tropism, and virulence but also a translational platform for the development of rationally attenuated live virus vaccines. in addition, we have constructed a recombinant pedv that bears an indicator gene, the rfp gene, which allows for rapid evaluation of antiviral efficacy and neutralizing antibody levels by means of high-throughput cell culture systems. recently, a molecular clone for a high-growth tissue culture variant of a thai isolate, designated pedv avct , was reported in the literature ( ) . in contrast to our findings, fulllength recombinant pedv avct could not be isolated unless orf expression had been ablated, either by naturally occurring deletions or by insertion of an indicator gene in this location. interestingly, naturally occurring deletions also removed amino acids from the c terminus of the s protein, similar to deletions described with other tissue culture strains, like pedv strain chm ( ) . at this time, the discrepancy between the two laboratory results is intriguing and most likely is directly related to the backbone sequence of the two isolates and/or the difficulties associated with culturing clinical isolates of pedv in vitro. tissue culture pedv avct replicates to logs more efficiently than wild-type pedv pc a and icpedv in culture, and trypsin is also required to culture the latter isolates in vitro. future studies may well reveal the emergence of similar tissue culture adaptations during serial passage of our highly virulent pedv pc a and icpedv in culture. importantly, pathogenic outcomes in vivo were not evaluated using the heavily tissue culture-adapted pedv avct strain, so the utility of this recombinant virus in evaluating pathogenic outcomes and/or the role of tissue cultureadaptive mutations in virulence are uncertain at best. although an exact infectious dose of our recombinant viruses was not determined from these studies, Ͻ pfu of pedv pc a is sufficient to cause disease in piglets ( ) . little information is available regarding the molecular mecha- a pigs , , , and were exposed by indirect contact to the pigs which were housed in the same isolator through small holes drilled into the stainless steel divider. the panel was located between the pigs in the shared pig tub isolator unit. b nd, not done because pig was used for hyperimmune serum production; na, not available; vh/cd, villous height/crypt depth. c fecal scores were as follows: , normal; , pasty stool; , semiliquid diarrhea; and , liquid diarrhea. nisms governing efficient coronavirus pathogenesis and transmission between hosts. importantly, icpedv and icpedv-⌬orf -rfp are efficiently transmitted to cohoused littermates, providing a potential platform for investigating the genetic mechanisms regulating efficient transmission between hosts. while similar studies using highly pathogenic influenza viruses in ferrets are highly controversial because of potential human-pandemic concerns ( ) , identifying genetic factors that attenuate transmission frequency offer a powerful tool to improve the safety and efficacy of live attenuated coronavirus vaccines, especially given the highanimal-density manufacturing approaches used in the swine industry. such studies may also provide significant insights into the fundamental principles and genetic functions that influence the transmission efficiency of other highly pathogenic human coronaviruses, like severe acute respiratory syndrome coronavirus (sars-cov) and middle east respiratory syndrome coronavirus (mers-cov). pedv infection is most devastating in neonatal and suckling piglets, necessitating vaccines that target lactogenic immunity through the vaccination of pregnant sows and gilts. piglets do not attain passive immunity preparturition but instead receive iggand iga-based lactogenic immunity from colostrum and milk, respectively. with both tgev and pedv, sows infected with live virulent virus transferred more protective immunity against viral challenge in their nursing piglets than sows infected with attenuated or inactivated virus ( ) . the usda has granted conditional licenses to two pedv manufacturers to date. the harris vaccine uses an attenuated venezuelan equine encephalitis virus (veev) vaccine strain replicon particle expressing the pedv spike protein ( ) . the second is a parenterally killed virus vaccine made by zoetis ( ) . both are used to immunize pregnant sows and gilts. their efficacy and ability to protect against various circulating u.s. strains are still under evaluation. we note that the vrp platform described in our paper was based on a biosafety level (bsl ) nonselect agent, a veev strain designated , which has been used in animal and human trials ( ) ( ) ( ) . in contrast to other veev replicon platforms, veev retains wild-type e and e glycoproteins, which efficiently target dendritic cells ( , ) , but lacks e sequences. this deletion of e confers an attenuated phenotype in vivo. veev expressing appropriate s glycoprotein genes provides robust protection against other coronaviruses, like sars-cov, mers-cov, and hku -s ( - ). using veev structural genes allows for recovery of high-titer vrp encoding pedv-s and/or -n under bsl conditions. it remains unclear whether the vrp platform will prove sufficiently robust to induce in sows lactogenic immunity capable of protecting suckling piglets ( ) . future experiments will have to be designed and implemented to test the relative efficacies of vrp vaccines, killedwhole-virus vaccines, or future live attenuated-virus vaccines. importantly, no live-virus vaccine is currently available in the united states, and historical live vaccines have not been effective in combating current u.s. or asian strains ( ) . robust studies using sars-cov have identified several viral genes, including the e protein, the exon nsp rna proofreading machinery, and the -o-methyltransferase nsp replicase, as high-priority targets for rational attenuation of coronaviruses ( ) ( ) ( ) . because coronavi- ruses undergo rna recombination at high frequency and encode an exonuclease function ( , ) , recombination repair and reversion to wild-type virus is a pressing concern when designing live attenuated coronavirus vaccines. however, our laboratory has developed strategies to prevent recombination repair that limit the capacity of rationally designed live attenuated virus to revert to the wild-type virus sequence ( ) . effective vaccines are increasingly important, as new strains are identified in the united states and circulating strains continue to devastate herds. the infectious clone platform allows for rapid construction of genetically modified pedv variants to evaluate the function of antigenic variation on neutralization phenotypes and can be used for the rational design of a live virus vaccine. this platform also allows for incorporation of genetic changes to enhance the replication of the virus in vitro for more efficient production of attenuated vaccines. globally, humans have experienced coronavirus outbreaks with increasing frequency, including outbreaks of two new human coronaviruses in the last years, notably, sars-cov and mers-cov ( ) . human and animal coronaviruses share similar structural proteins and replication dynamics. currently, no transmission model for these important human pathogens is available. the neonatal pig model described in this study can provide a surrogate transmission model for human coronaviruses. separately, the study of coronavirus transmission in its original host in a bsl climate affords the opportunity to safely and accurately research a cohoused with an inoculated piglet(s) to determine transmission. all animals succumbed to illness or were euthanized due to illness at their final time points (pedv, dpi; icpedv, dpi; icpedv-⌬orf -rfp, dpi; contact control, dpi). all images show representative histological slides of jejunum specimens from mock-infected or infected animals. histology of mock-, icpedv-, or icpedv-orf -rfp-infected animals, showing h&e staining and immunohistochemistry (ihc) staining (b) for pedv nucleocapsid (n) protein using mouse anti-pedv-n. a specimen from a contact piglet infected by icpedv is also shown. cell fusion and vacuolation were noted at the villus tips (arrows). ihc staining of icpedv-or icpedv-⌬orf -rfp-infected animals whose results are represented in panel a is shown. icpedv icpedv-⌬orf -rfp a the ihc signal of pedv antigen was scored as to according to the percentage of villous enterocytes within the section showing a positive signal. a score of means that there were no positive cells, and scores of , , and mean that Ͻ %, to %, and Ͼ % of villous enterocytes showed a positive signal, respectively. family of viruses that is devastating animal and human populations. it is possible that genetic manipulation of recombinant pedvs will enable studies that can significantly enhance our understanding of the role of coronavirus genetics in the transmission, virulence, and pathologies that are central to both animal and human health and disease prevention. in this article, we describe a reverse-genetics platform for a highly virulent u.s. pedv strain that causes lethal disease in newborn piglets, allowing for the future identification of attenuating mutations and virulence alleles. in parallel, we have developed indicator viruses that can be used for high-throughput neutralization assays or to evaluate the impact of antivirals. this reversegenetics system will allow for quick and robust pedv genetic manipulation in a clinical north american isolate, allowing for in-depth study of viral replication and pathogenesis, which are essential for the development of safe and robust live attenuatedvirus vaccines. the wild-type pc a strain of pedv (passage ) was cultured on vero cells, as described previously ( ) . cells were grown in growth medium containing dulbecco's modified eagle medium (dmem) (life technologies) supplemented with % fetal bovine serum (life technologies) and % antibiotic-antimycotic (gibco). virus was grown in vero cells in maintenance medium, which was dmem supplemented with g/ml trypsin (life technologies), . % tryptose phosphate broth (sigma), and % antibiotic-antimycotic (life technologies). cells were kept in a humidified incubator at °c and % co . assembly of full-length recombinant pedv. the icpedv clone was designed using six separate fragments (ordered from bio basic) flanked with unique flanking class ii restriction sites that leave nonpalindromic overhangs. sequences were ordered based on pc a passage sequence (genbank accession number km . ). all cdna subclones were grown in the pcxl-topo vector. in fragment e, a naturally occurring bsai site was removed by introducing a silent mutation in order to prevent interference with assembly of the full-length infectious clone. all pedv fragments were sequenced after transfection into bacterial culture to ensure sequence fidelity. the pedv fragments were digested using restriction sites designated in fig. , run on a % agarose gel, excised, and purified using a qiaquick gel extraction kit (qiagen). the pedv fragments were mixed and ligated overnight at °c using t dna ligase (roche). ligated fragments were phenol-chloroform extracted, and fulllength t rna transcripts were generated as described in the mmessage mmachine manufacturer protocol (ambion), but we allowed the reaction to run at °c for h and then at room temperature for h. in addition, sp pedv-n gene transcripts were generated from the pcr-purified pedv-n gene sample using a : ratio of cap to gtp (ambion). to generate the orf deletion rfp construct, the tdtomato gene was amplified by pcr with flanking pedv sequence and then inserted using native restriction sites into pedv-f. pedv-f-⌬orf -rfp was cultured and sequenced to ensure seamless replacement of orf with rfp containing the orf trs. in vitro transfection. genome-length and n rna transcripts were mixed with l of vero-bi cells ( ϫ cells/ml) in phosphatebuffered saline (pbs) and then added to an electroporation cuvette. three pulses of v at f were used to transfect the cells with a gene pulser ii electroporator (bio-rad). the cells were allowed to recover for min at room temperature and then were transferred to a -cm flask in growth medium at °c for h, after which time the cells were washed and incubated in cell culture medium. trypsin was added to the culture at g/ml h postelectroporation to assist in virus recovery and spread. sequence analysis identification of marker mutations. virus harvested from small intestinal contents was grown in vero-bi cell culture for h. virion rna was harvested from the supernatant using the qiaamp viral rna minikit (qiagen). after purification, viral cdna was generated with superscript ii reverse transcriptase (life technologies) as previously described by our group ( ) . to demonstrate the presence of the marker mutation, the icpedv bsai mutation site was amplified by pcr using primers = tccaagccatttctagttctatt = and = tgacacaac aaagatgagaaca =. pcr amplicons were gel purified and then sequenced using primers = tcaggctagcaggaagttag = and = ag gtcaactagtgtgttgttgatat =. western blot and transcript analysis. virus from infected animals was cultured in vero-bi cell culture for h and washed with pbs, and intracellular rna was harvested from cells using np- buffer ( mm nacl, % triton x- , mm tris, ph . ) for western blots or trizol (life technologies) for rna analysis. cdna from viral rna transcripts was generated using superscript ii reverse transcriptase (life technologies) and pcr amplified using primer pairs from the pedv leader sequence and nucleocapsid gene. pcr products were separated on a % agarose gel and visualized on a dark reader transilluminator (clare chemical research). for western blot analysis, protein from infected cells was denatured in ϫ laemmli buffer (bio-rad) at °c for min and then separated on gradient to % mini-protean precast gels (bio-rad) prior to electrophoretic transfer of the proteins to polyvinylidene difluoride (pvdf) membranes (bio-rad). to detect pedv antigens, blots were first blocked with % milk in tris-buffered saline with tween (tbst) and then probed with a polyclonal mouse serum (diluted : ) from mice which had been immunized with venezuelan equine encephalitis virus replicon particles (vrp) expressing pedv nucleocapsid (n) or spike (s) glycoprotein. blots were developed using ge healthcare amersham ecl western blotting detection reagents and exposed to film for imaging. animal studies. four groups of -to -week-old gnotobiotic (gn) pigs were used to examine the replication and pathogenesis of icpedvand icpedv-⌬orf -rfp-derived viruses in vivo and compared with pc a-and mock-infected positive and negative controls, respectively (table ) . piglets were orally inoculated with ml of icpedv or icpedv-⌬orf -rfp culture supernatants after transfection (p ) (Ͻ . ϫ pfu/ml) or with the tissue culture-adapted pc a strain at passage level (p ) at a dose of . log pfu/pig. to investigate transmission, pigs , , , and were cohoused in the same isolator as infected pigs but were separated by a stainless steel divider that contained small holes which allowed only indirect contact between the groups. animals were monitored daily for clinical signs of disease, including diarrhea and vomiting. rectal swabs were collected for scoring fecal denseness (scores: ϭ normal; ϭ pasty stool; ϭ semiliquid diarrhea; and ϭ liquid diarrhea) and for enumerating fecal viral rna shedding by rt-quantitative pcr (rt-qpcr). except for one pig in the icpedv group, which was kept long term for the production of hyperimmune serum, the gn pigs were euthanized at acute infection phase (within days postinoculation [dpi] or days postcontact with the inoculated pigs [dpc]) for histopathological examinations. at necropsy, small and large intestinal contents were collected and tested by rt-qpcr for viral rna levels and for infectious virus by plaque assay. the different sections of the small intestine (duodenum, jejunum, ilium) and large intestine (cecum and colon) were collected for histopathological examination and stained with hematoxylin and eosin (h&e) stain. the derivation and maintenance of gn pigs, sample collection and testing, and histopathology were performed as previously described ( , ) . all the animal use protocols employed in this study were reviewed and approved by the agricultural animal care and use committee of the ohio state university. ihc staining. the immunohistochemistry (ihc) staining procedure was optimized as described previously ( ) using the non-biotinpolymerized horseradish peroxidase (hrp) system (biogenex laboratories, san ramon, ca). briefly, intestinal tissue sections from each pig were deparaffinized and rehydrated in graded ethanol to pbs (ph . ). antigen retrieval and unmasking were performed by treatment with . % pronase e (sigma-aldrich, st. louis, mo) for min. the endogenous per-oxidase activity was quenched with % hydrogen peroxide (sigma) for min. then, the sections were incubated in power block solution (bio-genex) for min at room temperature. mouse monoclonal antibody anti-pedv nucleocapsid protein (n) (sd - ; a gift from e. nelson and s. lawson, south dakota state university) was applied to each section at °c overnight. after two washes in pbs, a commercial super sensitive ihc detection system (biogenex) was used. finally, these sections were counterstained with mayer's hematoxylin (biogenex) and dehydrated, and coverslips were added. the ihc signal of pedv antigen was scored as to according to the percentage of villous enterocytes within the section showing a positive signal. scores were as follows: indicated that there were no positive cells and , , and indicated that Ͻ %, to %, and Ͼ % of villous enterocytes showed a positive signal, respectively. identification of new respiratory viruses in the new millennium. viruses emergence of porcine epidemic diarrhea virus in the united states: clinical signs, lesions, and viral genomic sequences pathogenesis of porcine epidemic diarrhea virus isolate (us/ iowa/ / ) in -week-old weaned pigs comparative pathogenesis of us porcine epidemic diarrhea virus (pedv) strain pc a in conventional -day-old nursing piglets vs. -day-old weaned pigs swine enteric coronavirus diseases (secd), including porcine epidemic diarrhea virus (pedv) origin, evolution, and virulence of porcine deltacoronaviruses in the united states a novel pathogenic mammalian orthoreovirus from diarrheic pigs and swine blood meal in the united states distinct characteristics and complex evolution of pedv strains understanding human coronavirus hcov-nl porcine epidemic diarrhoea virus: a comprehensive review of molecular epidemiology, diagnosis, and vaccines outbreak of porcine epidemic diarrhea in suckling piglets origin, evolution, and genotyping of emergent porcine epidemic diarrhea virus strains in the united states us-like strain of porcine epidemic diarrhea virus outbreaks in taiwan porcine epidemic diarrhea: a review of current epidemiology and available vaccines receptor usage and cell entry of porcine epidemic diarrhea coronavirus porcine epidemic diarrhea virus uses cellsurface heparan sulfate as an attachment factor genome organization of porcine epidemic diarrhoea virus mutations in the spike gene of porcine epidemic diarrhea virus associated with growth adaptation in vitro and attenuation of virulence in vivo receptor-bound porcine epidemic diarrhea virus spike protein cleaved by trypsin induces membrane fusion porcine epidemic diarrhea virus e protein causes endoplasmic reticulum stress and up-regulates interleukin- expression porcine epidemic diarrhea virus n protein prolongs s-phase cell cycle, induces endoplasmic reticulum stress, and up-regulates interleukin- expression completion of the porcine epidemic diarrhoea coronavirus (pedv) genome sequence pedv orf encodes an ion channel protein and regulates virus production manipulation of the porcine epidemic diarrhea virus genome using targeted rna recombination cell culture isolation and sequence analysis of genetically diverse us porcine epidemic diarrhea virus strains including a novel strain with a large deletion in the spike gene reverse genetics with a full-length infectious cdna of the middle east respiratory syndrome coronavirus systematic assembly of a full-length infectious clone of human coronavirus nl development of mouse hepatitis virus and sars-cov infectious cdna constructs reverse genetics with a full-length infectious cdna of severe acute respiratory syndrome coronavirus strategy for systematic assembly of large rna and dna genomes: transmissible gastroenteritis virus model systematic assembly of a full-length infectious cdna of mouse hepatitis virus strain a live porcine reproductive and respiratory syndrome virus vaccines: current status and future direction transmissible gastroenteritis virus infection: a vanishing specter background paper. the appearance of the porcine respiratory coronavirus has created new problems and perspectives detection and genetic characterization of deltacoronavirus in pigs genetic manipulation of porcine epidemic diarrhea virus (pedv) recovered from a full-length infectious cdna clone determination of the infectious titer and virulence of an original us porcine epidemic diarrhea virus pc a strain avian influenza virus transmission to mammals resolution of the cellular proteome of the nucleocapsid protein from a highly pathogenic isolate of porcine reproductive and respiratory syndrome virus identifies parp- as a cellular target whose interaction is critical for virus biology usda licenses first vaccine for porcine epidemic diarrhea. usda animal and plant health inspection service usda grants zoetis a conditional license for porcine epidemic diarrhea vaccine improved mucosal protection against venezuelan equine encephalitis virus is induced by the molecularly defined, live-attenuated v vaccine candidate venezuelan equine encephalitis virus vaccine candidate (v ) safety, immunogenicity and efficacy in horses environmental hazard assessment of venezuelan equine encephalitis virus vaccine candidate strain v infected dendritic cells are sufficient to mediate the adjuvant activity generated by venezuelan equine encephalitis virus replicon particles role of dendritic cell targeting in venezuelan equine encephalitis virus pathogenesis rapid generation of a mouse model for middle east respiratory syndrome a mouse model for betacoronavirus subgroup c using a bat coronavirus strain hku variant successful vaccination strategies that protect aged mice from lethal challenge from influenza virus and heterologous severe acute respiratory syndrome coronavirus a live, impaired-fidelity coronavirus vaccine protects in an aged, immunocompromised mouse model of lethal disease attenuation and restoration of severe acute respiratory syndrome coronavirus mutant lacking =-omethyltransferase activity severe acute respiratory syndrome coronaviruses with mutations in the e protein are attenuated and promising vaccine candidates recombination, reservoirs, and the modular spike: mechanisms of coronavirus cross-species transmission coronaviruses: an rna proofreading machine regulates replication fidelity and diversity rewiring the severe acute respiratory syndrome coronavirus (sars-cov) transcription circuit: engineering a recombination-resistant genome molecular pathology of emerging coronavirus infections pathology of us porcine epidemic diarrhea virus strain pc a in gnotobiotic pigs we thank j. hanson, r. wood, and j. ogg for animal care assistance, and t.oka and x. wang for technical assistance. special thanks to steven lawson and eric nelson (department of veterinary and biomedical sciences, south dakota state university) for providing mouse anti-pedv nucleocapsid protein monoclonal antibody sd - .salaries and research support were provided by state and federal funds appropriated to ohio agricultural research and development center (oardc), the ohio state university.the authors acknowledge the key financial support from the national institutes of health, center for diagnostics and discovery (u ai ) to r.s.b. and grant - - from the usda to q.w., l.s., and r.s.b. key: cord- - zpw itb authors: pirofski, liise-anne; casadevall, arturo title: immune-mediated damage completes the parabola: cryptococcus neoformans pathogenesis can reflect the outcome of a weak or strong immune response date: - - journal: mbio doi: . /mbio. - sha: doc_id: cord_uid: zpw itb cryptococcosis occurs most frequently in immunocompromised individuals. this has led to the prevailing view that this disease is the result of weak immune responses that cannot control the fungus. however, increasingly, clinical and experimental studies have revealed that the host immune response can contribute to cryptococcal pathogenesis, including the recent study of l. m. neal et al. (mbio :e - , , https://doi.org/ . /mbio. - ) that reports that cd (+) t cells mediate tissue damage in experimental murine cryptococcosis. this finding has fundamental implications for our understanding of the pathogenesis of cryptococcal disease; it helps explain why immunotherapy has been largely unsuccessful in treatment and provides insight into the paradoxical observation that hiv-associated cryptococcosis may have a better prognosis than cryptococcosis in those with no known immune impairment. the demonstration that host-mediated damage can drive cryptococcal disease provides proof of concept that the parabola put forth in the damage-response framework has the flexibility to depict complex and changing outcomes of host-microbe interaction. c ryptococcosis is usually observed in hosts with impaired immunity. consequently, the agent causing cryptococcosis, cryptococcus neoformans, is often considered pathogenic only when the immune system is unable to control its growth. this has led to its characterization as an "opportunistic pathogen" ( ) . in their article in mbio, l. m. neal et al. ( ) report that the cd ϩ t cell-mediated response to c. neoformans is a major contributor to tissue damage in cryptococcal meningitis in mice even though it also mediates fungal clearance. this observation adds to increasing evidence that the host response can drive cryptococcal disease and highlights the ability of the damageresponse framework (drf) to guide our understanding of microbial pathogenesis. the drf was first put forth in ( ) to provide a theory of microbial pathogenesis that could incorporate the contributions of both host and microbe to host damage that stems from host-microbe interaction. prior to the drf, microbial pathogenesis was largely viewed as a singular outcome of either microbial factors or host factors. while such microbe-or host-centered views were able to explain the pathogenesis of certain infectious diseases, they could not explain others, especially those caused by microbes only rarely associated with disease. this shortcoming became glaring in the late s and early s as the hiv/aids pandemic led to the emergence of previously rare and unusual diseases, including cryptococcosis ( ). in the original formulation of the drf, the outcome of host-microbe interaction with different microbes was depicted by six curves that plotted host damage as a function of the strength of the immune response. these curves, referred to as pathogen classes, were based on what was known at the time about the outcome of infection with given microbes. the rationale for the pathogen classes was underpinned by the tenet that host damage can stem from microbial factors, host factors, or both. central to this tenet was the idea that host damage stemming from the immune response to a microbe can drive disease pathogenesis. at the time the drf was proposed, the host inflammatory response was not generally viewed as a causal factor in the pathogenesis of infectious diseases. however, this has changed. the occurrence of severe acute respiratory syndrome (sars) in young, previously well persons who presented with excessive pulmonary inflammation ( ), reminiscent of influenza epidemics that struck young, robust persons ( ), highlighted the role that host-mediated damage can play in viral disease pathogenesis. the ability of the drf to account for host damage due to inflammation stemming from the immune response to certain microbes ( ) highlighted its flexibility and capacity to incorporate new diseases and information. despite the ability of the drf to classify most microbes into one of the original six pathogen classes, new knowledge from clinical and experimental studies led to the conclusion that some of the original classifications were incorrect. again, lessons learned from the hiv/aids pandemic provided new insights into microbial pathogenesis, perhaps best exemplified by cryptococcosis. c. neoformans was first classified as a class pathogen, the definition of which was "pathogens that cause damage either in hosts with weak immune responses or in the setting of normal immune responses" ( ). this characterization was consistent with available knowledge in . however, the emergence of cryptococcus gattii in apparently healthy persons in the pacific northwest ( ) and the unexpected appearance of immune reconstitution inflammatory syndrome (iris)-associated cryptococcosis in patients with hiv/aids after initiation of antiretroviral therapy (art) ( , ) revealed that the host immune response itself can contribute to the pathogenesis of cryptococcosis. thus, classification of c. neoformans as a class pathogen needed to be revisited. another example of a pathogen needing reclassification is pneumocystis jirovecii, which was originally classified as a class pathogen, defined as "pathogens that cause damage only in the setting of weak immune responses" ( ). however, pneumocystis pneumonia is associated with intense inflammation in children and after initiation of therapy in patients with hiv/aids ( , ) . in fact, a major advance in treating pneumocystis pneumonia was the realization that corticosteroid therapy reduced morbidity and mortality, a finding that was counterintuitive, since this disease arose in the setting of profound hiv-associated immunodeficiency. thus, inflammation may drive pneumocystis-and cryptococcus-mediated damage, even in patients with severely impaired immunity, defying their initial classifications as class and pathogens, respectively. although one might propose that these microbes be reclassified as class pathogens, "pathogens that cause damage in the setting of appropriate immune responses and cause damage at both ends of the continuum of immune response" ( ), most persons with normal immune responses never exhibit clinically detected damage as an outcome of interaction with these microbes. these examples highlight limitations of the original drf pathogen classes and illustrate that each of the six classes is actually a derivative of a single parabola that can shift to the left, right, up, or down to depict the impact of the immune response on disease pathogenesis (fig. ) . the emergence of iris-associated cryptococcosis in the setting of art initiation provided clear evidence that host damage in patients with cryptococcal disease may be driven by inflammation ( ) . iris often occurred in patients with very low initial cerebrospinal fluid (csf) lymphocyte counts ( ) , and has been associated with chemokine dysregulation ( ) and activated cd ϩ t cells and macrophages ( , , ) . similar findings have also been reported in some non-hiv-infected patients with cryptococcal meningitis ( , ) . a role for cd ϩ t cells in the pathogenesis of cryptococcusassociated iris has also been demonstrated in experimental mouse models. eschke and colleagues induced iris in rag Ϫ/Ϫ mice by reconstituting them with naive (wild-type) cd ϩ t cells month after pulmonary infection with cfu of c. neoformans ( ) . in this model, mice developed wasting and systemic inflammation evidenced by high levels of th -type cytokines (interleukin [il- ], tumor necrosis factor alpha [tnf-␣], and gamma interferon [ifn-␥]), activated cd ϩ t cells, and granulomatous inflammation in the liver compared to control mice that did not receive cd ϩ t cells (and mice that received cd ϩ t cells and were not infected). reconstitution did not affect fungal clearance. this model demonstrated that cd ϩ t cell reconstitution induced systemic inflammation in c. neoformans-infected mice that closely resembled art-induced iris in hiv-infected patients with cryptococcosis. neal and colleagues ( ) describe another mouse model in which cd ϩ t cells mediate inflammation and host damage in the setting of c. neoformans infection. their study was undertaken to gain insight into the role that cd ϩ t cells may play in the pathogenesis of iris-and postinfectious inflammatory syndromes (piirs) in hivinfected and hiv-uninfected patients with cryptococcosis. mice infected intravenously with cfu of c. neoformans were monitored clinically or analyzed to determine their central nervous system (cns) fungal burden (cfu), inflammatory pathology, and cellular and cytokine responses. the number of c. neoformans cfu in the brains of mice increased from to weeks after infection and then decreased from to weeks thereafter. interestingly, the mice developed symptomatic disease and neurological deterioration to weeks after infection, corresponding to fungal clearance and surprisingly, mortality. cns pathology revealed cellular inflammation marked by leukocytes beginning weeks after infection that subsequently increased due to infiltration of cd ϩ and cd ϩ t cells, with cd ϩ t cells being the predominant population and both populations being antigen experienced. infiltrating cd ϩ and cd ϩ t cells exhibited a th -type bias, producing ifn-␥. depletion of cd ϩ t cells resulted in reduced mortality and inflammatory pathology, providing proof of concept that cd ϩ outcomes of host-cryptococcus neoformans interaction depicted by the basic parabola of the damage-response framework. the left side of the parabola, shaded in green and labeled weak, depicts the original circa concept that c. neoformans was a class pathogen that caused damage in the setting of a weak or normal immune response. here, the immune response fails to limit fungal growth, which results in host damage. the right side of the parabola, shaded in red and labeled strong, depicts information that has emerged since , which has revealed that c. neoformans can elicit strong immune responses that produce host damage stemming from inflammation. the portion of the parabola that rises above the black dotted line represents the threshold for clinical disease. the portion of the parabola that lies below the black dotted line represents the state of c. neoformans latency, which is not associated with clinically evident host damage. proposed therapeutic interventions, based on counteracting the main source of host damage are depicted by red (to enhance weak response) or green (to reduce strong response) arrows. some factors that contribute to weak and strong immune responses are shown along the y axis for each type of response, though immunity is likely multifactorial, and additional factors are also likely to contribute ( , - ) . commentary ® t cells were the principal mediators of inflammation and damage in this model. notably, survival of infected, cd ϩ t cell-depleted mice was improved despite an inability to induce fungal clearance and markedly elevated fungal burdens compared to mice with sufficient cd ϩ t cells. the data generated by neal et al. ( ) demonstrate that cd ϩ t cells can play dichotomous roles in c. neoformans infection. on one hand, they can mediate fungal clearance in the cns, but on the other hand, they can induce inflammation that leads to neurological deterioration and death. this suggests a possible explanation for why inflammation is often a prominent feature of cryptococcal meningitis in hiv-uninfected, but not hiv-infected patients, and raises the intriguing possibility that the profound cd ϩ t cell deficiency, which portends risk for cryptococcosis, may actually limit inflammatory pathology in hiv-infected persons not on art. this dichotomy may also provide insight into the failure of adjunctive steroid therapy to reduce mortality or improve morbidity in hiv-associated cryptococcosis ( ) and the fact that cryptococcosis may have a better prognosis in immunosuppressed patients than in nonimmunosuppressed patients ( ) ( ) ( ) . the detrimental effect of activated cd ϩ t cells in mice also suggests the possibility that antifungal therapy-induced fungal clearance and reduced levels of glucuronoxylomannan (gxm) (an immune suppressing molecule [ ] [ ] [ ] ), could enhance cd ϩ t cell activation and damage. this may help explain paradoxical worsening of cryptococcosis in certain patients after initiation of antifungal therapy ( ) . the aforementioned experimental models provide possible mechanistic explanations for the dichotomous role that the immune response may play in the pathogenesis of iris-and piirs-associated cryptococcosis while contributing to fungal clearance. thus, microbe and host both contribute to host damage and where an individual patient's immune response lies on the continuum of the drf parabola determines the nature of the disease process. at one end, on the left, a weak immune response can result in tissue damage due to fungal proliferation that results in compression of brain tissue with little or no inflammation in what is primarily microbe-mediated damage. at the other end, on the right, a strong cellular immune response can also damage brain tissue due to excessive inflammation in what is primarily host-mediated damage. the occurrence of host damage in the setting of weak and strong immune responses provides important clues as to why it has been so difficult to consistently use agents that modulate inflammation, such as corticosteroids and interferon, to improve the outcome of cryptococcosis. although adjunctive ifn-␥ therapy in hiv-associated cryptococcosis ( ) and corticosteroids can ameliorate iris ( ) in some patients, such immune-modulating agents may not be effective in other patients, because physicians do not usually know whether the preponderance of damage is due to host factors, microbial factors, or both. in this regard, the failure and detrimental effects of steroid therapy in patients with hiv-associated cryptococcosis ( ) probably represent additional suppression of weak immunity resulting in enhancement of fungus-mediated damage. hence, the effective use of immunotherapy is likely to require a rational approach that incorporates knowledge of where the immune response of an individual lies along the immune response co ntinuum depicted by the drf parabola. host-pathogen interactions: the attributes of virulence cd ϩ t cells orchestrate lethal immune pathology despite fungal clearance during cryptococcus neoformans meningoencephalitis host-pathogen interactions: redefining the basic concepts of virulence and pathogenicity what is a host? incorporating the microbiota into the damage-response framework a clinicopathological study of three cases of severe acute respiratory syndrome (sars) the pathogenesis of influenza virus infections: the contributions of virus and host factors spread of cryptococcus gattii into pacific northwest region of the united states paucity of initial cerebrospinal fluid inflammation in cryptococcal meningitis is associated with subsequent immune reconstitution inflammatory syndrome international network for the study of hiv-associated iris (inshi). . cryptococcal immune reconstitution inflammatory syndrome in hiv- -infected individuals: proposed clinical case definitions the expanding role of co-trimoxazole in developing countries anti-inflammatory treatment of acute and chronic pneumonia immune reconstitution inflammatory syndrome in hiv-associated cryptococcal meningitis: a prospective study chemokine levels and chemokine receptor expression in the blood and the cerebrospinal fluid of hiv-infected patients with cryptococcal meningitis and cryptococcosis-associated immune reconstitution inflammatory syndrome cellular immune activation in cerebrospinal fluid from ugandans with cryptococcal meningitis and immune reconstitution inflammatory syndrome the immunopathogenesis of cryptococcal immune reconstitution inflammatory syndrome: understanding a conundrum paradoxical immune responses in non-hiv cryptococcal meningitis fighting the monster: applying the host damage framework to human central nervous system infections a novel experimental model of cryptococcus neoformans-related immune reconstitution inflammatory syndrome (iris) provides insights into pathogenesis adjunctive dexamethasone in hiv-associated cryptococcal meningitis predictors of mortality and differences in clinical features among patients with cryptococcosis according to immune status different presentations and outcomes between hivinfected and hiv-uninfected patients with cryptococcal meningitis outcomes of central nervous system cryptococcosis vary with host immune function: results from a multi-center, prospective study effects of the capsular polysaccharides of cryptococcus neoformans on phagocyte migration and inflammatory mediators downregulation by cryptococcal polysaccharide of tumor necrosis factor alpha and interleukin- beta secretion from human monocytes purified capsular polysaccharide of cryptococcus neoformans induces interleukin- secretion by human monocytes an immune reconstitution syndrome-like illness associated with cryptococcus neoformans infection in organ transplant recipients adjunctive interferon-gamma immunotherapy for the treatment of hiv-associated cryptococcal meningitis: a randomized controlled trial central nervous system immune reconstitution inflammatory syndrome naive b cells reduce fungal dissemination in cryptococcus neoformans infected rag Ϫ/Ϫ mice igm ϩ memory b cell expression predicts hiv-associated cryptococcosis status susceptibility to cryptococcal meningoencephalitis associated with idiopathic cd ϩ lymphopenia and secondary germline or acquired defects fc gamma receptor a polymorphism and risk for hiv-associated cryptococcal disease study of common functional genetic polymorphisms of fcgr a, a and b genes and the risk for cryptococcosis in hiv-uninfected patients anti-gm-csf autoantibodies in patients with cryptococcal meningitis genotypes coding for mannose-binding lectin deficiency correlated with cryptococcal meningitis in hiv-uninfected chinese patients l.p. is supported in part by nih grant r ai . a.c. is supported in part by nih grants r hl , r ai , r ai , and r ai . key: cord- -y pvj l authors: patel, robin; babady, esther; theel, elitza s.; storch, gregory a.; pinsky, benjamin a.; st. george, kirsten; smith, tara c.; bertuzzi, stefano title: report from the american society for microbiology covid- international summit, march : value of diagnostic testing for sars–cov- /covid- date: - - journal: mbio doi: . /mbio. - sha: doc_id: cord_uid: y pvj l nan settings where potentially infected individuals (e.g., sick patients in hospitals) might otherwise pose a risk. unfortunately, these hypothetical scenarios are not reality. however, with this ideal situation as a guide, what we do have available as tests today should be carefully considered in terms of how they can be leveraged to move the current crisis closer to the ideal situation, especially in the absence of therapeutics or vaccines. although the virus can be cultured, this is dangerous and not routinely done in clinical laboratories. while detection of viral antigens is theoretically possible, this approach has not, to date, been a primary one, but one that those participating in the summit considered to deserve further research. most tests currently used for direct detection of sars-cov- identify viral rna through nucleic acid amplification, usually using pcr. an important consideration is exactly what gets tested for viral rna. tests that detect viral rna are contingent on viral rna being present in the sample collected. the most common sample types being tested are swabs taken from the nasopharynx and/or oropharynx, with the former considered somewhat more sensitive than the latter ( ); if both are collected, the two swabs may be combined and tested simultaneously in a single reaction to conserve reagents. today, health care professionals collect these swabs; however, evidence suggests that patients or parents (in the case of young children) might be able to collect their own swabs ( , ) . following collection, swabs are placed into a liquid to release virus/viral rna from the swabs into solution. then, viral rna is extracted from that solution and subsequently amplified (e.g., by reverse transcription-pcr). for patients with pneumonia, in addition to nasopharyngeal and oral secretions, lower respiratory tract secretions, such as sputum and bronchoalveolar lavage fluid, are tested. it should not be assumed that each of these (e.g., nasopharyngeal swab specimen, sputum, bronchoalveolar lavage fluid) will have the same chance of detecting sars-cov- ; detection rates in each sample type vary from patient to patient and may change over the course of individual patients' illnesses. some patients with pneumonia may have negative nasal or oropharyngeal samples but positive lower airway samples ( ), for example. accordingly, the true clinical sensitivity of any of these tests is unknown (and is certainly not %, as in the hypothetical scenario); a negative test does not therefore negate the possibility that an individual is infected. if the test is positive though, the result is most likely correct, although stray viral rna that makes its way into the testing process (for example, as the specimen is being collected or as a result of specimen cross-contamination or testing performed by a laboratory worker who is infected with sars-cov- [these are just some examples]) could conceivably result in a falsely positive result. also, we note that viral rna does not equate to live virus, and therefore, detection of viral rna does not necessarily mean that the virus can be transmitted from that patient. that said, viral rna-based tests are the best tests that we have in the setting of an acute illness. it is important to recognize that the accuracy of the test is affected by the quality of the sample, and thus it is critical that the sample be obtained in a proper (and safe) manner. testing patients for sars-cov- helps identify those who are infected, which is useful for individual patient management, as well as for implementation of mitigation strategies to prevent spread in health care facilities and in the community alike (fig. ) . there are numerous unanswered questions, challenges, and controversies surrounding testing for viral rna. rna may degrade over time. there are concerns that specimen collection for testing is exhausting the supply of critical personal protective equipment needed to care for infected patients. alternative strategies for specimen collection, including home collection, should therefore be considered either by a health care provider or patients themselves (or a parent in the case of young children); the use of alternative specimen types, such as oral fluid or nasal swabs (if they are shown to provide results equivalent to those from nasopharyngeal swabs) should also be considered. spread to health care workers and within health care and long-term-care facilities is a primary consideration for prioritization of testing; testing of patients likely to have sars-cov- who are in health care facilities or long-term-care facilities, alongside potentially ill workers critical to the pandemic response, including health care workers, public health officials, and other essential leaders, is a priority. that said, testing anyone who has symptoms compatible with covid- should be considered, since broad testing will help define who has this infection, allowing control of its spread. given that sars-cov- can infect anyone and result in transmission prior to the onset of symptoms or even possibly without individuals ever developing symptoms, testing asymptomatic patients could even be considered. unfortunately, little is known at this time about viral rna detection in asymptomatic patients, and such testing strategies may stretch available resources beyond realistic limits. some future therapeutics may work best if given early, which will demand early testing for sars-cov- to realize maximal efficacy. the questions of how many tests are needed and what kind should be performed on individual patients (for primary diagnosis if results of initial testing are negative and subsequently to document clearance of the virus to release patients from isolation) remain open. as the number of tests available for sars-cov- increases, new challenges, including the needs to (i) better understand variability in the performance characteristics of the various tests (e.g., sensitivity and specificity), including on different samples types, (ii) optimize assays from their original design (e.g., multiple targets to a single target) to improve reagent utilization while maintaining performance characteristics, and (iii) monitor test performance given the potential for the virus to mutate, are emerging. the last can be addressed by periodically sequencing the evolved virus to look for changes in primer and probe binding regions that might affect the performance of tests based on the detection of viral rna; periodic sequencing can also aid in tracking viral evolution. additionally, as testing increases, decreasing the time to results of testing will continue to be crucial to better manage both patients and health care workers. development of rapid, point-of-care diagnostics is a gap and should be a priority. measurement of viral levels may also be useful to monitor recovery, response to therapy, and/or level of infectivity. current rna-based diagnostic tests are primarily qualitative, and although they could be calibrated to provide viral loads, a standardized process does not currently exist. of note, there is no established threshold for interpretation of viral loads, which may vary in different hosts. although tests have become available, the huge demand for them has created supply chain challenges, compromising their very availability; this includes issues with the availability of nasopharyngeal swabs, rna extraction reagents and instruments, and pcr reagents and instruments. even with now-fda-approved/cleared commercial tests, there are delays with the installation of instruments and supply of reagents/kits to meet the demand at many sites. at the moment, extensive efforts are being made on multiple fronts to address the numerous supply challenges surrounding testing and a secure continuity of testing services. the other broad category of tests is those that detect igm, iga, igg, or total antibodies (typically in blood). development of an antibody response to infection can be host dependent and take time; in the case of sars-cov- , early studies suggest that the majority of patients seroconvert between and days postexposure to the virus, although some patients may develop antibodies sooner. as a result of this natural delay, antibody testing is not useful in the setting of an acute illness. we do not know for certain whether individuals infected with sars-cov- who subsequently recover will be protected, either fully or partially, from future infection with sars-cov- or how long protective immunity may last; recent evidence from a rhesus macaque study does suggest protective immunity after resolution of a primary infection (https://doi.org/ . / . . . ); however, further studies are needed to confirm this. antibody tests for sars-cov- may facilitate (i) contact tracing-rna-based tests can help with this as well; (ii) serologic surveillance at the local, regional, state, and national levels; and (iii) identification of those who have already had the virus and thus may (if there is protective immunity) be immune. assuming there is protective immunity, serologic information may be used to guide return-to-work decisions, including for individuals who work in environments where they can potentially be reexposed to sars-cov- (e.g., healthcare workers). serologic testing may also be useful to identify individuals who may be a source for (currently experimental) therapeutic or prophylactic neutralizing antibodies. in addition, antibody testing can be used in research studies to determine the sensitivity of pcr assays for detecting infection and be employed retrospectively to determine the true scope of the pandemic and assist in the calculation of statistics, including the case fatality rate. finally, serologic testing can possibly be used diagnostically to test viral rna-negative individuals presenting late in their illness. summit participants noted that testing for host markers might be needed to fully understand which patients are at risk of developing severe disease from their infection. in summary, both of the two categories of tests for sars-cov- should be useful in this outbreak. we are fortunate to have the technologies we do that have allowed diagnostics to be made rapidly available. there is likely to be a direct connection between understanding the level of virus/disease in individual communities and acceptance of control measures that require individual action, such as social distancing. now, we need to ensure systematic and coordinated efforts between the public, clinical, commercial, and industry sectors to ensure robust supply lines in the midst of the pandemic so that we can leverage the power of testing to address the pandemic confronting us. diagnostic stewardship: opportunity for a laboratory-infectious diseases partnership sars-cov- viral load in upper respiratory specimens of infected patients effectiveness of patient-collected swabs for influenza testing equal performance of self-collected and health care worker-collected pharyngeal swabs for group a streptococcus testing by pcr negative nasopharyngeal and oropharyngeal swab does not rule out covid- key: cord- -pd lt n authors: mathieu, cyrille; dhondt, kévin p.; châlons, marie; mély, stéphane; raoul, hervé; negre, didier; cosset, françois-loïc; gerlier, denis; vivès, romain r.; horvat, branka title: heparan sulfate-dependent enhancement of henipavirus infection date: - - journal: mbio doi: . /mbio. - sha: doc_id: cord_uid: pd lt n nipah virus and hendra virus are emerging, highly pathogenic, zoonotic paramyxoviruses that belong to the genus henipavirus. they infect humans as well as numerous mammalian species. both viruses use ephrin-b and -b as cell entry receptors, and following initial entry into an organism, they are capable of rapid spread throughout the host. we have previously reported that nipah virus can use another attachment receptor, different from its entry receptors, to bind to nonpermissive circulating leukocytes, thereby promoting viral dissemination within the host. here, this attachment molecule was identified as heparan sulfate for both nipah virus and hendra virus. cells devoid of heparan sulfate were not able to mediate henipavirus trans-infection and showed reduced permissivity to infection. virus pseudotyped with nipah virus glycoproteins bound heparan sulfate and heparin but no other glycosaminoglycans in a surface plasmon resonance assay. furthermore, heparin was able to inhibit the interaction of the viruses with the heparan sulfate and to block cell-mediated trans-infection of henipaviruses. moreover, heparin was shown to bind to ephrin-b and to restrain infection of permissive cells in vitro. consequently, treatment with heparin devoid of anticoagulant activity improved the survival of nipah virus-infected hamsters. altogether, these results reveal heparan sulfate as a new attachment receptor for henipaviruses and as a potential therapeutic target for the development of novel approaches against these highly lethal infections. required for cell-mediated trans-infection. as proteoglycans are used by several viruses for binding to target cells ( , ) , we investigated their potential role as a henipavirus attachment receptor. heparan sulfate (hs) is a complex and sulfated polysaccharide of the glycosaminoglycan (gag) family that is linked to ubiquitous core proteins of the cell surface and extracellular matrix of all eukaryotes. hs is composed of a repetition of a d-glucuronic acid/ n-acetyl-d-glucosamine disaccharide motif that can be further modified by addition of sulfate groups. hs has the ability to bind to a vast repertoire of proteins and is involved in many physiological as well as pathological processes ( ) . in addition, the long carbohydrate chains of hs provide easily accessible binding sites for a wide range of pathogens, including viruses, bacteria, and parasites ( , ) . commercial heparin (hp), which is widely used for its anticoagulant properties, is a gag that is chemically related to hs ( , ) and displays protein-binding properties similar to hs. in this study, we show that both niv and hev can use hs as an attachment receptor to mediate trans-infection. in addition, hs facilitates henipavirus infection in cis, and heparin can compete with hs for binding to the virus, thereby limiting both transinfection and infection itself. finally, heparin significantly reduced niv infection in hamsters, suggesting its potential use to treat henipavirus infections. we first asked whether leukocytes could capture hev and transmit it to susceptible cells in trans without becoming infected themselves (fig. a) . as we previously found for niv ( ) , peripheral blood lymphocytes (pbls) also transmit cell-attached hev to susceptible cells, indicating that trans-infection is a mechanism shared by both members of the henipavirus genus. the trans-infection by leukocytes can also be mimicked in cho cells ( ) , which are resistant to henipavirus infection due to the lack of entry receptors efn-b and -b ( ) . the treatment of cho-k cells with heparinase inhibited their trans-infection properties by Ͼ %. accordingly, cho-derived cell lines that lacked expression of hs because of their deficiency in gag biosynthesis enzymes ( ) were also highly deficient in their ability to mediate trans-infection of niv (fig. b) . to further characterize the niv-gag interaction, we compared the ability of virus particles pseudotyped with niv g and f glycoproteins or vesicular stomatitis virus g-protein (vsv-g) ( ) to bind surfaces coated with the hs analog heparin by using surface plasmon resonance (spr). the strong hyperbolic association shape observed during the injection phase of the sensorgram for niv, but not for vsv pseudoparticules, clearly indicated a much stronger binding affinity of niv-pseudotyped viral particles (fig. c) , underlining the importance of niv glycoproteins in the analyzed interaction. to assess the specificity of the niv-gag interaction toward hs, by using spr we compared the binding of niv-pseudotyped virus to surfaces displaying hs, hp, or dermatan sulfate (ds), another sulfated gag (fig. d) . although niv-pseudotyped virus efficiently bound to hp and hs, we did not detect any interaction either niv or hev, washed, cultured for h, and then transferred to vero cell monolayers, which were used for the determination of the viral titers after days of coculture, using infectious center assays. (b) cho-k cells, treated or not with heparinase , and three hs-deficient cho lines, pgsa- , pgsb- , and pgsd- , were incubated with niv and analyzed for their capacities to transmit infection to susceptible vero cells in trans. results are expressed as a percentage of inhibition compared to results with untreated cells Ϯ sd. *, p Ͻ . ; ***, p Ͻ . (mann-whitney u test). (c) spr analysis of the binding of mlv pseudotyped either with vsv-g (green) or with niv glycoproteins g and f (red) to hp-activated sensor chip surfaces. (d) spr analysis of the binding of niv g and f pseudoparticles to surfaces activated by either hs (blue), ds (green), or hp (red). the binding response, in ru, was recorded as a function of time; results from of experiments are presented. with the ds surface, suggesting an implication of specific carbohydrate features. altogether, these results suggested that niv could specifically use hs as an attachment receptor to promote efficient trans-infection. heparin is a competitive inhibitor of henipavirus transinfection. as niv-pseudotyped virus binds heparin in addition to hs (fig. d) , we next analyzed the effect of heparin on the ability of pbls and cho-k to mediate niv trans-infection. heparin reduced the trans-infection property of pbls and cho-k cells by % and %, respectively ( fig. a) . strikingly, heparin was also active when applied after contact with the virus, and both pretreatment and posttreatment with heparin were effective in inhibiting human pbl-mediated trans-infection of either niv or hev (fig. b ). this inhibition reflects the known capacity of heparin to bind multiple cell surface proteins due to its high negative charge ( ) . furthermore, in a pretreatment regimen, heparin also . trans-infection of vero cells was than determined as described for fig. . results are expressed as a percentage of inhibition compared to results in untreated cells, Ϯ the sd. *, p Ͻ . ; **, p Ͻ . ; ***, p Ͻ . (mann-whitney u test). (c) analysis of niv binding to cho-k and hs-deficient cho-pgsa- cells, pretreated or not with heparin ( . mg/ml). cells were put into contact with niv for h, and the number of viral rna (n gene) copies was determined by rt-qpcr. (d) spr analysis of niv pseudoparticle binding to an hp-activated surface in the absence or presence of soluble heparin at g/ml or g/ml. (e) spr analysis of the binding of niv pseudoparticles to hp-activated sensor chips, after preincubation with either pbs or g/ml of soluble heparin, cs-a, cs-c, or ds. (f) spr analysis of the binding of niv pseudoparticles, expressing either niv-g or niv-f protein, to hp-activated sensor chips. the binding response, in ru, was recorded as a function of time after removal of the background provided by nonpseudotyped particles. results are presented as the averages of separate experiments (Ϯ standard errors of the means) and reflect statistically significant binding of niv-g to hp. **, p Ͻ . (one-sample t test). prevented the binding of niv to cho-k cells by %, as measured by cell-associated viral rna, while the residual binding to the hs-defective pgsa- cho cell line remained unaffected (fig. c ). these results indicated the ability of heparin to elute viral particles from cell surface hs by competition. accordingly, soluble heparin was able to compete and inhibit niv-pseudotyped binding to immobilized hp or hs by spr ( fig. d and data not shown) with % and~ % reductions observed for g/ml and g/ml heparin, respectively. notably, other gags found on cell surface proteoglycans, such as chondroitin sulfate a (cs-a), cs-c, and ds, were devoid of inhibition properties (fig. e) , revealing the specificity of the inhibition of niv binding to hs by heparin. finally, to determine which of two niv membrane glycoproteins, f or g, was responsible for binding to heparin, we analyzed hp binding of pseudotyped niv expressing either niv-f or -g by spr (fig. f) . significantly more binding was obtained with niv-g than with niv-f (fig. f) , and results were further confirmed using hela cells transfected with either niv-g or -f (data not shown). we concluded from these results that niv trans-infection requires binding to cell surface hs via niv-g, a process that can be inhibited by competition with heparin. hs facilitates henipavirus infection in cis. as gags may play multiple roles in viral infection ( , ) , we next investigated whether the hs could be directly involved in henipavirus infection in cis, in addition to its role in trans-infection. permissive vero cells were treated with heparinase , to remove cell surface hs, prior to the infection with niv. this resulted in a modest but significant reduction in the number of infected cells at days postinfection (fig. a) . furthermore, the inhibition of hs sulfation in vero cells after treatment with sodium chlorate significantly reduced infection with both niv (fig. b) and hev (fig. c ), the latter being much more sensitive to sodium chlorate pretreatment than the former. the obtained results therefore suggest that hs sulfation status is determinant for virus attachment to cells and subsequent infectivity. we then tested whether soluble gags could interfere with henipavirus infection in cis. heparin pretreatment of vero cells reproducibly reduced the number of cells infected by either niv or hev (fig. a ). furthermore, pretreatment of either the cells or the virus was efficient, even with very low concentrations ( g/ml) (fig. b ). to distinguish the contribution of heparin-mediated inhibition of niv-g binding to cell surface hs from binding to an efn-b / entry receptor, hs-negative cho-pgsa- cells stably expressing either efn-b or -b ( ) were pretreated with . g/ml of heparin and infected with niv (fig. c ). heparin treatment modestly, but significantly, reduced the percentage of infected cells from both cell lines, indicating that this molecule may also directly inhibit or delay the binding of niv to its entry receptors efn-b and -b . this effect may be the consequence of direct heparin binding to efn-b and/or -b , as recently suggested ( ) . indeed, spr analysis suggested that both cho-pgsa- -efn-b and -b cells bound to heparin, in contrast to nontransfected cho-pgsa- cells (fig. d ), and proportionally to the level of expressed efn-b and -b transcripts (fig. e) . accordingly, soluble recombinant efn-b bound to heparin in the spr assay as well (fig. f) , fitting a : langmuir binding model and yielding a k d of Ϯ nm (mean Ϯ standard deviation [sd]) for the interaction. additional spr analysis showed interactions of efn-b with both hs and heparin, but not with cs (data not shown). thus, by its ability to compete with hs for binding to niv-g and to interfere with efn-b /b receptors, heparin can restrict both transand cis-infection by henipavirus. heparin treatment restricts nipah virus infection in animals. well known for its anticoagulant properties, heparin binds and activates anti-thrombin iii (at-iii) through a specific pentasaccharide sequence ( , ) . to test the antiviral effect of heparin during niv infection in vivo and to avoid potential hemorrhagic complications, we produced heparin lacking anticoagulant activity by using periodate oxidation (po-heparin), which alters the integrity of the at-iii-binding pentasaccharide motif ( ) . since po-heparin inhibited lymphocyte-mediated niv transinfection in vitro similarly to heparin (fig. a) , we tested its antiviral properties in the golden hamster model of niv infection, which closely reproduces the niv pathogenesis seen in humans ( ) . while all nontreated animals succumbed to infection in less than days, survival in the po-heparin-treated group increased moderately (p ϭ . ) (fig. b ), thus suggesting a biological relevance for niv-hs interaction and revealing potential antiviral properties of heparin-like molecules in vivo. the rapid dissemination of nipah virus and hendra virus in an infected host may play a critical role in the high pathogenicity of henipaviruses. our previous work demonstrated that human peripheral blood lymphocytes are not permissive to niv infection but that they could capture the virus and transmit it to susceptible target cells in trans ( ) . in contrast to human lymphocytes, specific subsets of porcine lymphocytes could be infected with niv and thus participate in the transmission of the virus in the swine host, also in cis ( ) . low levels of viral replication were detected in human dendritic cells, suggesting that this cell population could contribute to transmission of niv both in cis and in trans ( ) . recently, a cd -dependent trans-infection pathway in dendritic cells was demonstrated for henipaviruses ( ) ; however, the molecular mechanism of niv trans-infection mediated by lymphocytes, which do not express cd , has remained obscure. this report reveals hs to be a cell surface attachment receptor for both niv and hev that is implicated in the trans-infection properties of human leukocytes as well as in henipavirus infection in cis. hs has been shown to bind many different viruses by interacting with either one or more viral glycoproteins. interactions between hs and the hiv- envelope glycoprotein gp ( , ) and with vaccinia virus a l protein ( ) have been demonstrated. furthermore, hs can bind measles virus hemagglutinin (h) ( ) and the fusion (f) proteins of respiratory syncytial virus (rsv) ( ) and human metapneumovirus ( ) . in the cases of bovine rsv and canine distemper virus, both h and f seem to interact with hs ( , ) . our results suggest that niv-g interacts with hs. although vsv-g protein has been shown to interact with hs ( ), our spr analysis indicated that binding of niv-g to hs had a higher affinity. further studies using recombinant soluble viral g protein should give better insights into the biochemical characteristics of this interaction and identify binding sites on both ligands. hs has been previously suggested to capture, protect, and transmit hiv- ( ) and human t-cell lymphotropic virus type (htlv- ) ( ) in trans. similarly to what was shown for hiv- ( ), the other sulfated gags, such as cs or ds, were inactive, implying that structural features beyond charge density on the polysaccharide backbone could be important for the activity. the implication is that different hs-binding sites may be involved, as shown recently for papillomavirus, for which sequential engagement of three different hs-binding sites leads to virus attachment, interaction with the receptor, and entry into the cell ( ) . likewise, hs stabilizes varicella-zoster virus, making it more accessible for binding to its entry receptor ( ) . thus, similar to its role in other viral infections, hs could improve the contact of henipavirus with host cells so as to help the virus reach the cellular receptors efn-b and -b (summarized schematically in fig. ). we observed that heparin inhibits henipavirus trans-infection in vitro. owing to its capacity to bind multiple cell surface proteins via negatively charged sulfated groups ( ) , heparin could prevent henipavirus attachment to cell surface hs. as it is more sulfated than hs, it could also bind viruses with higher affinity than hs and thus act as an efficient competitive inhibitor for virus binding. furthermore, heparin significantly reduces direct virus infection in cis, in addition to affecting infection in trans. this effect may also result from the interaction between heparin and henipavirus receptors, as evidenced by spr for efn-b and in agreement with a recent report ( ) . these results allowed us to propose a model for the role of hs in henipavirus binding and entry and possible interference of heparin with viral infection (fig. ). in addition to the inhibition of henipavirus infection by binding to either viral g glycoprotein or viral entry receptors, heparin may significantly limit virus spread within the organism by blocking binding to the attachment receptor hs and consecutive trans-infection of permissive cells. the mechanism by which a heparin molecule devoid of anticoagulation properties ( ) restrains virus infectivity in vivo most likely depends on the combination of its different biological activities. in addition to affecting henipavirus infection in cis and in trans, heparin could constrain inflammation and consequent tissue damage by binding various inflammatory molecules. indeed, both heparin and hs bind to important immunoregulatory molecules, such as the chemokine cxcl ( ), which is strongly upregulated during niv infection ( ) . moreover, as heparin is a heavily sulfated electronegative molecule, it might enhance the antiadhesive properties of the endothelium against leukocytes ( ) . this effect may rely on the ability of heparin to bind directly to several adhesion molecules that are expressed during inflammation, including l-selectin ( ), cd b/mac ( ) , and p-selectin ( ) . finally, heparin-like derivatives can stabilize the endothelium ( ) and help in blocking the passage of both humoral and cellular immune factors, as well as viruses, through the blood-brain barrier ( , ) . this process may also contribute to the antiviral effect observed during in vivo experiments, together providing a "proof of concept" for further development of this antiviral approach. the heparin-mediated inhibition of henipavirus infection both in vitro and in vivo highlights the antiviral potential of this gag, which is well tolerated and has already been used in the clinical environment as an anticoagulant for more than years. indeed, heparin treatment reduces niv infection in a hamster animal model, thus opening interesting therapeutic perspectives to complement treatment of this highly lethal infection. additionally, the acute nature of henipavirus infection makes it more prone to the regulatory action of heparin, compared to some chronic infections, including hiv or htlv, where heparin showed in vitro antiviral activity ( , ) . the hs mimetic pi- has already been shown to have significant beneficial effect in vivo in the outcome of dengue virus and encephalitic flavivirus infections ( ) . the use of derivatives that mimic the heparin/hs structure ( ) , synthetic antilipopolysaccharide peptides that bind hs moieties on cell surfaces ( ) , or polyanionic compounds with longer halflives in vivo ( ) , devoid of anticoagulant activity and with potentially higher affinity to henipavirus g-protein, may further improve therapeutic effects. altogether, this study demonstrates a previously unrecognized hs-henipavirus interaction involved in both niv and hev infection and dissemination within the host. it may allow henipaviruses to bind to different circulating cells, to use them for transport for efficient spreading within the host organism, and for target cells, to allow accumulation on their surface, enhancing the efficacy of infection in cis. these results reveal heparan sulfate as a potential therapeutic target for the development of novel approaches against these highly lethal infections. in this context, heparin or its derivatives may be used in a metaphylaxis approach of virus-exposed and potentially infected animals, before the appearance of clinical symptoms, during regular hendra equine epizootics in australia ( ). ethics statement. venous blood from anonymous healthy human volunteers was obtained from the blood transfusion centre (etablissement francais du sang, lyon, france) in accordance with its guidelines, with informed written consent from each volunteer. all animals were handled in strict accordance with good animal practices as defined by the french national charter on the ethics of animals experiments, and all efforts were made to minimize suffering. animal work was approved by the regional ethical committee ceccapp (p _ _ ), and experiments were performed in the inserm jean mérieux bsl laboratory in lyon, france (french animal regulation commitee number a ). cell culture. the cho cell line k and cho pgsa- cells stably transfected with human ephrin-b and -b , (pgsa-efnb and pgsa-efnb ; all generously provided by b. lee [ucla, united states] [ ] ) were maintained in f- medium (invitrogen) supplemented with % fetal calf serum (fcs), u/ml penicillin, . mg streptomycin, mm hepes, and mm l-glutamine at °c in % co . vero e , hs-deficient cho-k cells cho pgsa- , cho pgsb- , and cho pgsd- cells ( ) , hela cells, and stable transfected hela cells with phcmv-nipah-g-neo and phcmv-nipah-f-neo, expressing niv-g and -f proteins, respectively (hela-g and hela-f), were maintained in dulbecco's modified eagle's medium (dmem; invitrogen) supplemented as described above. human peripheral blood was obtained from different healthy donors from the blood transfusion centre (lyon, france). peripheral blood mononuclear cells were isolated by density ficoll/hypaque gradient centrifugation and then centrifuged through a % percoll gradient (pharmacia fine chemicals) for min at ϫ g. pbls were recovered from the high-density fraction. for spr analysis, cells were detached by versene treatment and washed twice with hbs-p buffer ( mm hepes, mm nacl, . % surfactant p , ph . ). virus infection and titration. nipah virus (isolate ummc ; gen-bank accession number ay ) ( ), recombinant niv expressing enhanced green fluorescent protein ( ) , and hendra virus (australia/ horse/ ) obtained from porton down laboratory, united kingdom, were prepared on vero-e cells as described previously ( ) , and infection virus was used in the inserm jean mérieux bsl laboratory in lyon, france. all cells were infected at a multiplicity of infection (moi) of , for h at °c, washed twice, and observed by inverted and/or fluorescence microscopy daily or harvested for rna isolation or for use in transinfection assays. at the indicated times postinfection, l of cell culture supernatant was collected and frozen prior to viral titration. viral titration was performed by plaque assay as detailed elsewhere ( ) . the viral infection level in cocultures of leukocytes and cho cells with vero cells was determined using a previously described infectious center assay in -well plates ( ) , after min of formaldehyde fixation and crystal violet staining. alternatively, in heparin-mediated inhibition assays, titrations were directly performed by plaque assay, using vero cells or cho-pgsa -efn-b or -b cell monolayers. pseudotyped viral particles, containing either niv-g and/or -f or control vsv-g were produced using friend's murine leukemia virus (mlv) particles and t cells, as described previously ( ) . treatment of cells with sodium chlorate. vero cell monolayers were grown to confluence and maintained in dmem supplemented with % fcs and sodium chlorate (naclo ; , and mm) for h to inhibit hs sulfation in cells, as rapid turnover of proteoglycans in the cells ( ) required prolonged incubation with naclo . cells were then detached by using trypsin- . % edta, distributed into new -well tissue culture plates, and grown to confluence in the presence of the same range of concentrations of naclo and infected with either niv or hev ( or pfu/well). following h of incubation in cmc/dmem supplemented with % fcs and naclo , virus was titrated by using crystal violet staining. trans-infection assay. cells were put in contact with virus (moi, ) for h at °c, % co . after two washes with phosphate-buffered saline (pbs), cells were cultured at °c for h and then collected and washed, and -fold serial dilutions were added to cell monolayers of vero cells for determination of cell-associated infectious niv in a infectious center assay, as described previously ( ) . in some experiments, cells were incubated for h at °c with u/ml of heparinase (sigma) and washed times before contact with niv. treatment with heparin (porcine intestinal mucosa; sigma) of either cells or virus and pseudotyped viral particles was performed for min at °c. in heparin pretreatment experiments, after incubation with heparin, cells were thoroughly washed before adding the virus. in heparin posttreatment experiments, niv-resistant cells were initially put in contact with either niv or hev for h and incubated with heparin afterward, for min at °c, washed, and analyzed in a transinfection assay as described above. spr-based binding assays. spr experiments were performed on a biacore x apparatus (for analysis of cells and pseudotyped viral particles) or a biacore apparatus (for pseudotyped viral particles and recombinant ephrin-b ), using cm (for pseudotyped virus and recombinant efn-b ) and cm (for cells) sensor chips and hbs-p buffer. hs (celsus, cincinnati, oh, united states), hp (sigma), and ds (sigma) were biotinylated and immobilized on biacore sensor chips, as described before ( ) . briefly, two flow cells were activated with a mix of . m n-ethyl-n=-(diethylaminopropyl)-carbodiimide (edc) and . m n-hydroxysuccinimide (nhs). then, streptavidin ( g/ml in mm acetate buffer [ph . ]) was injected over the activated flow cells, to obtain an immobilization level of~ , response units (ru). one of these flow cells served as a negative control, while biotinylated hp, hs, and ds were injected on the other flow cells, to obtain suitable immobilization levels ( to ru for recombinant human ephrin-b fc and pseudotyped viral particles analysis; ru for cells). interaction assays were performed at a flow rate of l/min and involved -to -min injections of sample over the hp and negative-control surfaces, followed by a -min washing step with hbs-p buffer to allow dissociation of the complexes formed. at the end of each cycle, gag surfaces were regenerated by sequential injections of . % sds ( min) and m nacl ( . min). typical sample concentrations used were g/ml for recombinant human ephrin-b fc (r&d systems), ϫ to . ϫ particles/ml for pseudotyped viral particles and . ϫ to . ϫ cells/ml for cho-pgsa- and hela cell transfectants. the sensorgrams shown correspond to on-line subtraction of the negative-control signal from the gag surface signal. competition assays were performed by preincubating niv pseudoparticles with gags ( to g/ml, final concentration) for min prior to injection. rna isolation and rt-qpcr. rna was isolated from cells and plasma by using an rneasy minikit (qiagen) in rlt buffer, according to the manufacturer's instructions. reverse transcription (rt) was performed on . g of total rna by using oligo(dt) and random hexamer oligonucleotide primers (iscript cdna synthesis kit; bio-rad) and run in a biometra t-gradient pcr device, and cdnas were diluted / . quantitative pcr (qpcr) was performed with cdna samples by using platinum sybr green qpcr supermix-udg with a rox kit (invitrogen). qpcr was run on the stepone plus pcr system (applied biosystems) as follows: °c for min and cycles of °c for s and °c for min, followed by a melting curve of up to °c at . °c intervals. all samples were run in duplicate, and the results were analyzed using stepone software v . . the glyceraldehyde -phosphate dehydrogenase (gapdh) gene was used as a housekeeping gene to normalize the samples. gapdh and standard references for the corresponding genes were included in each run to check for rna integrity, rna load, and inter-pcr variation. after normalization, the results were expressed as the number of mrna copies of the gene of interest per microgram of analyzed rna. all calculations were done using the ⌬⌬ct model ( ) , and experiments were performed according to the miqe guideline ( ) . primer used included the following: niv n forward, ggcaggattcttcgcaaccatc, and reverse, ggctcttg ggccaatttctctg; murine gapdh forward, gcatggccttccgt gtcc, and reverse, tgtcatcatacttggcaggtttct; efn-b forward, caagttctgctggatcaa, and reverse, gatgttgttccccg aatg, efn-b forward, atggaaagagaccgaggg, and reverse, ga ggttgcattgctggtg. production of heparin devoid of anticoagulant activity (poheparin). to eliminate its anticoagulant properties, heparin was treated with periodate, as previously described ( ) . briefly, mg of heparin from porcine intestine ( . usp units/mg; sigma) was dissolved in l of . m naio in . m sodium acetate buffer (ph ) and stirred at °c for days. the unreacted naio was then neutralized by addition of glycerol ( l), dialyzed against h o, and lyophilized. the sample was resuspended in l of . m nabh in . m ammonium bicarbonate and incubated for a further h at °c. after acidification with glacial acetic acid (to eliminate any remaining nabh ), the reaction mixture was neutralized by addition of naoh ( m), dialyzed against water, and lyophilized. infection of hamsters. eight-week-old golden hamsters (mesocricetus auratus; janvier, france) were anesthetized and infected intraperitoneally with . ml containing niv ( % lethal doses [ld s] preincubated with heparin [ . mg/ml; min at °c]). groups of animals were treated daily subcutaneously with the po-heparin ( mg/kg of body weight) for days, starting from the day of infection. animals were followed daily for weeks. statistical analysis. data are expressed as means Ϯ sd or as the percentage of survival. statistical analyses were performed using mann-whitney u test, one-sample t test, and mantel-cox test within graph-pad's prism software. a morbillivirus that caused fatal disease in horses and humans nipah virus: a recently emergent deadly paramyxovirus hendra and nipah viruses: why are they so deadly? nipah virus-a potential agent of bioterrorism? ephrin-b ligand is a functional receptor for hendra virus and nipah virus ephrinb is the entry receptor for nipah virus, an emergent deadly paramyxovirus two key residues in ephrinb are critical for its use as an alternative receptor for nipah virus nipah virus uses leukocytes for efficient dissemination within a host syndecan captures, protects, and transmits hiv to t lymphocytes human t-cell leukemia virus type envelope glycoprotein gp interacts with cell surface heparan sulfate proteoglycans heparan sulphate proteoglycans and viral vectors: ally or foe? functions of cell surface heparan sulfate proteoglycans glycosaminoglycans and the regulation of blood coagulation novel drug development opportunities for heparin microbial adherence to and invasion through proteoglycans heparan sulfate: anchor for viral intruders ephrin-b binds to a sulfated cell-surface receptor the separation of active and inactive forms of heparin biosynthesis of heparin. o-sulfation of the antithrombin-binding region a golden hamster model for human acute nipah virus infection nipah virus infects specific subsets of porcine peripheral blood mononuclear cells virus particle release from glycosphingolipidenriched microdomains is essential for dendritic cell-mediated capture and transfer of hiv- and henipavirus human immunodeficiency virus type attachment to hela cd cells is cd independent and gp dependent and requires cell surface heparans heparan sulfate targets the hiv- envelope glycoprotein gp coreceptor binding site a l protein mediates vaccinia virus interaction with cell surface heparan sulfate heparin-like glycosaminoglycans prevent the infection of measles virus in slam-negative cell lines the fusion glycoprotein of human respiratory syncytial virus facilitates virus attachment and infectivity via an interaction with cellular heparan sulfate human metapneumovirus (hmpv) binding and infection are mediated by interactions between the hmpv fusion protein and heparan sulfate host range and receptor utilization of canine distemper virus analyzed by recombinant viruses: involvement of heparin-like molecule in cdv infection recombinant bovine respiratory syncytial virus with deletions of the g or sh genes: g and f proteins bind heparin cell surface heparan sulfate is a receptor for attachment of envelope protein-free retrovirus-like particles and vsv-g pseudotyped mlv-derived retrovirus vectors to target cells multiple heparan sulfate binding site engagements are required for the infectious entry of human papillomavirus type infection of cells by varicella zoster virus: inhibition of viral entry by mannose -phosphate and heparin heparin displaces interferon-gammainducible chemokines (ip- , i-tac, and mig) sequestered in the vasculature and inhibits the transendothelial migration and arterial recruitment of t cells lethal nipah virus infection induces rapid overexpression of cxcl heparan sulfate proteoglycans modulate monocyte migration across cerebral endothelium differential interactions of heparin and heparan sulfate glycosaminoglycans with the selectins. implications for the use of unfractionated and low molecular weight heparins as therapeutic agents heparin attenuates tnf-alpha induced inflammatory response through a cd b dependent mechanism gmp- binding to neutrophils is inhibited by sulfated glycans polyanionic drugs and viral oncogenesis: a novel approach to control infection, tumor-associated inflammation and angiogenesis contribution of proteoglycans to human immunodeficiency virus type brain invasion antiviral effect of the heparan sulfate mimetic, pi- , against dengue and encephalitic flaviviruses highly sulfated k escherichia coli polysaccharide derivatives inhibit respiratory syncytial virus infectivity in cell lines and human tracheal-bronchial histocultures a new class of synthetic peptide inhibitors blocks attachment and entry of human pathogenic viruses establishment of a nipah virus rescue system acute hendra virus infection: analysis of the pathogenesis and passive antibody protection in the hamster model specific detection of nipah virus using realtime rt-pcr (taqman) transgenic mice expressing human measles virus (mv) receptor cd provide cells exhibiting different permissivities to mv infections a kinetics and modeling study of rantes( - ) binding to heparin reveals a mechanism of cooperative oligomerization a new mathematical model for relative quantification in real-time rt-pcr the miqe guidelines: minimum information for publication of quantitative real-time pcr experiments periodatetreated, non-anticoagulant heparin-carrying polystyrene (nac-hcps) affects angiogenesis and inhibits subcutaneous induced tumour growth and metastasis to the lung this work was supported by inserm, anr- -mien- - , and anr-astrid- . c.m. was supported by cluster of infectiology rhône-alpes, and k.d. was supported by a dga/inserm research fellowship.we thank i. bally and n. thielens, from the ibs platform of the partnership for structural biology and the institut de biologie structurale in grenoble for assistance and access to the biacore facility and members of the group "immunobiology of viral infections" for their help during the realization of the study. in addition, we thank the labex ecofect (anr- -labx- ) of université de lyon, france, as well as i. grosjean from the cellulonet facility (sfr bioscience gerland-lyon-sud, ums /us ) for their help. we are also grateful to b. lee (ucla, usa) for providing us efn-b -and -b -expressing cho cells and to p. lawrence (ciri lyon) for english editing. we also thank d. cornec, f. jacquot, a. duthey, a. valve, l. barrot, and other biosafety team members from inserm bsl "jean mérieux" for their assistance. key: cord- -pkovyjo authors: li, yize; dong, beihua; wei, zuzhang; silverman, robert h.; weiss, susan r. title: activation of rnase l in egyptian rousette bat-derived roni/ cells is dependent primarily on oas and independent of mavs signaling date: - - journal: mbio doi: . /mbio. - sha: doc_id: cord_uid: pkovyjo bats are reservoirs for many rna viruses that are highly pathogenic in humans yet relatively apathogenic in the natural host. it has been suggested that differences in innate immunity are responsible. the antiviral oas-rnase l pathway is well characterized in humans, but there is little known about its activation and antiviral activity in bats. during infection, oass, upon sensing double-stranded rna (dsrna), produce ′- ′ oligoadenylates ( - a), leading to activation of rnase l which degrades viral and host rna, limiting viral replication. humans encode three active oass (oas to - ). analysis of the egyptian rousette bat genome combined with mrna sequencing from bat roni/ cells revealed three homologous oas proteins. interferon alpha treatment or viral infection induced all three oas mrnas, but rnase l mrna is constitutively expressed. sindbis virus (sinv) or vaccinia virus (vacvΔe l) infection of wild-type (wt) or oas -ko (knockout), oas -ko, or mavs-ko roni/ cells, but not rnase l-ko or oas -ko cells, induces robust rnase l activation. sinv replication is - to -fold higher in the absence of rnase l or oas than in wt cells. however, mavs-ko had no detectable effect on rna degradation or replication. thus, in roni/ bat cells, as in human cells, activation of rnase l during infection and its antiviral activity are dependent primarily on oas while mavs signaling is not required for the activation of rnase l and restriction of infection. our findings indicate that oas proteins serve as pattern recognition receptors (prrs) to recognize viral dsrna and that this pathway is a primary response to virus rather than a secondary effect of interferon signaling. b ats are reservoirs for many rna viruses that are highly pathogenic in humans yet attenuated in their natural host. these include filoviruses (ebola virus and marburg virus) ( ) ( ) ( ) ( ) , coronaviruses (severe acute respiratory syndrome coronavirus [sars-cov] and middle east respiratory syndrome coronavirus [mers-cov]) ( , ) , alphaviruses (venezuelan equine encephalitis virus [veev]) ( ) , and nipah/hendra viruses ( ) . several studies have suggested that bats can tolerate infection due to an enhanced innate immune response that enables early control of infection and prevents systemic dissemination ( ) ( ) ( ) ( ) ( ) ( ) . for example, three alpha interferon (ifn-␣) genes are constitutively expressed in black flying fox, pteropus alecto ( ) . other studies report that bats have a dampened host response, speculated to promote virus-host coexistence ( , ) . for example, the cgas-sting pathway is dampened in some bats species due to a mutation in sting ( ) and the inflammasome dna sensor aim is missing from almost all the known bat species ( ) . thus, there is a need for further investigation into the innate immune response in bats and how it impacts viral infection. double-stranded rna (dsrna)-induced innate immune responses play a critical role in limiting viral infection ( ) . one dsrna-induced and potent antiviral pathway is the oas-rnase l system, which has been well characterized in human cells and murine cells ( ) . the human oas family contains four members, three enzymatic active proteins (oas , oas , and oas ) and one oas-like (oasl) protein, lacking enzymatic activity ( ) . all three enzymatically active oass contain a core unit with dsrna binding and catalytic functions ( , ) . oas and oas duplicate one or two nonenzymatic units which are believed to enhance the binding affinity to dsrna. mice express homologous oas proteins that produce =- = oligoadenylates ( - a), including oas a/g, oas , and oas , as well as oasl and several catalytically inactive oas isoforms, oasl , and additional oas proteins ( , ) . after sensing dsrna, the catalytic domain of oass undergoes a conformational rearrangement to form the catalytic cavity and, from atp, synthesizes - a. - a binds to monomeric rnase l, leading to dimerization and activation to cleave viral and cellular single-stranded rnas, thereby blocking viral replication as well as protein synthesis ( ) . while all three oass (oas to - ) produce - a upon binding dsrna in vitro or when overexpressed ( ) , we showed previously, using a series of cells with oas gene knockouts (kos), that only oas was required for detectable activation of rnase l during infection of three human cell lines with diverse viruses ( ) . there is scant information in the scientific literature about activation or inhibition of the oas-rnase l pathway in bats and its contributions to bat innate immunity, although it was shown that oas-rnase l can be activated by poly(ri)·poly(rc) (pic) in p. alecto bat cells ( ) . we screened a group of bat cells (discussed further below) for activation of rnase l during sindbis virus (sinv) infection and chose to carry out further studies in egyptian rousette (er) bat (egyptian fruit bat)-derived roni/ cells. through bioinformatic analysis of the annotated genomic sequence of egyptian rousette bats from genbank ( ), we identified three oas genes (boas , boas , and boas ) and two oasl genes (boasl and boasl ). (we designate bat genes or proteins as boas to differentiate them from human [h] or mouse [m] genes or proteins.) we utilized crispr-cas gene-editing technology to generate boas-ko egyptian rousette-derived roni/ cells ( ) . we found that, as in human cells, the activation of rnase l is dependent on boas expression during infection with sinv. in addition, ko of boas or rnasel leads to greater than -fold more sinv replication, while ko of either boas or boas promotes more modest increases in replication. as in human cells, activation of rnase l by sinv is independent of mavs expression in bat roni/ cells, suggesting that rnase l may be activated either before or without virus-induced ifn. similar results were obtained with vaccinia virus, vacvΔe l, in bat roni/ cells. finally, our data indicate that the oas-rnase l pathway has a greater antiviral effect than mavs-dependent ifn signaling in roni cells as well as human cells. the bat genome contains three oas genes with interferon-stimulated response elements (isres) in the promoter region, two oasl genes, and one rnase l gene. using the genomic sequences of egyptian rousette bat from genbank, we analyzed the genes and protein sequences of bat oass, oasls, and rnase l ( ) . the genes were annotated as oas (genbank id ), oas -like (genbank id ), oasl (genbank id ), and oasl (genbank id ). no oas was noted. the egyptian rousette (er) boas gene encodes a protein that shares % and % amino acid sequence similarity to human oas and mouse oas , respectively ( table ) . the annotated er oas -like gene encodes a protein of , amino acids. amino acids to share % similarity with human oas ( , amino acids), and amino acids to share % similarity with human oas ( amino acids). these results suggested that the annotated er oas -like gene encoded an oas -oas fusion protein. since in most known species, oas and oas are two separate genes, we could not exclude the possibility that er oas and oas genes were falsely annotated as one gene. to clarify this question, we attempted to clone the mrna encoded by the boas and boas -like genes. we cloned the boas open reading frame (orf) successfully, but we could not clone the mrna predicted to encode a boas -boas fusion protein. instead, we cloned two cdnas which encode boas and boas open reading frames separately. these results suggested that er bats and humans share similar oas gene structures (fig. ). nd nd nd boas yes nd / nd nd nd boas yes nd nd / nd nd boasl no nd nd nd / boasl yes / nd nd / a protein catalytic activity was predicted by the conservation in the active site of the p-loop and aspartic acid triad. b amino acid consensus similarity. genbank protein accession numbers are shown in parentheses as follows: bat proteins, boas (xp_ ), boas (qct . ), boas (qct . ), boasl (xp_ ), and boasl (xp_ ); human proteins, hoas (np_ . ), hoas (np_ . ), hoas (np_ . ), and hoasl (q ); mouse proteins, moas a (p ), moas (e q a ), moas (q vi ), moasl (q vi ), and moasl (q z f ). vector nti was used to do the sequence alignment. c nd, not determined. human and mouse oas genes are induced by interferon (ifn) (i.e., they are interferon-stimulated genes [isgs]) and thus contain interferon-stimulated response elements (isres) in the promoter regions, required for interferon-inducible transcription. to determine whether er oas genes are isgs, we analyzed the genomic sequences for isres (a/gngaaanngaaact or agtttcnntttcnc/t) ( ) . we found that the er oas gene promoter region contains one isre with high sequence identity with that of human oas (fig. ) . upstream of the boas coding region, we found two adjacent isres in the opposite polarity, the first with homology to the one isre found in the human oas gene. the isre of er boas is identical to the first one of two found in the human oas gene. we did not find an isre in the promoter region of the er rnase l gene. boas , boas , and boas mrnas are induced by ifn-␣ or infection with sinv or sev in roni/ cells. roni/ cells were treated with human universal ifn-␣, the relative mrna levels were determined by quantitative reverse transcriptase pcr (qrt-pcr), and the fold increase in expression over mock-treated cells was calculated. induction of bat ifit (isg ) mrna was used as a positive control ( ) and was increased by -fold upon ifn-␣ treatment ( fig. a ). all three boas mrnas were induced by ifn-␣; the boas mrna level increased by -fold, while the mrna level of boas and boas increased by -and -fold, respectively ( fig. a) , consistent with the isres in their promoters. the mrna level of rnase l did not change upon ifn treatment in roni/ cells, indicating that, similarly to the human rnasel gene, bat rnasel is not an isg ( fig. a) , consistent with the lack of an isre in its promoter region (data not shown). to investigate induction of boas genes during viral infection, roni/ cells were infected with sinv or sendai virus (sev). while the relative mrna level of boas increased by -to -fold during sinv or sev infection, boas mrna was only weakly ( -to -fold) induced and boas mrna was induced somewhat more ( -or -fold) during sinv or sev infection, respectively ( fig. b and c), similar to induction by ifn-␣ ( fig. a to c) . amino acid sequence alignment indicates that the egyptian rousette boas , boas , boas , and boasl proteins are oligoadenylate synthetases with potential catalytic activity. the human and mouse oas genes and proteins have been extensively studied ( , ) . to investigate the bat oas gene family, we aligned the predicted amino acid sequences of bat, human, and mouse oas and oasl proteins. while boas protein sequences are highly homologous to human and mouse proteins, in each case boas , boas , and boas share more homology with human than with mouse proteins (table ) . we aligned the catalytic domain of each boas protein (full-length boas , domain ii of boas [boas . ], and domain iii of boas [boas . ]) with the corresponding protein or domains of human oas , oas , and oas (fig. ). based on sequence homology and previous studies of the structure of human ( ) and porcine ( ) oas , bat oas catalytic domains are composed of three parts, the n-terminal lobe (blue line in fig. ), the linker (red dotted line), and the c-terminal lobe (black line). three aspartic acid residues (asp , asp , and asp of hoas ) indicated with diamonds are essential for enzymatic function of oass and conserved among all the bat and human oass ( ) . the p-loop which contributes to the formation of the catalytic cavity is also conserved. the arg or lys residues (indicated by stars) participate in dsrna binding and are conserved among all bat or human oass. these results predict that boas , boas . , and boas . have the potential to recognize dsrna and synthesize - a. besides three oas genes, the egyptian rousette genome has two oas-like genes (oasl and oasl ). the boasl protein shares high sequence similarity with hoasl ( %) and moasl ( %), suggesting that, like these human and mouse proteins, it does not have oligoadenylate synthetase activity (table ; see also fig. s in the supplemental material). however, boasl is similar to moasl ( %) ( table ) and when aligned with human oas has a conserved catalytic domain, suggesting that it may have oligoadenylate synthetase activity (fig. s ) . egyptian rousette (er) rnase l shares high amino acid similarity to human rnase l with conserved - a binding sites and endoribonuclease catalytic sites. by sequence alignment, we found that brnase l shares % amino acid similarity with the human protein (fig. ). similar to human or porcine rnase l, brnase l contains three predicted domains, an n-terminal ankyrin-repeat domain (blue line in fig. ) , a pseudo-protein kinase domain (red dotted line), and an rnase domain (black line) ( ) . the amino acids which are responsible for - a binding (stars) and catalysis residues (diamonds) are conserved among bat and human rnase l sequences. protein expression levels of oas and oas in bat cells are upregulated by ifn treatment. we generated rabbit polyclonal antibodies against domain ii of boas (anti-boas ) or domains ii and iii of boas (anti-boas ). (attempts to raise antisera against boas were unsuccessful.) we used these antisera to determine both basal and induced oas protein expression levels in bat cells derived from several species of bats. activation of bat rnase l depends on oas but not mavs ® interestingly, antisera against either boas or boas detected both oas proteins. basal expression of oas was detected in roni/ cells and eptesicus fuscus skin fibroblasts, and the expression was upregulated upon ifn-␣ treatment in both cell types (fig. a ). while no to low basal oas expression was detected in myotis lucifugus skin fibroblasts, myotis lucifugus embryonic fibroblasts, and myotis velifer embryonic fibroblasts, the oas protein expression level became detectable upon ifn treatment (fig. a ). while basal oas protein expression was observed in eptesicus fuscus skin fibroblasts and weakly in myotis lucifugus skin fibroblasts, expression of boas was detected in all of these bat cells following ifn treatment (fig. a) . no oas -oas fusion protein was detected in these cells, consistent with our cdna cloning data. to assess activation of rnase l in these cells, we infected these bat cell lines with sinv; rnase l was activated in roni/ cells and eptesicus fuscus skin fibroblasts but not in other bat cells (fig. b) , likely because sinv failed to replicate. we next sought to determine whether the activation of rnase l in bat roni/ cells is dependent on expression of one or more oas genes. thus, we deployed the clustered regularly interspaced short palindromic repeat (crispr)-cas geneediting technology to ko expression of boas , boas , boas , and brnase l, each separately, from roni/ cells. the kos of boas and boas were confirmed by western blotting, while the disruptions of the oas and rnase l genes for which we lacked antibodies were confirmed by sequencing of the crispr-induced insertion (boas ) or deletion (rnase l) ( fig. a and b ). upon infection with sinv, degradation of rrna, as assessed by bioanalyzer, was detected in wild-type (wt) and boas -ko and boas -ko cells but not in boas -ko and brnase l-ko cells (fig. c) , indicating that the activation of rnase l during sinv infection in roni/ cells is dependent on boas expression, similar to our previous findings in human cells. we assessed viral replication in this set of ko cells at several time points postinfection. at and h postinfection (hpi), both brnase l-ko and boas -ko cells showed approximately -to -fold higher titers than the parental wt cells (fig. d ). viral titers from boas -ko and boas -ko cells were more modestly elevated than in wt cells at and h postinfection, -to -fold for boas -ko and -to -fold for boas -ko (fig. d ). when the second boas -ko and activation of bat rnase l depends on oas but not mavs ® boas -ko clones were infected with sinv, similar data were obtained for activation of rnase l by bioanalyzer assay (fig. s a ) and for virus replication ( fig. s b and c) . the second boas -ko clone examined displayed lower levels of virus replication than the clone originally used, closer to levels observed in wt cells (fig. ), but neither clone showed increased rnase l activation as assessed by bioanalyzer, compared to wt cells (fig. s ). it is important to note that while boas and boas mrna and protein expression levels are upregulated during sinv infection ( fig. a and fig. e ), this is not sufficient to activate rnase l in the absence of boas expression (fig. c) . to verify that oas -ko cells were competent to activate rnase l, we introduced flag-tagged human oas into boas -ko roni/ cells. (we used human oas rather than bat oas because they are about % homologous and we did not have a full-length boas clone assembled.) expression of ϫflag-hoas in boas -ko roni/ cells was verified by western blotting (fig. a) . wt, boas -ko, and ϫflag-hoas expressing cells were infected with sinv. degradation of rrna, as assessed by bioanalyzer, was detected in wt and ϫflag-hoas cells but, as expected, not in boas -ko cells (fig. b) , confirming that these cells are competent to activate rnase l when they express oas . we assessed viral replication in this set of cells at several time points postinfection. at , , and h postinfection, ϫflag-hoas -ko cells produced the same titers as parental wt cells, approximately -to -fold lower than boas -ko cells (fig. c) , confirming as expected that hoas expressed in boas -ko cells restores the antiviral effects of rnase l activation and thus restricts virus replication to the same extent as in wt cells. to determine to what extent activation of rnase l is dependent on ifn signaling, we investigated the role of mavs in oas-rnase l activation. thus, we used crispr/cas editing to generate bmavs-ko roni/ cells. the ko was validated by sequencing (fig. a ) in the roni/ mavs gene. (fig. b) , consistent with rnase l activation. while sinv replicated to more than -fold-higher titers in brnase l-ko cells than in the parental wt cells (fig. d and fig. c ), replication in bmavs-ko cells did not show significant differences at h postinfection and showed a slightly higher titer ( -fold) at h postinfection (fig. c) . similar data were obtained for activation of rnase l by rrna degradation assay by bioanalyzer (fig. s a) and for virus replication when a second bmavs-ko clone was infected with sinv (fig. s b) . while as expected ko of mavs from roni/ cells reduced boas and boas induction during sinv infection (fig. d) , there was no effect on the activation of rnase l. similar results were observed when we used human a cells. during sinv infection, rrna degradation was observed in mavs-ko a cells (fig. e ). in addition, no significant titer differences were observed at or h postinfection in mavs-ko a cells, while rnase l-ko cells showed -fold-higher titers than wt cells (fig. f) at h postinfection. rna was harvested from cells at h postinfection for rrna analysis. degradation of rrna, as assessed by bioanalyzer, was observed in the infected wt, boas -ko, boas -ko, and bmavs-ko cells but not in infected boas -ko or brnase l-ko cells (fig. a ). in addition, cells were lysed at h postinfection for determination of viral replication by plaque assays. consistent with the rrna degradation data, viral titers were -and -fold higher in the brnase l-ko or boas -ko cells, respectively, than in the boas , boas , bmavs, and wt cells (fig. b) . thus, activation of rnase l during vacvΔe l infection of bat roni/ cells was dependent on rnase l and oas but not oas , oas , or mavs, similar to our findings with sinv. since rna viruses are often lethal in humans but relatively apathogenic in the natural bat host, it is important to understand host-virus interactions and how these may differ among hosts. we have focused on understanding expression and activation of the dsrna-induced antiviral oas-rnase l system, a pathway our group has investigated in detail in human and murine cells, but which had not been characterized in the bat. when we analyzed the annotated egyptian rousette genomic sequences in genbank, we noted three genes, boas , boas , and boas , homologous to human oas genes, each with one or two isres in their promoter regions indicative of isgs. we cloned and sequenced the open reading frames (orfs) from the mrnas encoding boas , boas , and boas . our findings are in contrast to the annotated egyptian rousette genomic sequences, which indicate two boas genes, boas , which is predicted to encode boas , a homologue of human oas , and a boas -like gene which was predicted to encode a long oas -oas fusion protein. interestingly, oas -oas fusion genes and proteins are predicted also by annotated genbank sequences for two additional bat species, eptesicus fuscus and myotis brandtii, and three other nonbat species (donkey, ferret, and marmot). using antiserum to detect oas and oas expression from bat cells, we did not detect a fusion protein for any of the bat species. it is likely that the oas -oas fusion genes and resulting fusion protein predicted arise from annotation mistakes in genbank. in addition to the oas genes, two oasl genes encoding oasl and oasl proteins were detected from genomic sequencing. alignment of the boas and boasl amino acid sequences with human and mouse protein sequences showed that the bat proteins are more similar to human homologues than to mouse proteins (table and fig. ) . the crystal structure of human ( ) and porcine ( ) oas has been well studied, and essential amino acids which correspond to dsrna binding and - a production have been identified. all three boas proteins contain highly conserved dsrna binding amino acid residues as well as catalytic amino acids, suggesting that the bat oass have the ability to recognize dsrna and synthesize - a. bats encode boasl and boasl , with homology to mouse and human oas proteins. boasl shares high sequence similarity with mouse oasl (see fig. s in the supplemental material), suggesting that activation of bat rnase l depends on oas but not mavs ® like the mouse protein, boasl may have catalytic activity. in contrast, humans encode only one enzymatically inactive oasl protein. a recent study suggested that human oasl (enzymatically inactive) and mouse oasl (enzymatically active) suppress the function of cgas-sting pathways ( , ) . this is surprising since hoasl is more similar to mouse oasl than to mouse oasl (fig. s ). nevertheless, it will be interesting to determine if boasl or boasl has a similar function. xie et al. showed that a point mutation of sting from many bats species attenuates the function of sting ( ). thus, it is possible that bats may use two mechanisms to suppress the cgas-sting pathway to make the host become more tolerant of viral infection. analysis of the promoter region of the boas genes revealed isre sequences in all the three genes, consistent with our qpcr data, showing that each boas mrna is induced by ifn treatment and during viral infection ( fig. and ). bat and human oas genes have one similar isre, and oas mrna expression is moderately inducible by ifn or virus infection ( -to -fold) in bats (fig. ) , similar to our findings in human cells ( ) . interestingly, while the boas gene has two adjacent isres and boas mrna expression is induced only -to -fold (fig. ) , the human oas has one isre and yet the expression of oas mrna is highly induced ( , -fold) during infection of human cells ( ) . similarly, while boas has one isre, human oas has two isres, and oas mrna induction was about -to -fold in both bat and human cells with ifn. these findings suggest that the number of isres by itself does not have a major influence on the fold induction of oas gene expression, with the caveat that extent of gene induction will vary with basal expression levels in different cell types. interestingly, we detected basal oas protein expression in roni/ bat cells and basal oas and oas protein expression in eptesicus fuscus skin fibroblasts. it is important to note that we cannot directly compare basal expression levels of oas and oas among cells from the various species of bats because the antisera were raised against roni/ cell proteins and may preferentially react to the homologous proteins. since we were unable to detect boas protein, it is possible, albeit unlikely, that oas is not expressed in roni/ cells. however, it is more likely that the oas and oas antibodies just do not cross-react with oas . this is not surprising as boas shares lower sequence homology ( %) with the catalytic domains of boas and boas than they do with each other. importantly, further evidence that oas protein is expressed is that the oas gene is intact (fig. ) , the mrna is clearly expressed and induced by ifn or infection (fig. ) and contains an orf encoding a protein very similar to human oas with predicted active site intact (fig. ; table ), and oas -ko cells have increased sinv replication (fig. ) . in contrast to bat cells, we did not detect basal oas protein expression in any human cells in our previous studies (fig. a) ( , ) . while this may suggest that basal oas could be important in enhancing bat resistance to viruses, it could also be due to the different cell types examined and differences in the sensitivity of detection of bat and human oas proteins by western blotting. there are clearly many more unanswered questions about the regulation of boas gene expression. infection of a series of roni/ cells engineered using crispr/cas to ko each boas gene and brnase l showed that boas is essential for activation of rnase l during sinv or vacvΔe l infection as assessed by rrna degradation. (please note that oas is induced in boas -ko cells as well as in wt cells.) these data are similar to our previous findings in several human cell lines, with diverse viruses. this was due to a higher affinity of oas for long dsrna compared to the shorter oas and oas proteins ( , ) , which is likely the case for the bat homologues as well. also consistent with our previous findings in human cells, sinv replication is markedly increased ( -to -fold in the boas -ko cells as well as brnase l-ko cells compared to wt cells). interestingly, in boas -ko or boas -ko cells sinv also replicates to a higher level than in wt cells, although not to the extent observed in boas -ko or brnase l-ko cells, and rrna degradation was observed in cells lacking expression of either boas or boas . these data suggest that boas and boas may exert some antiviral activity by activating rnase l in the absence of boas . in human cells, we previously observed increased sinv replication in oas -ko cells at some time points. however, we observed rrna degradation in oas -ko cells, as well as accumulation of - a to the same extent as in wt human cells ( , ) , suggesting that this antiviral activity mediated by oas in human cells may be at least in part independent of rnase l activation. consistent with this observation, previous studies concluded that human oas has an antiviral function which is rnase l independent ( ) . however, during infection of roni/ cells with vacvΔe l, as we previously found in human a cells, degradation of rrna occurred to a similar extent in the absence of oas and oas as in wt cells and virus replicated to the same extent in oas -ko, oas -ko, and wt roni cells (fig. ) , suggesting that only oas played a role in antiviral activity. further investigation needs to be carried out to understand the individual roles of oas or oas with different viruses and specifically whether there may be oas-dependent antiviral activities independent of rnase l in bat or human cells. oas genes are isgs, and as such, it has been generally thought that activation of rnase l is a downstream effect of ifn signaling. in this model, viral dsrna would first need to be recognized by pattern recognition receptors (prrs), such as rig-i/mda , leading to ifn induction and subsequent upregulation of oass, which would then be activated by viral dsrna ( ) . however, we found previously that the oas-rnase l pathway can be activated in the absence of murine coronavirus-induced ifn in mouse bone marrow-derived macrophages ( ) . here, we show that activation of rnase l during infections with either sinv (fig. ) or vacvΔe l (fig. ) is independent of mavs expression in bat roni/ cells, similar to findings for sinv in human a cells, indicating that basal oas levels which are weakly induced in the absence of mavs (fig. d) are sufficient for activation of rnase l. we recently reported similar results during zika virus infection of a cells ( ) . in addition, mavs plays little if any role in limitation of sinv replication in bat or human cells (fig. b and e) or of vacvΔe l in bat cells (fig. b ). this is in contrast to rnase l, which, as discussed above, dramatically limits viral replication (fig. , , and ) ( ) . these results imply that the oas-rnase l pathway plays a significant role in limiting viral infection before ifn induction or in the absence of ifn by a virus that antagonizes ifn induction or signaling. indeed, a recent study showed robust rnase l activation as early as h after pic transfection in human a cells ( ) . thus, whereas ifn induction of oass may enhance the activation of the pathway, it is not always required. these studies suggest that oass act in parallel to other prrs, including rig-i-like receptors, and the oas-rnase l system is a separate pathway rather than a secondary pathway of ifn induction/signaling. importantly, oas-rnase l is a potent antiviral pathway, activated by infection in bat roni/ cells. egyptian rousette (rousettus aegyptiacus) kidney-derived roni/ cells ( ) ( ) ( ) were obtained from marcel müller (charité-universitätsmedizin, berlin, germany), and myotis lucifugus skin fibroblasts ( ) , myotis lucifugus embryonic fibroblasts, myotis velifer embryonic fibroblasts, and eptesicus fuscus skin fibroblasts ( ) were obtained from cedric feschotte (cornell university). the cells were cultured in dulbecco's modified eagle's medium (dmem; gibco catalog no. ) supplemented with nonessential amino acids, % fetal bovine serum (fbs), u/ml of penicillin, and mg/ml streptomycin. african green monkey kidney vero cells (atcc ccl ) were cultured in dmem (gibco ), supplemented with % fbs, mm hepes, mm sodium pyruvate, u/ml of penicillin, mg/ml streptomycin, and g/ml gentamicin. baby hamster kidney (bhk- ) cells were cultured in dmem supplemented with % fbs, % tryptose phosphate broth (sigma-aldrich), u/ml penicillin, and mg/ml streptomycin. human hek t cells were cultured in dmem supplemented with % fbs and mm sodium pyruvate. human a cells were cultured in rpmi medium (gibco) supplemented with % fetal bovine serum (fbs), u/ml of penicillin, and mg/ml streptomycin. human hek t and a cells have been authenticated by the atcc. the roni/ cells were authenticated by cloning and sequencing of oas and rnase l mrnas, which matched the sequences derived from the egyptian rousette (rousettus aegyptiacus) bat genes deposited in genbank. rnase l or mavs-ko a cells were generated by crispr/cas technology and were described in previous studies ( , ) . sindbis virus girdwood g (sinv), expressing mcherry, was obtained from mark heise (university of north carolina, chapel hill) and was prepared in bhk cells as previously described ( ) . sendai virus (sev) strain cantell ( ) was obtained from carolina b. lopez (university of pennsylvania). mutant vaccinia virus (vacvΔe l) was obtained from bertram jacobs (arizona state university, tempe, az) and was grown as described previously ( ) . cloning and sequencing of bat oas cdnas. boas , boas , and boas cdnas were cloned from roni/ cells. briefly, the cells were treated with , u of ifn-␣ overnight, and total cellular rna was extracted using an rneasy plus mini kit (qiagen). cdna was synthesized by reverse transcription using superscript iii (invitrogen) and mrna-specific reverse primers. boas -rev and boas -rev (table ) primers were used for reverse transcription to synthesize boas and boas cdna, respectively. three cdna fragments were cloned to assemble the full length of the boas open reading frame. boas -f rev, boas -f -rev, and boas -f -rev (table ) were used for reverse transcription reactions to synthesize three cdna fragments (boas -f , boas -f , and boas -f , respectively). the dna fragments of boas , boas , boas -f , and boas -f were amplified by using accuprime dna polymerase (invitrogen) with the forward primers and reverse primers (see table s in the supplemental material), and were cloned into pcr . topo vectors (invitrogen). the dna fragment of boas -f was amplified by using q dna polymerase (neb) and was cloned into pcr ii blunt-topo vectors (invitrogen). the cloned dnas were sequenced and analyzed. the oas sequence matched the predicted mrna sequence in genbank (id ). construction of plasmids and pseudolentivirus. the oligonucleotide sequences to be used for generation of small guide rnas (sgrnas) for kos of boas , boas , boas , brnase l, and bmavs genes are shown in table s . a pair of forward and reverse oligonucleotides for generation of each sgrna (synthesized by idt) were annealed by published methods ( ) and were inserted into plenti-crispr (addgene) between bsmbi restriction sites. the resulting plasmids are named plenti-sgbo (targeting the boas gene), plenti-sgbo (targeting the boas gene), plenti-sgbo (targeting the boas gene), plenti-sgbrl (targeting the brnase l gene), and plenti-sgbma (targeting the bmavs gene). for packaging of pseudolentiviruses, ϫ hek t cells were plated in one well of a -well plate, and the next day were transfected with g plenti-crispr (with sgrna), . g pspax , and . g of pcmv-vsv-g (obtained from paul bates, university of pennsylvania) using lipofectamine (invitrogen) ( l in l of dmem). the supernatants were harvested at and h posttransfection and stored at Ϫ °c, and the -h supernatants were used for further ko experiments. construction of boas , boas , boas , brnase l, and bmavs gene knockout roni/ cells. for construction of roni/ ko cells using lenti-crispr, ϫ cells were plated into one well of a -well plate and were transduced with l of pseudolentiviruses. forty-eight hours postransduction, cells were cultured in medium containing . g/ml of puromycin for days. the resistant cells were further cultured for week in medium without puromycin before being cloned by limited dilution. briefly, cells were diluted to cells/ml and l of cells was added to one well of -well plates. single cells were selected for further amplification and genotyping. genomic dna was extracted using a dneasy blood and tissue kit (qiagen). dna fragments covering the region targeted by sgrna were amplified by pcr using gene-specific primers (table s ) mixed with gotaq master mix (promega), and the pcr products were sequenced. cells with frameshift mutations (deletion or insertion) in targeted genes were selected for further experiments. generation of ؋flag-hoas knock-in cells. boas -ko cells were electroporated (btx) with plasmid pcmv - xflag-hoas ( ) and plated without antibiotics. at h postelectroporation, cells were selected with g/ml of g (gibco) for weeks and then cloned by limiting dilution. clones were screened by western immunoblotting using anti-flag m antibodies. cell clones expressing ϫflag-hoas were maintained in medium containing g/ml of g . antibody. rabbit anti-boas and -boas antibodies were generated by immunizing rabbits with purified mbp-boas -d and mbp-boas -f / . briefly, boas domain (catalytic domain) was amplified by pcr using primers boas -d -for (acgtcgactacccccgggcatcttctggataaattc) and boas -d rev (gctctagatcactcgaggagcccccaacttctgaac), and boas fragment f -f was amplified by pcr using primers boas -f / -for (acgtcgactgatctgtctcagatccccgccaatgag) and boas -f / rev (gccaagctttcagacgtgtccaaggggagggaccacatg). the fragments were cloned into pmal (neb) vectors between sali and xbai restriction sites. pmal-boas -d and pmal-boas -f / were transformed into neb-express competent escherichia coli, and iptg was added to the bacterial cultures to induce expression of the proteins. the proteins were purified by elution from the protein bands cut from polyacrylamide gels with the pierce zinc reversible stain kit (thermo fisher). the eluted proteins were concentrated and showed a single band on a polyacrylamide gel by staining. rabbits were injected with g protein each times over about months before bleeding. mouse anti-gapdh ga r ( : , ; thermo fisher) was used to detect bat gapdh. mouse anti-flag m antibodies ( : , ; sigma-aldrich) were used to detect ϫflag-hoas . secondary antibodies goat anti-mouse antibody ( : , ; santa cruz) and goat anti-rabbit antibody ( : , ; cell signaling), conjugated to horseradish peroxidase (hrp), were used to detect mouse-or rabbit-derived primary antibodies. western blotting. confluent cells in -well plates were treated or mock treated with , u of ifn-␣ overnight or infected with sinv at an moi of at h postinfection. cells were harvested, washed in pbs, and lysed with np- buffer with protease inhibitor cocktail (roche). cell lysates were mixed with ϫ laemmli buffer, boiled at °c for min, and analyzed by electrophoresis on to % or to % gradient sds gels. proteins were transferred to polyvinylidene difluoride membranes, which were treated with % nonfat milk in tbst (tris-hcl-buffered saline with . % tween ) blocking buffer for h, followed by incubation overnight at °c with antibodies diluted into tbst. membranes were then washed three times with tbst, incubated with secondary antibodies for h at room temperature, again washed three times with tbst, and then incubated with supersignal west dura extended-duration substrate (thermo fisher), and the signal was detected using an amersham imager (ge). virus growth kinetics. cells were plated in -well (roni/ ) or -well (a ) plates, ϫ cells per well. the next day, cells were infected with the indicated viruses at an moi of , three parallel wells per virus. at , , and h postinfection, l of supernatant was harvested and stored at Ϫ °c for titration. plaque assays. sinv was diluted in dmem, and l was added to confluent vero monolayers in -well plates. the plates were incubated for h at °c and were rocked at -min intervals. cells were then overlaid with ml warm dmem containing % fbs and . % agar. vaccinia virus (vacvΔe l) titers were determined in bhk- cells as described previously ( ) . rrna cleavage assay. cells were infected with sinv at an moi of (bat cells) or an moi of (a cells). at (bat cells) or (a cells) h postinfection, cells were harvested in rlt buffer (rneasy minikit; qiagen). bat cells were infected with vacvΔe l at an moi of and at h postinfection were harvested in rlt buffer (bio basic, inc.). total rna was extracted and was resolved on rna chips using an agilent bioanalyzer ( ) . mrna quantification by quantitative reverse transcriptase pcr (qrt-pcr). cells were infected with sinv (moi ϭ ) or sev (moi ϭ ) or treated with , u of ifn-␣ in triplicate in -well plates. cells were lysed at (sinv) or (sev) h postinfection (hpi) or h after ifn treatment in rlt plus rna lysis buffer (qiagen), and rna was isolated using the rneasy plus mini kit (bio basic, inc.) as previously described ( ) . cycle threshold (c t ) values were normalized to ␤-actin. oas mrna levels were calculated relative to ␤-actin mrna and expressed as fold over mock-treated or mock-infected with the formula ϪΔ(Δct) (Δc t ϭ c tgene of interest Ϫ c tactin ). primer sequences are listed in table s in the supplemental material. software and statistical analysis. sequences were analyzed by vector nti. sequence alignment figures were created by vector nti. all other figures were created by graphpad. all analyses were performed in graphpad prism version . a. plaque assay data were analyzed by two-way anova. significance is shown as follows: ns, not significant; *, p Ͻ . ; **, p Ͻ . ; ***, p Ͻ . ; ****, p Ͻ . . data availability. the nucleotide sequences of boas and oas orfs were deposited in genbank with accession numbers mk and mk , respectively. supplemental material for this article may be found at https://doi.org/ . /mbio . - . oral shedding of marburg virus in experimentally infected egyptian fruit bats (rousettus aegyptiacus) experimental inoculation of egyptian fruit bats (rousettus aegyptiacus) with ebola virus virological and serological findings in rousettus aegyptiacus experimentally inoculated with vero cells-adapted hogan strain of marburg virus human betacoronavirus c emc/ -related viruses in bats, ghana and europe a sars-like cluster of circulating bat coronaviruses shows potential for human emergence seroepidemiology of selected alphaviruses and flaviviruses in bats in trinidad. zoonoses public health pteropid bats are confirmed as the reservoir hosts of henipaviruses: a comprehensive experimental study of virus transmission type iii ifn receptor expression and functional characterisation in the pteropid bat, pteropus alecto type iii ifns in pteropid bats: differential expression patterns provide evidence for distinct roles in antiviral immunity bat mx and oas , but not pkr are highly induced by bat interferon and viral infection contraction of the type i ifn locus and unusual constitutive expression of ifn-alpha in bats type i interferon reaction to viral infection in interferon-competent, immortalized cell lines from the african fruit bat eidolon helvum innate immune responses of bat and human cells to filoviruses: commonalities and distinctions dampened sting-dependent interferon 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of rnase l is dependent on oas expression during infection with diverse human viruses interferon-stimulated genes: roles in viral pathogenesis the egyptian rousette genome reveals unexpected features of bat antiviral immunity cell lines from the egyptian fruit bat are permissive for modified vaccinia ankara virus-activated interferon regulatory factor upregulates expression of the interferon-regulated bst gene independently of interferon signaling structural basis for cytosolic double-stranded rna surveillance by human oligoadenylate synthetase crystal structure of the =-specific and double-stranded rna-activated interferoninduced antiviral protein =- =-oligoadenylate synthetase the nature of the catalytic domain of =- =-oligoadenylate synthetases dimeric structure of pseudokinase rnase l bound to - a reveals a basis for interferon-induced antiviral activity interactome and proteome dynamics uncover immune modulatory associations of the pathogen sensing factor cgas oligoadenylate-synthetase-family protein oasl inhibits activity of the dna sensor cgas during dna virus infection to limit interferon production viruses in bats and potential spillover to animals and humans replication defective viral genomes exploit a cellular pro-survival mechanism to establish paramyxovirus persistence extracellular =- = oligoadenylate synthetase stimulates rnase l-independent antiviral activity: a novel mechanism of virus-induced innate immunity interferon-inducible antiviral effectors activation of rnase l by murine coronavirus in myeloid cells is dependent on basal oas gene expression and independent of virus-induced interferon zika virus production is resistant to rnase l antiviral activity real-time - a kinetics suggest that interferons beta and lambda evade global arrest of translation by rnase l comparative analysis of ebola virus glycoprotein interactions with human and bat cells differential sensitivity of bat cells to infection by enveloped rna viruses: coronaviruses, paramyxoviruses, filoviruses, and influenza viruses establishment of fruit bat cells (rousettus aegyptiacus) as a model system for the investigation of filoviral infection comparative biology of mammalian telomeres: hypotheses on ancestral states and the roles of telomeres in longevity determination identification of adult mouse neurovirulence determinants of the sindbis virus strain ar the ebola virus vp protein inhibits activation of interferon regulatory factor blockade of interferon induction and action by the e l double-stranded rna binding proteins of vaccinia virus genome engineering using the crispr-cas system antagonism of the interferon-induced oas-rnase l pathway by murine coronavirus ns protein is required for virus replication and liver pathology research reported in this publication was supported by the national institute of allergy and infectious diseases of the national institutes of health (nih) under award r ai to s.r.w. and r.h.s. and r ai to s.r.w. z.w. was supported in part by the chinese scholarship council.we thank marcel muller for roni/ cells and cedric feschotte for myotis lucifugus skin fibroblasts and eptesicus fuscus skin fibroblasts (originally from woody wright, university of texas southwestern), myotis lucifugus embryonic fibroblasts (originally from mario capecchi, university of utah), and myotis velifer embryonic fibroblasts (originally from david ray, texas tech university). we also thank cedric feschotte and rachel cosby, as well as ravi sachidanandam, for bat transcriptome analysis. we also thank melanie hoffner and earl poptic in the hybridoma core (lerner research institute) for making the rabbit anti-bat oas polyclonal antibodies and christina gaughan for expert technical assistance. key: cord- -vmtfn x authors: li, kun; li, zhuo; wohlford-lenane, christine; meyerholz, david k.; channappanavar, rudragouda; an, dong; perlman, stanley; mccray, paul b.; he, biao title: single-dose, intranasal immunization with recombinant parainfluenza virus expressing middle east respiratory syndrome coronavirus (mers-cov) spike protein protects mice from fatal mers-cov infection date: - - journal: mbio doi: . /mbio. - sha: doc_id: cord_uid: vmtfn x middle east respiratory syndrome coronavirus (mers-cov) can cause severe and fatal acute respiratory disease in humans and remains endemic in the middle east since first being identified in . there are currently no approved vaccines or therapies available for mers-cov. in this study, we evaluated parainfluenza virus (piv )-based vaccine expressing the mers-cov envelope spike protein (piv /mers-s) in a human dpp knockin c bl/ congenic mouse model (hdpp ki). following a single-dose intranasal immunization, piv -mers-s induced neutralizing antibody and robust t cell responses in hdpp ki mice. a single intranasal administration of ( ) pfu piv -mers-s provided complete protection against a lethal challenge with mouse-adapted mers-cov (mers(ma) . . ) and improved virus clearance in the lung. in comparison, single-dose intramuscular immunization with ( ) pfu uv-inactivated mers(ma) . . mixed with imject alum provided protection to only % of immunized mice. intriguingly, an influx of eosinophils was observed only in the lungs of mice immunized with inactivated mers-cov, suggestive of a hypersensitivity-type response. overall, our study indicated that piv -mers-s is a promising effective vaccine candidate against mers-cov infection. m iddle east respiratory syndrome (mers) emerged as a significant illness on the saudi arabian peninsula in mid- , and the causative agent was identified as a novel coronavirus (cov), mers-cov ( ) . mers has a high mortality rate (ϳ %) associated with severe lung disease that can advance to acute respiratory distress syndrome (ards). mers-cov, similarly to sars-cov, which caused a similar epidemic in , has been a global cause for concern due to its high fatality rate. epidemiologic studies established that mers-cov is zoonotic in origin, with transmission occurring from dromedary camels on the arabian peninsula ( ) ( ) ( ) . spread from camels to people is documented ( ) , as well as person-to-person spread among health care workers in hospital settings ( ) . to date, mers-cov has spread to countries and caused deaths in , confirmed cases ( february , world health organization [who]), including a large travel-related outbreak in south korea in ( ) . mers-cov is an enveloped positive-stranded rna virus whose entry into target cells is mediated by the viral envelope s protein. the s protein consists of an s subunit responsible for binding to the virus receptor, dipeptidyl peptidase (dpp or cd ), via a receptor-binding domain (rbd), and an s subunit that mediates membrane fusion ( ) ( ) ( ) . thus, the s protein, particularly the rbd, is an important target for mers-cov vaccine development ( , , ) . there is currently no vaccine or antiviral therapeutic against mers-cov. a number of candidate mers-cov vaccines, including those based on recombinant virus, viral vectors (e.g., mva, adenovirus, and measles virus), nanoparticles, dna, and dna/protein, as well as subunit vaccines, are under development ( , ) . none are approved; thus, the need remains for an effective and broadspectrum vaccine against mers-cov infection ( ) . piv , formerly known as simian virus (sv ), is a nonsegmented, negative-strand, rna virus (nnsv). it is a member of the rubulavirus genus of the family paramyxoviridae, which includes mumps virus (muv) and human parainfluenza virus type (hpiv ) and type (hpiv ) ( ) . piv encodes eight known viral proteins ( ) . nucleocapsid protein (np), phosphoprotein (p), and large rna polymerase (l) protein are important for transcription and replication of the viral rna genome. piv is an excellent viral vector candidate for vaccine development; it is safe and infects a large number of mammals without being associated with any diseases, except kennel cough in dogs ( ) ( ) ( ) ( ) ( ) . because piv does not have a dna phase in its life cycle, its use avoids the possible unintended consequences of genetic modifications of host cell dna through recombination or insertion. in comparison to positive-strand rna viruses, the genome structure of piv is stable. a recombinant piv expressing f of respiratory syncytial virus (rsv) has been generated, and the f gene was maintained for more than generations ( ) . piv can be grown to ϫ pfu/ml, indicating its potential as a costeffective and safe vaccine vector that may be used in mass production. we have discovered that piv -based influenza, respiratory syncytial virus (rsv), and rabies vaccines are efficacious ( ) ( ) ( ) ( ) ( ) ( ) ( ) . in studies of influenza, we previously reported that that a piv vector expressing influenza virus na provided sterilizing immunity (no mortality, no morbidity, and no virus detected in the lungs of challenged mice at days postchallenge) and piv expressing np protected % of mice against lethal influenza virus h n challenge in mice ( ) , demonstrating that piv is an excellent vector for developing vaccines for respiratory pathogens. here we investigate the utility of a piv -based vaccine expressing the mers s protein in a robust humanized mouse model of lethal mers-cov infection. construction of a piv vector expressing mers-cov spike glycoprotein. previously, we inserted the ha gene of influenza a virus at different locations within the genome of piv and found that the insertion at sh and hn generates the best immune responses ( ) . thus, we inserted the full-length gene of s of mers at the sh and hn junction. a plasmid containing full-length piv cdna with the s gene insertion at sh and hn junction was constructed using standard molecular cloning techniques (fig. a) . the plasmid was transfected into bhk cells along with plasmids expressing t rna polymerase, np, p, and l of piv , and infectious virus piv -mers-s was rescued as described before ( ) . the rescued virus was plaque-purified and then expanded to large quantity in mdbk cells for further analysis. the viral genome was sequenced and confirmed to contain the desired input dna sequence. to verify s protein expression in piv -mers-s-infected cells, the cells were infected at different mois and then lysed for immunoblotting using anti-s antibody. the full-length s and cleaved s fragments were observed in piv -mers-s-infected cells, suggesting that the s protein was properly processed (fig. b) . expression of s protein in piv -mers-s-infected cells was further confirmed by immunofluorescence assay (fig. c) . interestingly, piv -mers-s caused massive syncytium formation in vero cells. piv -mers-s had a similar growth kinetics as wild-type piv (fig. d) . to determine whether piv -mers-s can generate immune responses in mice, c bl/ mice were immunized with a single dose of piv -mers-s or control piv -gfp virus at pfu or pfu per mouse via intranasal route. while both doses generated antibody responses, neutralizing titers were modest at : and : for the and doses, respectively ( fig. a and b) . it is known that c bl/ and balb/c mice generate th -and th -dominant immune responses, respectively, following immunization. a single dose of pfu of piv -mers-s resulted in a neutralization antibody titer as high as : , in balb/c mice (fig. c) , consistent with a th dominant response in balb/c mice. to assess the primary cd t cell response generated by piv -mers-s immunization, hdpp -ki mice were intranasally immunized with pfu piv -mers-s. four weeks later, lungs were harvested and examined for mers-cov-specific lung-resident cd t cells (fig. a) . as shown in fig. b to d, we observed a significant increase in the immunization with piv expressing mers-cov spike ® percentage and total number of cd ϩ -ifn-␥ ϩ cells in the lungs of piv -mers-simmunized mice in comparison to those infected with piv -gfp virus, consistent with a mers-s-specific primary cd t cell response in the lungs. further, to examine recall response of mers-s-specific cd t cells, we challenged piv -mers-s-and piv -gfpimmunized mice with pfu of mers-cov ma . . . our results show a significant increase in the recall cd t cell response at day p.i. in comparison to piv -gfpimmunized mice ( fig. e to g). we also observed -fold increase in mers-s-specific cd t cells compared to the primary cd t cell response ( fig. b to g). collectively, these results indicate that mers-s immunization induces a significantly increased mers-s-specific cd t cell response upon piv -mers-s immunization. to determine the efficacy of piv -mers-s in preventing or modifying a mers-cov infection, hdpp ki mice on the c bl/ background were immunized with pfu piv -mers-s via the intranasal route. at weeks after immunization, mice were challenged with a mouseadapted mers-s strain (fig. a ). all piv -mers-s-immunized mice survived this lethal challenge and lost little weight ( fig. b and c). in contrast, piv -gfp-or pbs-immunized mice all died following challenge ( fig. b and c), indicating that piv -mers-s completely protected mice against lethal challenge. while piv -mers-s-immunized mice had a higher rate of virus clearance from the lungs, this did not provide sterilizing immunity (fig. d) . histopathology of lung tissues. histopathology studies of lungs after challenge with mers ma . . indicated that piv -mers-s-immunized mice had significantly less cellular debris present and greater mononuclear infiltrates ( fig. a and b ). piv -mers-s-immunized mice exhibited robust cellular infiltration of leukocytes (mostly mononuclear cells) and less evidence of lesions (edema, hyaline membranes, necrotic cellular debris, etc.) indicative of severe disease (fig. ) . to investigate the protective responses elicited by piv -mers-s and the inactivated mers-cov, hdpp ki mice were immunized with pfu of piv -mers-s or pbs via i.n. or uv-inactivated mers-cov with adjuvant via i.m. route. while piv -mers-s provided % protection against lethal challenge, inactivated mers-cov protected % of mice from mortality (fig. ). it has been reported that mice immunized with inactivated sars-cov and mers-cov developed a hypersensitivity-type response after respective sars-cov and mers-cov challenge, manifested by increased il- and il- expression and an influx of eosinophils ( ) . we examined the lungs of mice that were immunized and then challenged with mers-cov (fig. ) . we observed more eosinophils in the lungs of mice immunized with inactivated mers-cov than in pbs-or piv -mers-s-immunized mice following mers-cov challenge. compared to pbs, perivascular eosinophilic infiltration was significantly increased in the inactivated mers-cov group, but no statistical difference was seen many strategies have been considered to develop vaccines for both sars-cov and mers-cov. a live attenuated sars-cov with rationally introduced mutations was efficacious in golden syrian hamsters ( ) . however, the development of a live attenuated vaccine for a positive-stranded rna virus like sars-cov has often been hampered by safety concerns. several mers vaccine candidates are under investigation. a dna-based vaccine expressing the full-length s protein is the most advanced to date ( ) ; it is well tolerated in humans, as shown in a phase i clinical trial. the prime-boost regimen of mva (modified vaccinia ankara) expressing mers s protein induced neutralizing antibodies and t cell responses in mice and limited viral replication after challenge in mice and camels. however, mva-s did not provide sterilizing immunity, immunization with piv expressing mers-cov spike ® and infectious mers-cov and genomic rna were detected after challenge in mice and camels ( , ) . the prime-boost regimen of measles virus (mv) expressing mers s or soluble s induced both humoral and cellular immune responses. after mers challenge, infectious mers-cov or genomic rna significantly decreased, but these two vaccines did not provide sterilizing immunity, and signs of inflammation were observed in mouse lung tissue ( ) . an inactivated rabies virus (rabv) expressing mers s provided complete protection from mers challenge in mice but three -g doses of vaccine were needed ( ) . furthermore, the ad /hdpp -transduced mouse model used in these studies has limitations. adenovirus (ad ) expressing mers s or s also induced a neutralizing antibody in mice ( ) . ad , an enteric adenovirus, may induce enhanced mucosal immunity when administered via an oral or intragastric (i.g.) route ( , ) . however, i.g. immunization of both ad -s and ad -s failed to generate mucosal immunity. although ad -s induced humoral immunity in serum, it was significantly less than ad -s ( ). chimpanzee adenovirus-based vector systems have also been used ( ) . in our work, we demonstrated that a single dose as low as pfu of piv -mers-s was sufficient to provide % protection against lethal mers-cov challenge. the low dose is especially advantageous in a situation where a mass immunization program is needed in a short period of time. to the best of our knowledge, this is the most efficacious mers-cov vaccine tested in a relevant animal model. the protective mechanism of piv -mers-s vaccine in c bl/ mice is likely due to robust cellular immune responses after piv -mers-s immunization. while neutralizing antibody was generated in c bl/ mice after a single-dose immunization with piv -mers-s, titers were modest at : and : with pfu and pfu of piv -mers-s, respectively ( fig. a and b) . consistent with protective cellular immune responses protecting the mice, a significant influx of cd ϩ ifn-␥ ϩ cells was detected in lungs of c bl/ mice following piv -mers-s immunization (fig. ) . furthermore, the observation that piv -mers-s-immunized mice had a higher rate of mers virus clearance (fig. c ) suggests a role for t cell-based immunity in protecting c bl/ mice against lethal challenge. interestingly, in balb/c mice, piv -mers-s generated neutralizing antibody titers as high as : , (fig. c) . it is possible that the higher neutralizing antibody titers in balb/c mice may be protective. unfortunately, the only available small animal model is a humanized mouse model on the c bl/ background. it is known that the s protein is a major protective antigen for coronaviruses. it may be possible to improve our vaccine efficacy by expressing additional mers-cov proteins such as n and m using piv as a vector. however, a parainfluenza virus (piv )-based sars-cov vaccine candidate expressing n, m, or e without the s protein failed to protect hamsters from sars-cov challenge ( ) . the ability of piv -mers-s to generate cellular and humoral immune responses in mice may be in part attributed to the ability of piv to express the mers s protein in its native conformation. as shown in fig. c , piv -mers-s caused massive syncytium formation in vero cells, indicating the s protein was functional in promoting cell-to-cell fusion. thus, we reasoned that the s protein produced in piv -mers-s-infected cells maintains a native conformation. the mers s protein has , amino acid residues. the entire insertion of the s gene with proper regulatory sequences is over , nucleotides in length. this is the longest single gene we have inserted into the piv genome. since we inserted this gene between sh and hn, and the sh gene is not essential, it may be possible to remove sh to allow insertion of longer sequences. thus, we speculate that the piv genome can accommodate sequences longer than , nucleotides. it has been reported that inactivated sars-cov-immunized mice generated a hypersensitive-type lung pathology after virus challenge, raising the concern of vaccine-enhanced disease ( , ) . previously, a formalin-inactivated, whole-virus respiratory syncytial virus (rsv) vaccine caused enhanced disease in vaccinated children, leading to vaccine-related deaths ( ) . similarly, inactivated mers-cov has been reported to generate a th -type immunopathology after mers-cov challenge in mice ( ) . in the case of a piv -based rsv vaccine, extensive studies indicate that piv -based rsv vaccine does not cause enhanced diseases ( ) . thus, as a viral vector, piv is not known to cause any enhanced diseases, and in our experiment, we observed no abnormal immune responses in piv -mers-s-immunized mice after mers-cov challenge, suggesting that piv -mers-s is unlikely to be associated with enhanced disease. immunization with piv expressing mers-cov spike ® lung tissues of mice immunized with inactivated mers-cov had an influx of eosinophils after mers-cov challenge, indicative of a hypersensitivity-type response. this result is consistent with a previous report that inactivated mers-cov immunization caused increased il- and il- expression and an influx of eosinophils in lungs after challenge ( ) . understanding whether immunization with inactivated mers-cov can cause enhanced disease is critical for developing a safe and effective vaccine. while mers-cov has a high morbidity and mortality, it has very a low prevalence in human populations. dromedary camels are considered the intermediate host that transmits mers-cov to humans. thus, it may be possible to control the spread of mers-cov in humans by controlling infection in dromedary camels. perhaps virus transmission from camels to humans can be blocked, with concomitant immunization of high-risk human populations, as proposed by cepi (the coalition for epidemic preparedness innovations) and who. as a vaccine vector, piv has been effective in mice, cotton rats, hamsters, guinea pigs, ferrets, dogs, and nonhuman primates ( , ( ) ( ) ( ) ( ) . it will be worthwhile to test piv -mers-s in camels in the future. recently, sars-cov- ( -ncov) was identified in wuhan, china, in late . this is a novel zoonotic cov related to the sars-cov that can cause severe respiratory disease . to date, this virus resulted in a significant disease burden, with more than , cases reported in countries and an estimated case fatality rate of~ %. the finding that piv expressing mers s protected mice against lethal mers-cov challenge at a single low dose of pfu suggests its potential use as a vaccine vector for emerging viruses such as sars-cov- . further studies of using piv expressing the s protein from sars-cov- as a vaccine candidate are ongoing. cells. vero cells were maintained in dulbecco's modified eagle medium (dmem) supplemented with % fetal bovine serum (fbs), iu/ml penicillin, and g/ml streptomycin ( % p/s; mediatech inc., manassas, va, usa). bhk cells were maintained in dmem containing % tryptose phosphate broth (tpb), % fetal bovine serum (fbs), iu/ml penicillin, and g/ml streptomycin. mdbk cells were grown in dmem containing % fbs and % p/s. cells were prepared day prior to infection, achieving approximately % confluence by the following day. viruses. the plasmid containing the cdna clone of piv with mers-s inserted between sh and hn was constructed using previously described methods ( , , ) . primer sequences are available upon request. infectious virus was rescued in bhk cells as previously described ( ) . recombinant piv or piv -mers-s was propagated in mdbk cells as previously described ( , ) . piv plaque assays were performed as previously described ( ) . briefly, -fold serial dilutions were prepared in dmem with % bsa. one hundred microliters of each dilution was transferred to -well plates of bhk cells, in a total infection volume of ml. after adsorption for to h at °c, % co , the inocula were aspirated, and cell monolayers were overlaid with dmem containing % tryptose phosphate broth (tpb), % fbs, % p/s, and % low-melting-point agarose. after days, the cells were fixed with % formaldehyde, overlays were removed, and the cells were stained with crystal violet to visualize the plaques. to obtain virus titers in lung tissues, lungs of infected mice were removed at the indicated days after challenge and homogenized in pbs using a manual homogenizer. virus titer was determined in vero cells by plaque assay. infected vero cells were fixed in % formaldehyde and stained with . % crystal violet to delineate plaques. to determine growth rates of piv and piv -mers-s, mdbk cells were infected with piv or piv -mers-s at an moi of . . after adsorption for to h at °c, % co , dmem with % fbs and % p/s was added to the plates. one-hundred-microliter samples of supernatant were collected daily for days and frozen at Ϫ °c. virus titers in the samples were quantified by plaque assay. immunization and infection of mice. specific-pathogen-free -week-old c bl/ and balb/c mice were purchased from charles river laboratory (cr). specific-pathogen-free human dpp knockin (hdpp ki) mice were generated on a c bl/ background as previously reported ( ) . all mice were bred and maintained in the university of iowa animal care facility. all protocols were reviewed and approved by the university of iowa institute animal care and use committee. six-to -week-old male and female mice were used for these studies. mouse-adapted mers-cov strain mers ma . . was generated as reported earlier ( ) . mice were anesthetized with xylazine-ketamine ( . mg/kg of body weight ketamine, . mg/kg xylazine) and infected intranasally with pfu or pfu piv -mers-s or piv -gfp in l dmem. the mouse-adapted mers ma . . strain was inactivated by exposure to uv light for h using a wattage of , w/cm . then pfu uv-inactivated viruses were : (vol/vol) mixed with imject alum (thermo, catalog no. ) and delivered to mice intramuscularly. four weeks postimmunization, mice were infected intranasally with pfu mers ma . . in l dmem. for passive immunization, sera were collected from hdpp ki mice that received pfu piv -mers-s or piv -gfp intranasally at weeks postimmunization. two hundred microliters of sera were transferred into hdpp ki mice intraperitoneally day before challenge with pfu mers ma . . . infected mice were examined daily, and weights were recorded. all work with mers-cov was performed in the biosafety level (bsl ) laboratory of the university of iowa. immunization with piv expressing mers-cov spike ® histology. at the indicated days postchallenge, mice were anesthetized and perfused with pbs by intracardiac injection followed by perfusion with zinc formalin. lungs were removed, fixed in zinc formalin overnight, and paraffin embedded. lung sections (ϳ -m thickness) were stained with hematoxylin and eosin. tissues were evaluated by board-certified veterinary pathologists and scored using a postexamination masking method ( ) . lungs were scored for edema, hyaline membranes, cellular debris, and hemorrhage, with scores of , , , , and representing detection in %, less than %, % to %, % to %, and more than % of lung fields, respectively. lungs were scored for mononuclear infiltrates, with scores of representing values within normal parameters, representing small aggregates in peribronchial and perivascular areas, representing perivascular and periairway aggregates filling perivascular space, and representing a score of plus expanding sheets of infiltrates into septa and consolidation lesions in regions of the lung, respectively. lungs were scored for granulocytic infiltrates, with scores as follows: , within normal parameters; , scattered pmns sequestered in septa; , a score of plus solitary pmns extravasated in airspaces; , a score of plus small aggregates in vessels and airspaces, respectively. lung tissues were evaluated for perivascular eosinophil infiltration. briefly, vessels with cellular infiltration (n ϭ /lung) were randomly selected by a masked pathologist, and the number of eosinophils was enumerated and averaged for a final score for each lung ( ) . neutralizing antibody assay. four weeks postimmunization, sera from immunized mice were collected. all serum samples were heat inactivated by incubation at °c for min. heat-inactivated serum was serially diluted by -fold in -well plates before the same volume of mers-cov pseudovirus was added and incubated at °c for h. the mixture was added into vero cells in -well plates and incubated at °c for h to allow virus binding. then the mixture was removed, and cells were rinsed with pbs. the next day, the neutralization results were measured by luciferase assay and plotted relative to the value for serum-free wells. immunoblotting and immunofluorescence. vero cells were infected with piv -mers-s at different mois or mock infected at °c for h. cell lysates were collected at days postinfection and applied to western blotting. the expression of mers-cov spike protein was detected by a rabbit anti-mers-cov s antibody (sino biological, catalog no. -t ) and colocalized using a mouse anti-␣-tubulin antibody (sigma, catalog no. t ). indirect immunofluorescence assays were performed to detect expression of the s protein in piv -mers-s-infected cells. vero cells were infected with piv or piv -mers-s. forty-eight hours later, cells were fixed with % formaldehyde in phosphate-buffered saline (pbs) and permeabilized by adding . % saponin to the immunostaining buffers. anti-mers-s from sino biological was used (catalog no. -t ). the cells were imaged using a nikon a r confocal microscope. analyses of mers-cov-specific cd t cell response. hdpp ki mice were immunized with pfu piv -gfp or piv -mers-s via intranasal route. at weeks postimmunization, lung cells were harvested and mers-cov-specific cd ϩ t cells were stimulated with m s and s peptides as described previously ( ) in the presence of golgi-plug ( l/ml) for h. cells were then washed and stained for cell surface cd , cd , and cd markers followed by intracellular ifn-gamma staining. to examine the recall cd t cell response, hdpp ki mice immunized with pfu piv -gfp or piv -mers-s via intranasal route were challenged with pfu mers ma . . . four days after mers ma . . infection, lungs were harvested and cd t cells were stimulated with s and s peptides in the presence of golgi-plug ( l/ml) for h. cells were then washed and stained for cell surface cd , cd , and cd markers followed by intracellular ifn-gamma staining. the following monoclonal antibodies were used: pecy anti-cd ( -f ), anti-cd (rm - ), and anti-cd ␣ ( - . ), all from bd bioscience, and ifn-␥ (xmg . ) from ebioscience. isolation of a novel coronavirus from a man with pneumonia in saudi arabia prevalence of middle east respiratory syndrome coronavirus (mers-cov) in dromedary camels in abu dhabi emirate tropism and replication of middle east respiratory syndrome coronavirus from dromedary camels in the human respiratory tract: an in-vitro and ex-vivo study acute middle east respiratory syndrome coronavirus infection in livestock dromedaries evidence for camel-to-human transmission of mers coronavirus ksa mers-cov investigation team. . hospital outbreak of middle east respiratory syndrome coronavirus mers-cov outbreak following a single patient exposure in an emergency room in south korea: 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incomplete protection in mice and induces increased eosinophilic proinflammatory pulmonary response upon challenge respiratory syncytial virus vaccine development parainfluenza virus expressing wild-type or prefusion respiratory syncytial virus (rsv) fusion protein protects mice and cotton rats from rsv challenge a single-dose recombinant parainfluenza virus -vectored vaccine expressing respiratory syncytial virus (rsv) f or g protein protected cotton rats and african green monkeys from rsv challenge efficacy of a parainfluenza virus (piv )-based h n vaccine in mice and guinea pigs: antibody titer towards ha was not a good indicator for protection evaluating a parainfluenza virus -based vaccine in a host with preexisting immunity against parainfluenza virus vaccination with recombinant parainfluenza virus expressing neuraminidase protects against homologous and heterologous influenza virus challenge mouse-adapted mers coronavirus causes lethal lung disease in human dpp knockin mice principles and approaches for reproducible scoring of tissue stains in research nonglycosylated g-protein vaccine protects against homologous and heterologous respiratory syncytial virus (rsv) challenge, while glycosylated g enhances rsv lung pathology and cytokine levels rapid generation of a mouse model for middle east respiratory syndrome we thank the members of the perlman, mccray, and he laboratories for their helpful discussion and technical assistance. key: cord- -uz vqq r authors: davis, c. todd; chen, li-mei; pappas, claudia; stevens, james; tumpey, terrence m.; gubareva, larisa v.; katz, jacqueline m.; villanueva, julie m.; donis, ruben o.; cox, nancy j. title: use of highly pathogenic avian influenza a(h n ) gain-of-function studies for molecular-based surveillance and pandemic preparedness date: - - journal: mbio doi: . /mbio. - sha: doc_id: cord_uid: uz vqq r nan z oonotic influenza viruses circulating in poultry and swine pose an ever present threat to human health. in particular, the rapid geographical expansion of highly pathogenic avian influenza (hpai) a(h n ) throughout asia and then into europe, the middle east, and africa during the s galvanized the global community in an attempt to control this rapidly growing threat. despite successful control efforts in some countries, the virus remains endemic in poultry in at least six countries and continues to cause human illness and deaths as well as countless outbreaks in birds. during the past decade, cases and deaths were detected and reported to the world health organization (who) ( ) . during the years since human infections with hpai a(h n ) were first identified in hong kong, special administrative region, people's republic of china, in , these viruses have evolved substantially through mutation and reassortment, resulting in multiple divergent genotypes and clades ( ) . ongoing h n circulation has appropriately resulted in a focus on sequencing viral genomes to understand the evolution of these viruses and the significance of observed genetic changes. expanded laboratory capacity for high-throughput sanger sequencing and recent technological advances, such as nextgeneration sequencing and parallel computing, have revolutionized the quantity, quality, and availability of gene sequences and our ability to quickly and accurately analyze these data ( ) . consequently, the number of animal and human influenza virus sequences available in publically accessible databases has dramatically increased over the years, as have the bioinformatics tools required for efficient investigation ( , ) . these advances in laboratory and analytical methods provide strong incentives to utilize molecular data for pandemic risk assessment of zoonotic influenza viruses at the animal-human interface ( ) . however, examination of influenza sequence data alone does not allow us to assess the pandemic potential of a virus. pandemic risk assessment that utilizes sequence data can take place only after critical genetic signatures are identified through laboratory research into the consequences for relevant biological properties (or phenotypes). these critical genetic features include those that based on previous experimental validation are predicted to confer virulence and/or have the ability to transmit efficiently in mammals. in this context, genomes are sequenced, mutations are detected relative to earlier viruses and prototype strains, significance is appraised based on prior knowledge of genetic markers, and phenotypes are tested using a variety of in vitro and in vivo experiments. viruses possessing phenotypes of interest or concern often become candidates for reverse-genetics studies, which are essential to elucidate the precise molecular correlate(s) of a given phenotype (fig. ) . from a molecular epidemiological perspective, this process is at the heart of how the public health community makes informed decisions about the threat posed by zoonotic influenza viruses and which interventions might be most effective ( ) . laboratories worldwide have employed reverse genetics to study the mechanisms by which hpai h n and other zoonotic influenza viruses evolve and how these mechanisms influence host receptor specificity, antigenic variation, replication, pathogenesis, drug susceptibility, and transmission ( ) ( ) ( ) ( ) . besides being used to create vaccine viruses for the development of live, attenuated ( ) and inactivated prepandemic h n influenza vaccines ( ), reverse-genetics methodologies also have been used for many years to study the phenotypic consequences of particular mutations, including genetic changes that confer a gain of function (gof). influenza virus gof studies have focused on several research areas: in vitro and/or in vivo replication in mammalian cell culture or animal hosts, adaptive mutations conferring changes in host susceptibility, alteration of receptor binding profiles and/or tropism for mammalian airway tissues, enhanced polymerase activity, changes in host antiviral response (e.g., cell signaling pathways), susceptibility to antiviral drugs, and pathogenesis and/or transmissibility in mammalian animal models. such gof experiments have elucidated key biological principles and provided the scientific basis for genomic sequence-based risk assessment of zoonotic viruses with pandemic potential. for example, the molecular basis for avian versus mammalian influenza virus receptor binding (␣ , versus ␣ , sialylated glycans) has been elucidated largely through gof experiments, and some recent studies that identified specific ha mutations conferring a switch from avian to mammalian host receptor specificity also demonstrated the impact of these mutations on the ability of h n virus to more efficiently infect the human upper respiratory tract ( ) ( ) ( ) ( ) ( ) ( ) . mutations conferring enhanced virulence in mammalian models or inhibition of the host antiviral response with the potential to cause more serious human illness have been described in other studies ( ) ( ) ( ) . still other gof work characterized mutations that confer resistance to neuraminidase (na) inhibitors ( ) ( ) ( ) ( ) . these data are critical to make effective drug treatment decisions and to inform stockpiling of antiviral medications. finally, many publications have described mutations that confer adaptation of h n viruses to mammalian hosts and transmissibility in guinea pigs or ferrets ( , , ) . it should be noted that in many cases, this research can demonstrate loss of function, which is also valuable for risk assessment. these types of studies provide vital data with which to monitor circulating viruses for features that may suggest increased capability for human-to-human transmission or more long-term adaptation of h n viruses in humans or other mammalian hosts, such as pigs. reverse-genetics experiments using the well-established ferret model to measure h n respiratory droplet-mediated transmission were, until recently, unable to identify specific mutations involved in this process because the laboratory-derived viruses, like their wild-type counterparts, demonstrated a lack of or limited capacity for aerosol transmission ( , , ) . however, studies from by herfst et al. ( ) and imai et al. ( ) were able to demonstrate that genetically modified h n viruses could be transmitted relatively efficiently via respiratory droplets in a mammalian host following acquisition of specific mutations in the hemagglutinin (ha) and/or in combination with the presence of lysine at position in the pb protein sequence. the controversy sparked by these studies continues to reverberate ( , ) . some have argued that benefits associated with h n gof transmission studies outweigh the risks ( - ), while others have stated that public health derived little to no benefit to justify the research ( ) ( ) ( ) . some gof research resumed following the end of a voluntary global moratorium ( ) . however, a new moratorium on funding certain types of gof research on influenza, severe acute respiratory syndrome (sars), and middle east respiratory syndrome (mers) viruses is now in place in the united states ( ) . this pause provided an opportune time to describe how the molecular markers identified by the controversial ferret transmission studies and many other gof studies have provided important information for public health risk assessment of naturally occurring viruses detected in animals and humans. here we outline how we utilize a molecularly based surveillance approach focused on knowledge and insights gained from gof research to inform influenza pandemic risk assessment, as well as risk management and pandemic preparedness. we present the dramatic increase in the number of human cases caused by hpai h n in cambodia and an outbreak of influenza a(h n ) in china during as examples of how the results of gof studies supported a more rapid and accurate risk assessment and response to these situations. in both instances, molecularly based surveillance identified naturally occurring mutations in avian influenza viruses isolated from humans that had been demonstrated by gof studies to increase transmissibility in the ferret model, prompting the public health actions described below. to keep up with the large body of data from h n gof publications, the centers for disease control and prevention (cdc) in collaboration with domestic and international researchers compiled published amino acid mutations and protein motifs that were tested experimentally and shown to alter relevant functional aspects of h n virus phenotypes in the h n genetic changes inventory ( ) . while this type of data had been used at cdc for many years to conduct molecularly based surveillance of naturally occurring influenza viruses, the end result of the h n genetic changes inventory was to provide a single point of reference to facilitate the efficient identification of specific mutations or motifs in viral proteins that might signal adaptation to mammalian species, changes in susceptibility to antiviral medications, or changes in viral pathogenesis and transmissibility. by focusing on mutations specifically identified as conferring gof phenotypes, veterinary and public health experts have used the genetic changes inventory to help assess the relevance of molecular markers identified in naturally occurring viruses. during pandemic risk assessment, viral sequence data are weighed with respect to other virologic and epidemiological traits of a newly identified virus. results from more than years of h n gof studies were compiled to assist researchers at institutions worldwide in their risk assessment of naturally occurring influenza viruses to facilitate an early response in the face of emerging zoonotic influenza virus threats. individuals or institutions tasked with risk assessment and pandemic planning must weigh the significance of the mutations detected via molecular surveillance. for example, mutations associated with some human h n fatalities (e.g., pb k) ( , ) may carry more weight than those found to enhance virulence only in a mouse model (e.g., the presence of the ns pdz binding domain motif) ( ) . similarly, mutations previously found to alter the receptor-binding preferences of h n viruses from an ␣ , (avian) to ␣ , (human) preference would be of particular concern. of greater concern would be the identification of mutations associated with enhanced mammalian transmissibility, including the specific amino acid residue combinations identified by herfst et al. and imai et al. to confer h n respiratory droplet transmission in ferrets ( , ) . a recent instance of enhanced surveillance and response by the cdc and regional partners occurred following the abrupt rise in human cases of h n that occurred in cambodia in . at the same time that increased case numbers were detected, public sequence database mining by researchers identified viruses from several human infections that possessed the same mutations shown by gof studies to alter receptor-binding specificity toward an ␣ , preference (k r and q l) and enhanced respiratory droplet transmission of a clade virus in ferrets (n k with q l) ( ) . additional amino acid sequence comparisons of these viruses to those of previously circulating cambodian clade viruses revealed three other ha substitutions conserved in all viruses. these three mutations were also shown by gof experiments to increase binding of h viruses to mammalian host cell sialic acid receptors in ␣ , linkage either alone (s a and s n) or in combination with other mutations (s p) ( ) ( ) ( ) . these sequence findings directly led the cdc to dispatch a team of three subject matter experts to cambodia to conduct an epidemiological investigation of sources of exposure to poultry for these human cases, case contact trace-back, including serologic analysis for h antibody, and retrospective investigations of poultry deaths and outbreaks in locations where cases were discovered. in addition, an intensive effort was undertaken to consolidate and analyze human and animal epidemiological and sequence data through collaboration across public health and veterinary sectors, as well as local, regional, and global agencies ( ) . although comparisons of the viral genomes of poultry and environmental samples to human samples demonstrated that the k r, n k, and q l mutations (i) were absent in poultry viruses, (ii) were likely to have arisen during human infection, and (iii) did not transmit from person to person, the enhanced surveillance, improved lab-oratory capacity, and financial resources that resulted from this investigation highlight the utility of gof data for pandemic preparedness ( ) . because of the finding that these gof mutations likely occurred during replication in humans and the possibility that they might arise again, a candidate vaccine virus (cvv) was developed against a/cambodia/x / , the virus that possessed two of the markers described by imai et al. as enhancing ferret aerosol transmission and three mutations shown to alter avian receptor-binding specificity ( , ) . while a vaccine stockpile was not manufactured using this particular cvv, having a vaccine virus available that has been developed for human use and excluded from select agent regulations reduces the time required for vaccine development and testing by at least month, thus enhancing global pandemic preparedness. this reduction in the time required for vaccine development is the basis for the creation of a library of cvvs for emerging influenza pandemic threats, an approach taken by who's global influenza surveillance and response system for many years ( ) . due to the continuous evolution and antigenic drift detected in many subtypes of avian and swine influenza viruses, prioritization of cvv development is required. to meet this need and prioritize other research decisions, the influenza risk assessment tool (irat) was developed by the influenza division at the cdc together with a global consortium of animal and public health experts to offer a standardized set of considerations to be applied when evaluating viruses with pandemic potential ( ) . the tool uses an additive model, based on multiattribute decision analysis, to integrate weighted elements from both laboratory and field observations. collectively, assessment of the molecular characteristics of circulating virus, together with factors such as incidence of human infections, population immunity, geographic or host distribution, antigenic variation, and/or extent of overall genetic diversification, provides an objective approach to measuring potential risk of a given strain, subtype, or group of viruses ( ) . the recent emergence of low-pathogenicity avian influenza (lpai) h n virus causing human infections in china, like the emergence of pandemic influenza a(h n ) virus in , was instructive from a molecular surveillance perspective because the ha and neuraminidase (na) genes were not closely related to those of previously recognized influenza a viruses ( ) . despite the lack of high sequence identity of this h n virus to other known viruses, many structural and functional motifs between this lpai h n and hpai h n viruses remain conserved, particularly the amino acid domains making up the three major structural elements of the receptor binding site (rbs) ( ) . in addition, na enzymatic active sites, which are targets of na inhibitors, share homology with na proteins from h n and other influenza a subtype viruses ( ) . finally, the internal genes described for h n viruses share common ancestry with h n lineage viruses, ancestry that is retained in the internal genes of many h n genotypes. thus, many of the gof mutations described for both surface and internal protein sequences of h n viruses were assessed in the context of h n virus sequences as they were deposited in databases during the early wave of human infections in china. within hours of the posting of the h n sequence data, cdc and other investigators using the h n genetic changes inventory identified several of the mutations described previously in h viruses shown to possess gof phenotypes, such as increased respiratory droplet transmission in ferrets (using h numbering: ha t a and ha q l), mammalian host adaptation (pb e k and pb d n), and enhanced virulence in mice (m n d and m t a) ( , ) . early detection of these molecular markers in h n viruses isolated from humans gave public health authorities evidence that these viruses posed an immediate pandemic threat. based on h n sequence data alone, development of a candidate vaccine virus began within a day using synthetic biology ( ) . h n viruses obtained from human cases in china were subsequently shared with international partners within weeks so that additional virologic characterization could be performed. over a period of approximately to weeks from the initial reports of human infection, numerous laboratories identified h n viruses (and the responsible mutations) with binding affinity to ␣ , host cell receptors ( , ) , replication without prior adaptation in mouse and ferret models ( ) , limited respiratory droplet transmission in ferrets ( ) , and reduced susceptibility to antiviral drugs ( ) , all substantiating inferences obtained from molecularly based surveillance using knowledge gained from gof studies. the irat was used, as data accumulated, to assess and reassess the risk posed by h n viruses compared with other zoonotic influenza viruses, and it was determined that the risk was greater than for h n subtype viruses. more recently, gof studies have focused specifically on h n viruses and have identified other mutations (i.e., pb k r) associated with enhanced mammalian replication ( ) . researchers also assessed the antigenic relationships of the novel viruses with existing h prepandemic vaccine candidates. these collective virologic findings, along with the molecular markers identified, led who collaborating centers for influenza and vaccine manufacturers to rapidly develop candidate vaccine viruses ( ) and then led the department of health and human services to perform human clinical trials ( ) and to stockpile h n vaccine in the united states ( ) for pandemic preparedness, shaving months off the time required to deploy this vaccine, should it be needed. as coordination of international surveillance activities and global sharing of viruses improve (especially in the wake of the pandemic and emergence of h n viruses in china), molecularly based surveillance has great potential for rapid risk assessment of samples collected as part of active and passive surveillance systems. real-time feedback to investigators in the field or authorities making policy decisions related to poultry outbreak containment, clinical intervention, and diagnostic methodology, to cite a few examples, will remain critical in the future. in addition, as building laboratory capacity is prioritized in countries impacted the most by endemic h n virus circulation and the higher incidence of human infection, the ability to screen viruses for amino acid markers or lineage-associated molecular determinants by sequencing, pyrosequencing, mass spectrometry, and real-time reverse transcription (rt)-pcr assays will become the reality for more and more laboratories. in recent years, both the range and speed of molecular surveillance for h n and other avian influenza viruses have continued to improve. notwithstanding, gof studies are needed to inform our interpretation of genetic data obtained from naturally occurring viruses. despite recent gains in our understanding of the molecular basis for phenotypic properties of hpai h n and lpai h n viruses, more data are required to fully elucidate the mechanisms by which influenza viruses with pandemic potential cause severe disease and how they evolve during replication in mammalian hosts. this is especially true for studies that offer insight into the virologic and molecular changes associated with increased capacity for mammalian transmission, a hallmark of pandemic influenza viruses. as outlined above, gof studies have provided critical information for molecularly based surveillance, as well as for research groups sequencing, characterizing, or experimentally testing these viruses. besides answering fundamental questions about the molecular basis for key phenotypic characteristics of h n and other avian influenza viruses, gof data have been used to launch outbreak investigations and allocate resources (e.g., h n in cambodia), to develop criteria for the influenza risk assessment tool, and to make difficult and sometimes costly pandemic planning policy decisions, such as preparing cvvs and purchasing prepandemic vaccine stockpiles (e.g., h n in china). the detection of gof mutations in hpai h n and lpai h n viruses prompted immediate public health responses that differed from the actions that would have occurred with a rise in case numbers alone because concurrent detection of gof mutations with an increase in human cases could be a signal that human-to-human transmission had begun, a situation where rapid response is paramount. by coupling results obtained from gof studies with enhanced surveillance and preparedness, we as a community of scientists, veterinary and public health experts, regulators, and policy advisers have an opportunity to use the most advanced methodologies available to address the continuing threat posed by influenza viruses with pandemic potential. the findings and conclusions in this report are those of the authors and do not necessarily represent the views of the centers for disease control and prevention. cumulative number of confirmed human cases of avian influenza a(h n ) reported to who world health organization/world organisation for animal health/ food and agriculture organization (who/oie/fao) h n evolution working group life technologies promises $ , genome label: fast and accurate lineage assignment with assessment of h n and h n influenza a hemagglutinins influenza preparedeness for the next wave prediction and prevention of the next pandemic zoonosis improving pandemic influenza risk assessment detection of amantadine-resistant variants among avian influenza viruses isolated in north america and asia natural variation can significantly alter the sensitivity of influenza a (h n ) viruses to oseltamivir evolution of drug resistance in multiple distinct lineages of h n avian influenza neuraminidase inhibitor sensitivity and receptor-binding specificity of cambodian clade highly pathogenic h n influenza virus. antimicrob many ways to make an influenza virus-review of influenza virus reverse genetics methods generation and characterization of candidate vaccine viruses for prepandemic influenza vaccines human-like receptor specificity does not affect the neuraminidaseinhibitor susceptibility of h n influenza viruses recent avian h n viruses exhibit increased propensity for acquiring human receptor specificity in vitro assessment of attachment pattern and replication efficiency of h n influenza a viruses with altered receptor specificity glycosylation at n of the hemagglutinin protein and receptor binding specificity synergistically affect the antigenicity and immunogenicity of a live attenuated h n a/vietnam/ / vaccine virus in ferrets effect of receptor binding domain mutations on receptor binding and transmissibility of avian influenza h n viruses in vitro evolution of h n avian influenza virus toward human-type receptor specificity polygenic virulence factors involved in pathogenesis of hong kong h n influenza viruses in mice experimental adaptation of an influenza h ha confers respiratory droplet transmission to a reassortant h ha/h n virus in ferrets differential contribution of pb -f to the virulence of highly pathogenic h n influenza a virus in mammalian and avian species selection of influenza virus mutants in experimentally infected volunteers treated with oseltamivir selection of h n influenza virus pb during replication in humans susceptibility of highly pathogenic a(h n ) avian influenza viruses to the neuraminidase inhibitors and adamantanes efficacy of oseltamivir therapy in ferrets inoculated with different clades of h n influenza virus identification of amino acids in ha and pb critical for the transmission of h n lack of transmission of h n avian-human reassortant influenza viruses in a ferret model airborne transmission of influenza a/h n virus between ferrets the pause on avian h n influenza virus transmission research should be ended ferret-transmissible influenza a(h n ) virus: let us err on the side of caution mammalian-transmissible h n influenza: the dilemma of dual-use research rethinking biosafety in research on potential pandemic pathogens gain-of-function research: unproven technique gain-of-function research: unknown risks transmission studies resume for avian flu government gain-of-function deliberative process and research funding pause on selected gain-of-function research involving influenza, mers, and sars viruses. us government, washington h n genetic changes inventory: a tool for influenza surveillance and preparedness isolation of a genotypically unique h n influenza virus from duck meat imported into japan from china pathogenicity of highly pathogenic avian h n influenza a viruses isolated from humans between and in northern vietnam a new influenza virus virulence determinant: the ns protein four c-terminal residues modulate pathogenicity identification of molecular markers associated with alteration of receptorbinding specificity in a novel genotype of highly pathogenic avian influenza a(h n ) viruses detected in cambodia in haemagglutinin mutations responsible for the binding of h n influenza a viruses to human-type receptors immunization by avian h influenza hemagglutinin mutants with altered receptor binding specificity viremia associated with fatal outcomes in ferrets infected with avian h n influenza virus a(h n ), a(h n ) and variant influenza viruses and candidate vaccine viruses developed for potential use in human vaccines. world health organization pandemic preparedness and the influenza risk assessment tool (irat) human infection with a novel avian-origin influenza a (h n ) virus structures of receptor complexes of a north american h n influenza hemagglutinin with a loop deletion in the receptor binding site infectivity, transmission, and pathology of human-isolated h n influenza in ferrets and pigs synthetic generation of influenza vaccine viruses for rapid response to pandemics pathogenesis and transmission of avian influenza a (h n ) virus in ferrets and mice r k substitution and drug susceptibility of influenza a(h n ) viruses the k r substitution in viral protein pb enhances the effect of e k on influenza replication a(h n ), a(h n ) and variant influenza viruses and candidate vaccine viruses developed for potential use in human vaccines. world health organization serological responses to an avian influenza a/h n vaccine mixed at the point-of-use with mf adjuvant: a randomized clinical trial hhs h n vaccine response. us department of health and human services, washington the views expressed in this perspective do not necessarily reflect the views of the journal or of asm key: cord- -efo f c authors: vaillancourt, mylene; jorth, peter title: the unrecognized threat of secondary bacterial infections with covid- date: - - journal: mbio doi: . /mbio. - sha: doc_id: cord_uid: efo f c coronavirus disease (covid- ) is the greatest pandemic of our generation, with million people affected and , deaths worldwide so far. one of the risk factors associated with covid- is secondary bacterial pneumonia. in recent studies on covid- patients, secondary bacterial infections were significantly associated with worse outcomes and death despite antimicrobial therapies. in the past, the intensive use of antibiotics during the severe acute respiratory syndrome coronavirus (sars-cov) pandemic led to increases in the prevalence of multidrug-resistant bacteria. the rising number of antibiotic-resistant bacteria and our decreasing capacity to eradicate them not only render us more vulnerable to bacterial infections but also weaken us during viral pandemics. the covid- pandemic reminds us of the great health challenges we are facing, especially regarding antibiotic-resistant bacteria. t he outbreak of severe acute respiratory syndrome coronavirus (sars-cov- ), which causes coronavirus disease (covid- ), is the greatest pandemic of our generation, with million people infected and , deaths worldwide so far ( ) . one of the great mysteries in this pandemic is why some people become critically ill while others exhibit relatively mild symptoms, even when the patients share similar risk factors. it is becoming apparent that secondary bacterial infections occur in many covid- patients and can be associated with worse outcomes. in a multicenter study that included covid- patients, secondary bacterial infections were significantly associated with outcome severity ( ) . in that study, patients were divided into groups (moderately ill, severely ill, and critically ill). the critically ill patients had the highest percentage of bacterial coinfection ( . %) compared to patients in the moderately ill and severely ill groups ( . % and . %, respectively) ( ). more concerning, this higher rate of coinfections in critical patients happened although the majority of them ( . %) received antibiotic treatments compared to . % and . % in the moderately ill and severely ill groups. zhou and colleagues ( ) also found that among covid- patients, bacterial coinfections occurred in % of all cases, including % of nonsurvivors, even though % of patients received antibiotics. even more troubling, / covid- patients with coinfections succumbed ( ) . in both studies, other comorbidities were also associated with mortality; thus, it is difficult to determine the exact impact of coinfections. a third study ( ) used real-time pcr to detect specific pathogens causing covid- coinfections. they found that ( . %) patients were coinfected with at least of different pathogens. bacterial coinfections were predominant ( . %) over viral ( . %) and fungal ( . ) infections. although the authors found no significant association between coinfection rates and outcome severity or mortality, they described interesting coinfection patterns in different clinical groups (asymptom-atic and mildly, moderately, and severely/critically ill). for instance, streptococcus pneumoniae, klebsiella pneumoniae, haemophilus influenzae, escherichia coli, staphylococcus aureus, aspergillus, and epstein-barr (eb) virus were detected in all four clinical groups, while pseudomonas aeruginosa, human adenovirus, human rhinovirus, and herpes simplex virus were detected only in symptomatic patients regardless of disease severity. interestingly, coinfections with influenza a virus, influenza b virus, or coronavirus were not common in these covid- patients, though samples were collected during the flu season ( ). altogether, these early data suggest that the specific coinfecting pathogens may worsen disease prognosis and warrant further investigation. while it is unclear whether coinfections definitively worsen covid- patient outcomes, historical data from pandemics and seasonal flu suggest that bacterial coinfections can worsen viral diseases ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . during the first sars-cov outbreak in , up to % of patients were diagnosed with secondary bacterial infections and coinfection was positively associated with disease severity ( , ) . bacterial coinfections are also present during regular influenza seasons in % to % of cases and are associated with morbidity and mortality ( ) ( ) ( ) we are using more antibiotics in our fight to save covid- patients from bacterial coinfections, and it is important to consider how this could affect the prevalence of antibiotic-resistant bacteria globally. during the first sars-cov outbreak, analyses of isolates collected from patients in the intensive care unit (icu) in prince of wales hospital (hong kong) from march to may showed that rates of methicillinresistant s. aureus acquisition drastically increased during the outbreak from . % pre-sars to . % during the sars outbreak, despite extensive infection control precautions ( ) . other pathogens were found in postmortem lung specimens of patients from hong kong and singapore, including s. aureus, p. aeruginosa, klebsiella spp., and s. pneumoniae, all of which are well known for their high resistance to a broad spectrum of drugs ( , ) . it is not clear whether the covid- outbreak will lead to increased rates of antibiotic-resistant bacteria since the use of antibiotics does not always result in increased rates of drug-resistant strains ( ) , yet it will be important to continue monitoring rates of antibiotic-resistant bacterial infections. these data from the current covid- pandemic, previous pandemics, and seasonal influenza raise important questions that need to be investigated. first, are there synergic interactions between the sars-cov- virus and certain coinfecting bacteria? second, does coinfection with antibiotic-resistant bacteria affect disease severity? indeed, some of the pathogens detected in covid- patients can be antibiotic resistant, which could reduce the efficacy of treatments administered to patients. unfortunately, in the first two studies, where coinfections were associated with worse outcomes ( , ), the specific coinfecting pathogens detected were not described and no studies thus far have analyzed rates of coinfection by antibiotic-resistant bacteria. thus, it is impossible to determine from the available data whether certain bacterial species or whether antibiotic-resistant strains correlate with outcome severity or mortality. however, the presence of antibiotic-resistant bacteria could potentially explain the high rates of bacterial coinfections in critically ill patients despite extensive antibiotic treatments in these cohorts. finally, the battle with covid- may accelerate the worsening of our already dire situation with respect to antibiotic-resistant pathogens. the rising number of multidrug-resistant bacteria and our decreasing capacity to eradicate them not only render us more vulnerable to bacterial infections but also weaken us during viral pandemics. to tackle this serious issue, we urgently need to investigate the effects of bacterial coinfections during viral infections and find new antimicrobial compounds to eradicate multidrug-resistant pathogens. who. . covid- situation reports covid- with different severities: a multi-center study of clinical features clinical course and risk factors for mortality of adult inpatients with covid- in wuhan, china: a retrospective cohort study co-infection with respiratory pathogens among covid- cases risk of ruling out severe acute respiratory syndrome by ruling in another diagnosis: variable incidence of atypical bacteria coinfection based on diagnostic assays a major outbreak of severe acute respiratory syndrome in hong kong bacterial coinfection in influenza: a grand rounds review the frequency of influenza and bacterial coinfection: a systematic review and meta᎑analysis influenza-associated pediatric mortality in the united states: increase of staphylococcus aureus coinfection increase in methicillin-resistant staphylococcus aureus acquisition rate and change in pathogen pattern associated with an outbreak of severe acute respiratory syndrome streptococcus pneumoniae coinfection is correlated with the severity of h n pandemic influenza factors associated with death in hospitalized pneumonia patients with h n influenza in shenyang pediatric acute lung injury and sepsis investigator's network and the national heart, lung, and blood institute ards clinical trials network lung pathology of severe acute respiratory syndrome (sars): a study of autopsy cases from singapore pulmonary pathological features in coronavirus associated severe acute respiratory syndrome (sars) correlation between antimicrobial consumption and incidence of health-careassociated infections due to methicillin-resistant staphylococcus aureus and vancomycin-resistant enterococci at a university hospital in taiwan from to our contributions were as follows: literature search, m.v.; writing, m.v. and p.j. we declare no competing interests. p.j. was supported by grants from the nih (k ai and ul tr ) and the cystic fibrosis foundation (jorth f , promise, jorth i , jorth p ). key: cord- -wxiazglk authors: li, ji lian; cornman, r. scott; evans, jay d.; pettis, jeffery s.; zhao, yan; murphy, charles; peng, wen jun; wu, jie; hamilton, michele; boncristiani, humberto f.; zhou, liang; hammond, john; chen, yan ping title: systemic spread and propagation of a plant-pathogenic virus in european honeybees, apis mellifera date: - - journal: mbio doi: . /mbio. - sha: doc_id: cord_uid: wxiazglk emerging and reemerging diseases that result from pathogen host shifts are a threat to the health of humans and their domesticates. rna viruses have extremely high mutation rates and thus represent a significant source of these infectious diseases. in the present study, we showed that a plant-pathogenic rna virus, tobacco ringspot virus (trsv), could replicate and produce virions in honeybees, apis mellifera, resulting in infections that were found throughout the entire body. additionally, we showed that trsv-infected individuals were continually present in some monitored colonies. while intracellular life cycle, species-level genetic variation, and pathogenesis of the virus in honeybee hosts remain to be determined, the increasing prevalence of trsv in conjunction with other bee viruses from spring toward winter in infected colonies was associated with gradual decline of host populations and winter colony collapse, suggesting the negative impact of the virus on colony survival. furthermore, we showed that trsv was also found in ectoparasitic varroa mites that feed on bee hemolymph, but in those instances the virus was restricted to the gastric cecum of varroa mites, suggesting that varroa mites may facilitate the spread of trsv in bees but do not experience systemic invasion. finally, our phylogenetic analysis revealed that trsv isolates from bees, bee pollen, and varroa mites clustered together, forming a monophyletic clade. the tree topology indicated that the trsvs from arthropod hosts shared a common ancestor with those from plant hosts and subsequently evolved as a distinct lineage after transkingdom host alteration. this study represents a unique example of viruses with host ranges spanning both the plant and animal kingdoms. virus-negative colonies consumed virus-contaminated foods. this discovery raised concerns about a possible role of pollen in spreading viruses and suggested that viruses could possibly contribute to the observed pollinator decline around the world. in order to advance our understanding of the role of pollen in virus transmission of honeybees, we carried out a study to screen bees and pollen loads of bee colonies for the presence of frequent and rare viruses. our study resulted in the serendipitous detection of a plant virus, tobacco ringspot virus (trsv), in honeybees and prompted us to investigate whether this plant-infecting virus could cause systemic infection in exposed honeybees. generally, the majority of plant viruses are dependent upon herbivorous insects for their spread from one host plant to another in nature but cause infection only in plants that the insect vectors feed upon. to date, only a few plant viruses are known that also infect their insect vectors. rhabdoviridae, a family of arboviruses carried by arthropods, has long been recognized to have a broad range of hosts throughout the animal and plant kingdoms ( ) . flock house virus (fhv), a positive-stranded rna virus of insect origin belonging to the family nodaviridae, has been shown to replicate in plants as well as in yeast (saccharomyces cerevisiae) and mammalian cells ( , ) . a recent study ( ) showed that a plant-pathogenic virus, tomato spotted wilt virus (tswv), which is a member of the family bunyaviridae, could directly alter the behavior of thrips that vector it. the phenomenon of viral host range spanning the plant and animal kingdoms adds an additional layer to the already complex plant-pathogen-pollinator interactions and could have important epidemiological consequences. trsv is a type species of the genus nepovirus within the family secoviridae ( ) . trsv infects a wide range of herbaceous crops and woody plants, some of considerable economic importance. the infected plants show discoloration, malformation, and stunted growth, accompanied by reduced seed yield or almost total seed loss due to flower and pod abortion. of a number of plant diseases caused by trsv, bud blight disease of soybean (glycine max l.) is the most severe. it is characterized by necrotic ring spots on the foliage, curving of the terminal bud, and rapid wilting and eventual death of the entire plant, resulting in a yield loss of to % ( ) . like other members of the genus, trsv has a bipartite genome of positive-sense, single-stranded polyadenylated rna molecules, rna- and rna- , which are encapsidated in separate virions of similar size. both rna molecules possess a genome-linked protein (vpg) covalently bound at their = ends. rna- encodes a large polyprotein precursor that is proteolytically processed into protease cofactor (p a), putative atpdependent helicase (hel), picornain c-like protease (pro), and rna-directed rna polymerase (pol). rna encodes a virion capsid protein (cp), a putative movement protein (mp), and an n-terminal domain involved in rna- replication (p a). proteins encoded by rna- are required for rna replication, while proteins encoded by rna- function in cell-to-cell movement and viral rna encapsulation. rna- is capable of replication independently of rna- , but both are required for systemic infection. transmission of trsv can occur in several ways. the numerous vectors include a dagger nematode ( ) , aphids, thrips, grasshoppers, and tobacco flea beetle ( ) ( ) ( ) ; however, vertical transmission through seeds is important for long-distance dispersal of the virus ( ) . it has also been shown that honeybees transmit trsv when they move between flowers and transfer virusborne pollen from infected plants to healthy ones ( ) ( ) ( ) ( ) . it was, however, unknown prior to our study whether honeybees could become infected by plant viruses they physically encounter or consume. in the present study, we provide evidence that trsv is present in honeybees and the infection can be widespread through the body of honeybees. trsv in honeybees does not fit a circulativepropagative model of insect-vectored plant viruses, in which virions are ingested by an insect vector, replicate, and disperse to salivary glands for reinfection of the plant host. instead, our data indicate that the replication of trsv occurs widely in the honeybee body but not in the gut or salivary gland and that trsv in conjunction with other bee viruses is correlated with winter colony level declines. further, virus was found in a common ectoparasite mite of honeybees, varroa destructor, but was restricted to the gastric cecum. this study presents a unique example of viruses that cause infection in both plants and animals. sequence identity of trsv genomic segments and morphology of the virus isolates. sequence analysis of cdna libraries from purified virus preparation revealed overlapping and nonoverlapping clones of different lengths. about % of the clones (n ϭ ) matched the genome sequences of common honeybee viruses, including bqcv, dwv, and israeli acute paralysis virus (iapv). unexpectedly, about % of the clones (n ϭ ) matched the sequences of trsv for two genomic segments in the ncbi database. by assembling sequence fragments from different cdna clones, we obtained a , -bp length of nucleotide sequences encoding the rna helicase and covering~ % of the coding region of the polyprotein gene of genomic rna- . we also obtained a , -bp long sequence encoding the complete capsid protein. a blast search of the helicase sequence showed highest identity with a trsv strain isolated from bud blight disease of soybean (genbank accession no. u ), with % homology at the nucleotide level and % homology at the amino acid level. a blast search of the dna fragment encoding the capsid protein showed strongest similarity to a trsv strain from bean (genbank accession no. l ), with % homology at the nucleotide level and % homology at the amino acid level. the cdna sequences were used to design two primer sets, trsv-f /r and trsv-f /r (fig. ) , for the subsequent studies of trsv replication and distribution in honeybees and varroa mites. electron microscopy showed no obvious contamination from host cellular material. negatively stained viral particles had a diameter of to nm and an icosahedral shape, typical morphological features of secoviruses (fig. ) , and rt-pcr assay confirmed the presence of trsv in the viral preparation for em analysis. the purity of the virus preparation in our study was confirmed by electron microscopy. electron microscopy showed no obvious contamination from host cellular material. negatively stained viral particles had a diameter of to nm and an icosahedral shape, typical morphological features of secoviruses (fig. ) . however, the viral preparation was determined by rt-pcr to contain not only trsv but other bee viruses as well, including bqcv, dwv, and iapv. it was not possible to definitely distinguish trsv viral particles morphologically from these other bee viruses. distribution and replication of trsv in infected honeybees. although no apparent disease symptoms were observed in examined bees, trsv was widespread in honeybee tissues, which was confirmed by the amplification of a -bp pcr fragment with the trsv-f /r primer set. except for the compound eyes, trsv was found in all tissues examined, including hemolymph, wings, legs, antennae, brain, fat bodies, salivary gland, gut, nerves, tracheae, and hypopharyngeal gland. although there was the same amount of input cdna, the intensity of the pcr signals varied between samples. tissues of the gut and muscle had weaker pcr bands than other tissues, indicating a relatively lower level of trsv infection (fig. ) . it is unclear if the absence of pcr amplification in the compound eye was due to pcr inhibition previously reported for that tissue ( ) . trsv is a positive-stranded rna virus replicating through the production of a negative-stranded intermediate; therefore, the presence of negative-stranded rna constitutes proof of active viral replication. to investigate the replication of trsv in bees, negative-stranded rt-qpcr was performed using a tagged primer system ( ) . amplification and sequence analysis of a -bp negative-strand-specific product in different tissues showed that active replication of trsv occurs in most tissues (fig. ) . a single peak on the melting curve analysis corroborated the specificity of the amplicon. the lack of amplification following rt-qpcr of total rna without primers in the reverse transcription reaction mixture ruled out any nonspecific effect from self priming due to the secondary structure of viral rna or false priming by antigenomic viral rna or cellular rnas. among tissues with detectable levels, the relative abundance of negative-stranded trsv varied significantly (p Ͻ . ; one-way analysis of variance [anova]). the brain had the lowest detectable level of negativestranded trsv and was chosen as the calibrator. the abundance of trsv in other tissues relative to the brain ranged from -fold to -fold. the concentration of trsv in additional body tissues showed the following ranking: muscle Ͼ hypopharyngeal gland Ͼ leg Ͼ fat body Ͼ trachea Ͼ hemolymph Ͼ antenna Ͼ nerve Ͼ wing. the replication of trsv was not evident in the salivary gland, gut or compound eye (fig. ) , although the presence of pcr inhibitors in the latter is a possibility ( ) . localization of trsv in the ectoparasitic varroa mite of honeybees. in situ hybridization showed that trsv could also be detected in the ectoparasitic mite, v. destructor, collected from the same trsv-infected bee colonies. sections hybridized with a digoxigenin (dig)-labeled trsv rna probe had strong staining within the storage organs of the mite, the upper and lower gastric ceca (fig. a ), although histopathological signs were not evident in these areas. no positive signal of trsv was observed in other mite tissues, and no signal was observed with the negative-control probe (fig. b) . prevalence of trsv infection in honeybee colonies. of ten bee colonies included in this study, six were classified as described in materials and methods as strong colonies and four were classified as weak colonies. both trsv and iapv were absent in bees from strong colonies in any month, but both were found in bees from weak colonies. as with other detected viruses, trsv showed a significant seasonality. the infection rate of trsv increased from spring ( %) to summer ( . %) and autumn ( . %) and peaked in winter ( . %) before colony collapse. of viruses detected in weak colonies, dwv was the most commonly detected, with an average annual infection rate of %, followed by bqcv, iapv, and trsv. additionally, a low incidence of sbv and chronic bee paralysis virus (cbpv) infections was also detected in bees from weak colonies. while dwv and bqcv were detected in both healthy and weak colonies all year round, the prevalence of dwv and bqcv in weak colonies was significantly higher than that in strong colonies. the bee populations in weak colonies that had a high level of multiple virus infections began falling rapidly in late fall. all colonies that were classified as strong in this study survived through the cold winter months, while weak colonies perished before february. in fig. a and b, the seasonal prevalence of trsv along with other bee viruses in both weak and strong colonies is presented. phylogenetic characterization of trsv isolates. figure illustrates the phylogenetic relationship among our trsv isolates and viruses with existing genbank trsv sequence records, based on the partial capsid protein sequence amplified with primers. trsv isolates infecting plants constitute the early lineages of the phylogenetic tree, and trsv isolates from honeybees, bee pollen, and varroa mites clustered together, branching next from the early lineage. there is no obvious sequence divergence among trsv isolates from bees, mites, and bee pollen. among major pathogen groups, rna viruses have the highest rate of mutation, because the virus-encoded rna polymerases lack =¡ = exonuclease proofreading activity ( ) . the consequence of such high mutation rates is that populations of rna viruses exist as "quasispecies," clouds of genetically related variants that might work cooperatively to determine pathological characteristics of the population ( ) . these sources of genetic diversity coupled with large population sizes facilitate the adaptation of rna viruses to new selective conditions, such as those imposed by a novel host. rna viruses therefore are the most likely source of emerging and reemerging infectious diseases, such as human immunodeficiency virus (hiv), severe acute respiratory syndrome (sars), type a avian influenza a (h n ), and swine origin influenza a (h n ), that have engendered worldwide public health concern because of their invasiveness and ability to spread among different species ( ) ( ) ( ) ( ) ( ) . honeybees carry a strong electrostatic charge that ensures the adherence of pollen to their bodies, and they also actively store pollen in specialized pollen baskets on their hind legs. it is therefore not unexpected that the foraging behavior of honeybees could move virus-contaminated pollen to the flowers of healthy plants ( , ) . however, this study represents the first evidence that honeybees exposed to virus-contaminated pollen could also be subsequently infected and that the infection could be systemic and spread throughout the entire body of honeybees. about % of known plant viruses are pollen transmitted, and the genomes of the majority of plant viruses are made of rna ( , ) , providing a large set of potential host-jumping viruses. the finding from this study illustrates the complexity of relationships between plant pathogens and the pollinating insects and emphasizes the need for surveillance for potential host-jumping events as an integrated part of insect pollinator conservation. for a virus to successfully establish infection in a novel host, the virus must overcome three major hurdles. first, it must have the opportunity to come into contact with a prospective host for the viral particles to gain entry into the host cells. second, the virus must undergo genetic changes that mediate the entry of virus into host cells, typically through host receptors on the cell surface. the virus must also undergo genetic changes that can lead to the ability to bypass the host's immune defense and replicate its genome using the host's cellular machinery. finally, the virus must gain the ability to spread horizontally between individuals of the same gen- food-borne transmission is one of the most important routes for virus transmission in honeybees. infections of several honeybee viruses occur through ingestion of virus-contaminated food followed by dissemination of the viruses from the midgut into other tissues through the hemolymph ( ) . since trsv is a known pollen-borne plant virus, we initially believed that the presence of trsv was restricted to the bees' digestive tract. however, titers of trsv in our study were unexpectedly low in the gut. viral replication was not detected in either the gut or the salivary gland. instead, high titers of negative-stranded virus were found in the wing, nerve, antenna, trachea, hemolymph, and fat body, indicating replication in those tissues. the absence of virus replication in the tissues of the gut and salivary gland excludes the possibility of trsv as a persistent-propagative virus which must first replicate in epithelial cells of the midgut and then migrate to the salivary glands to be ejected together with saliva. our quantitative analysis suggests that trsv is neurotropic in honeybees, with more extensive infection detected in the nervous system than in other internal tissues, and therefore it is conceivable that severe trsv infection can cause functional impairment of the nerve and muscle in honeybees. the low levels of trsv in the gut suggests a possible result of sloughing off of infected epithelial cells from midgut as a host defensive mechanism or the possibility that trsv might utilize some alternative invasion routes such as the neural or tracheal route. further investigation of the virus transmission and pathogenicity is warranted. the circulation of trsv in bee hemolymph was further proven by the presence of trsv in varroa mites. varroa is an obligate parasite of the honeybee and has been catastrophic for the beekeeping industry. both adult mites and nymphs use their piercing mouth parts to penetrate the body wall of the bees and suck out the hemolymph. in addition to its direct detrimental effects on host life span and colony vigor ( ) ( ) ( ) ( ) ( ) ( ) , the feeding of mites on bees provides an entry for microbial pathogens ( ) . indeed, the roles played by varroa mites in acquiring and transmitting honeybee viruses have been experimentally demonstrated in several studies ( ) ( ) ( ) ( ) . the observation of the positive signal of trsv within the storage organ of the mites suggests that the varroa mite is not merely a mechanical vector that physically transports viruses from host to host with its mouthparts. more work is needed to confirm whether varroa mites can act as a biological vector to support trsv replication. trsv isolates from honeybees, varroa mites, and bee pollen clustered together phylogenetically, indicating that they descended from a common ancestor. it is likely that varroa mites obtained the virus from their hosts during the blood feeding and that the virus-infected bees contaminated the bee pollen when they mix plant pollen with their glandular secretions and honey to produce "bee bread." the finding that trsv isolates from honeybees appeared to be derived more recently on the evolutionary timeline than trsv from plants suggests that life cycles of the virus involving arthropod hosts evolved after host expansion. however, it remains to be determined whether trsv possesses the ability to maintain persistent infection in honeybee colonies in the absence of newly inoculated viruses from visited plants or whether infected bees can subsequently inoculate healthy plants. it will similarly be helpful to screen other pollinator species for the presence of trsv, since it is known that honeybees and other pollinators share some viral species ( ) ( ) ( ) ( ) ) . sequence comparison of the trsv isolates from this study with isolates with other accession numbers suggests that the capsid protein region is much more conserved than the rna helicase region at the nucleotide level. the relatively high level of sequence similarity at the amino acid level for both capsid protein and helicase indicates a high level of structural and functional conservation. nevertheless, substitution of a single or a few amino acids at the surface of virus particles can be sufficient to alter receptor recognition and thereby alter host range ( ) . thus, the few amino acid polymorphisms observed in trsv strains infecting honeybees may still be associated with cell tropism and host adaptation. it would be helpful to further characterize the complete genome of trsv isolates from honeybees as well as from varroa mites to deepen our understanding of genetic diversity of this virus. more work is needed to elucidate the molecular basis of cell tropism and host range modifications and to investigate the roles of the honeybee as a newly identified host in the epidemiology of trsv. the evidence of systemic spread and propagation of a plantpathogenic virus in honeybees raises awareness of the potential impact of new viral disease emergence on bee health. while findings from this study have important implications for understanding trsv transmission and pathogenesis, much remains to be learned about the intracellular life cycle, species-level genetic variation, and pathogenesis of the virus in honeybee hosts. although the cause(s) of ccd and the decline in the worldwide bee population is not yet fully understood ( ), a growing body of evidence has indicated that parasites and pathogens are key culprits involved in widespread disappearance/death and population declines of honeybees ( , , ( ) ( ) ( ) ( ) ( ) . the observation that increasing prevalence of trsv in conjunction with other bee viruses in infected colonies is associated with gradual decline of host populations and winter colony collapse supports the argument that virus infections could have a significant negative impact on colony survival. while the simultaneous presence of multiple viruses and asymptomatic viral infections in honeybees as well as lack of a cell culture system for virus production ( , ) makes koch's postulates of disease causality difficult to fulfill, the observed negative correlation between the level of trsv infections and size of host populations suggests that trsv, in combination with other viruses, is likely a contributing factor to poor survivorship of honeybee colonies. honeybee colonies and sample collection. honeybee colonies used for this study were maintained in the research apiaries of the usda-ars bee research laboratory in beltsville, md. for viral particle purification and tissue dissection, fifty adult worker bees were collected by removing a central frame filled with brood and covered with adult bees from a hive and gently scraping worker bees into a -ml conical tube. in addition, bee pollen that was processed by bees and stored in combs around the brood was collected using a spatula and transferred into -ml conical tubes. individual varroa mites that had crawled from brood cells onto the tops of brood frames were collected with forceps and transferred into . -ml microcentrifuge tubes. for assessing the effects of viruses on honeybees, the seasonal prevalence of virus infections was determined in ten colonies for a period of year starting in march and finishing in february of the following year. bee colonies were classified as strong or weak based on the size of adult populations, amount of sealed brood, and presence of food stores, as previously described ( ) . bee colonies that had more than ten frames covered with adult workers and more than six frames filled with brood and food stores were defined as strong colonies, while bee colonies that had a small number of foraging bees flying in and out, fewer than ten frames of adult bees, fewer than six combs with brood, and small patches of food stores were defined as weak colonies. for each colony, samples of adult workers were collected every month and stored at Ϫ °c until subsequent rna isolation for virus analysis. virus purification and electron microscopy. thirty worker bees were frozen in liquid nitrogen, ground to a fine powder, and homogenized in -ml extraction buffer ( . m potassium phosphate buffer [ph . ], . % diethyldithiocarbamate, / volume of diethyl ether). the mixture was emulsified with ml carbon tetrachloride and centrifuged at , ϫ g at °c for min to remove tissue debris. supernatant containing viruses was centrifuged once more at , ϫ g at °c for min and then filtered through a -m filter to remove small tissue debris. the filtrate was then centrifuged at , ϫ g for h at °c to pellet the viral particles. the pellet was resuspended in ml of . m phosphate-buffered saline (pbs) buffer. a -l portion of viral solution was examined for the presence of virus particles in an electron microscope. the rest of the viral solution was saved for subsequent viral rna isolation and cdna library construction. virus particles were negatively stained with % uranyl acetate on a formvar-coated ni grid and viewed in a hitachi h- electron microscope at magnifications between ϫ , and ϫ , . cdna library construction and virus-specific primer design. total rna was extracted by homogenizing the viral solution with trizol ls reagent (invitrogen), a solution of phenol and guanidine isothiocyanate used for isolating total rna from liquid samples according to the manufacturer's instructions. the resultant rna pellets were resuspended in dnase-and rnase-free water (invitrogen) in the presence of ribonuclease inhibitor (invitrogen). the quantity and purity of rna were measured with a nanodrop spectrophotometer (nanodrop technologies). the cdna library was constructed using a cloneminer cdna library construction kit (invitrogen) per the manufacturer's protocol. first-strand cdna was synthesized from extracted rna using superscript ii reverse transcriptase with a biotin-conjugated attb oligo(dt) primer. after cdna synthesis, the products were size fractionated by column chromatography to remove excess primers, adapters, and small cdnas and cloned into an attp-containing donor vector, pdonr . the bp (recombination between attb and attp sites) reaction products were transformed into electromax dh b t phage-resistant cells, and the transformed cells were plated onto lb agar medium supplemented with kanamycin ( g/ml). the positive clones were purified using the wizard plus miniprep dna purification system (promega). a total of cdna clones were randomly selected and sequence analyzed to confirm the presence of the insert. primers specific for trsv rna segments and were designed based on the nucleotide sequences obtained from cdna clones of this study. the sequences of primers for amplifying a -bp region of helicase (hel) of rna segment were trsv-f ( =-catgaatgttgttatccaat- =) and trsv-r ( =-tcctcagtaaatttcatttg- =). the sequences of primers for amplifying a -bp region of capsid protein (cp) region of rna segment were trsv-f ( =-gtgtgctgtgacggttgttcc- ') and trsv-r ( =-tgccagaccacccaagattcc- =). figure illustrates the positions of primers. bee tissue dissection. twenty adult worker bees were individually fixed on the wax top of a dissecting dish with steel insect pins. under a dissecting microscope, about l of hemolymph was collected from each bee with a micropipette tip by making a small hole on the roof of the bee's thorax with a needle to make it bleed. following hemolymph collection, the legs, wings, antennae, and compound eyes were cut off with a pair of fine scissors. the body was opened by cutting along the dorsal midline from the tip of the abdomen to the head with scissors. tissues of the brain, fat body, salivary gland, gut, muscle, nerve, trachea, and hypopharyngeal gland were individually removed using a pair of fine forceps under a dissecting microscope. in total, thirteen tissues were collected from each bee, and a total of thirty bees were dissected. the scissors and forceps were wiped between tissues once with a cotton pad soaked with % bleach and once with a cotton pad soaked with % alcohol followed by a final rinse in sterile water. to prevent possible contamination with hemolymph, all tissues were rinsed once in ϫ phosphate-buffered saline (pbs) and twice in nuclease-free water. the washing solution was changed every time for each tissue to prevent cross-contamination. all freshly dissected tissues were subjected to subsequent rna extraction immediately. total rna extraction and conventional rt-pcr. total rna was isolated from disserted tissues, adult bees, bee pollen, and varroa mites using invitrogen trizol reagent according to the manufacturer's instructions. conventional rt-pcr was performed on rna samples extracted from adult bees, varroa mites, different tissues, and bee bread collected from the same colony for the presence and distribution of trsv. the promega one-step access rt-pcr system was used for virus detection as previously described ( ) . pcr products were purified and sequenced to confirm the specificity of the primers. to determine the seasonal prevalence of trsv in honeybee colonies, bee samples collected every month were subject to rt-pcr analysis individually for trsv as well as other seven common honeybee viruses, including acute bee paralysis virus (abpv), bqcv, chronic bee paralysis virus (cbpv), dwv, israeli acute paralysis virus (iapv), kashmir bee virus (kbv), and sbv. the primer pair trsv-f /trsv-r was used for rt-pcr amplification of trsv. the primer sets used for rt-pcr amplification of common honeybee viruses have been reported previously ( , ) . putative trsv amplification products were purified and sequenced to confirm the specificity of the rt-pcr assay. the infection rate of each virus ( workers) and strength of individual colonies were recorded every month throughout the year. strand-specific rt-qpcr. in order to determine the ability of trsv to replicate in different tissues of honeybees, rna samples were further analyzed for the presence and abundance of negative-stranded rna, a replicative intermediate, using strand-specific reverse transcription coupled with quantitative pcr (rt-qpcr). for each tissue sample, the firststrand cdna was synthesized from total rna using superscript iii reverse transcriptase (invitrogen) with tag-trsv-f ( =-agcctgcgca cgtggcatgaatgttgttatccaat- =), where the capitalized sequence corresponding to tag was published by yue and genersch ( ) . the synthesized cdnas were then purified using a minelute pcr purification kit (qiagen) followed by a minelute reaction clean kit (qiagen) to remove short fragments of oligonucleotides and residue of enzymatic reagents to prevent amplification of non-strand-specific products ( ) . cdna derived from negative-stranded rna was amplified using the brilliant sybr green qpcr master mix (stratagene) with a . m concentration each of the tag ( =-agcctgcgcaccgtgg- =) and trsv-r primers in a -l volume according to the manufacturer's protocol. to normalize the qpcr result, amplification of a housekeeping gene, the ␤-actin gene, was performed for each sample with a previously reported primer set ( ) . the amplification for both trsv and ␤-actin was carried out following the manufacturer's recommended protocol for thermal profile parameters for three-step pcr. after amplification, a melting curve analysis was performed to determine the specificity of the pcr products. each sample was run in triplicate, and the qpcr assay was repeated twice. the amplification efficiencies of the sybr green real-time rt-qpcr assay for both trsv and ␤-actin were proved to be approximately equal (data not shown). the output of rt-qpcr assays for trsv in different tissues was interpreted by using the comparative cycle threshold method (⌬⌬c t method). the average c t value (⌬c t ) of trsv in each tissue was normalized using the c t value corresponding to the endogenous control, ␤-actin, with the following formula: ⌬c t ϭ average c t (trsv) Ϫ average c t (␤actin). the tissue that had the lowest level of trsv was chosen as a calibrator. the ⌬c t value of each tissue was subtracted from the ⌬c t value of the calibrator to yield ⌬⌬c t . the concentration of trsv in each tissue was calculated using the formula Ϫ⌬⌬ct and expressed as n-fold difference relative to the calibrator. in situ hybridization. purified amplicons corresponding to the region flanked by the trsv-f and trsv-r primer set were incorporated into a pcr . ta cloning vector upstream of a t promoter (invitrogen, carlsbad, ca) following the manufacturer's protocol. recombinant plasmid dnas with the trsv insert were linearized by restriction enzyme bamhi (new england biolabs, ipswich, ma) at °c for h. the linearized dnas were extracted once with an equal volume of phenolchloroform-isoamyl alcohol ( : : ), precipitated by ethanol, and dissolved in nuclease water. the dig-labeled rna probe complementary to trsv genomic rna was synthesized using a dig-rna labeling kit (t ) (roche applied science, indianapolis, in) following the manufacturer's protocol. live varroa mites were fixed in % paraformaldehyde in mm pbs (ph . ) overnight at °c, rinsed in nuclease-free water three times, and then stored in % ethanol ( proof) at °c until used. tissue dehydration was carried out by successive incubations in ethanol ( %, %, and %) and xylol (twice for min each) and embedded in paraffin. paraffin sections were cut to micrometers thick and mounted on poly-llysinated slides and stored at °c overnight. the sections were then rehydrated through a descending concentration of ethanol ( %, %, and %), dewaxed in xylol, treated with proteinase k ( g/ml) for min, and acetylated with . % (vol/vol) acetic anhydride in . m triethanolamine-hcl (ph . ) for min prior to hybridization. the sections were prehybridized in prehybridization solution ( % formamide, ϫ ssc [ ϫ ssc is . m nacl plus . m sodium citrate], g/ml salmon sperm) at °c for h and incubated in hybridization buffer with dig-labeled trsv probe solution to a concentration of to ng/ml probe in prehybridization solution at °c overnight. after hybridization, the sections were washed twice in low-stringency wash solution ( ϫ ssc, . % sds) at room temperature for min and washed twice in high-stringency wash solution ( . ϫ ssc, . % sds) at °c for min. the hybridization signals were detected with alkaline phosphatase (ap)-labeled sheep anti-dig antibody conjugate (roche applied science). the conjugate solution was added to the dry sections and incubated at °c for h in a humid chamber. the slides were rinsed three times with washing buffers. the color development was performed by adding the buffer solution containing nitroblue tetrazolium (nbt) and -bromo- -chloro- -indolyl phosphate (bcip) to the tissue sections and incubating for to h at room temperature with protection from light. the color reaction was stopped by a -min wash in tris-edta ( . mm, ph . ). the nonspecific staining was removed in % ethanol overnight. the sections were rehydrated through successive incubation in ethanol ( %, %, and %) and xylol (twice for min each) and mounted in eukitt resin. negative control reactions included regular dutp instead of dig-labeled trsv probe. in situ hybridization slides were observed under a light microscope (eclipse te ; nikon) and photographed with a nikon digital camera (dxm ). dark blue coloring indicates where the dig-labeled probe bound directly to the viral rna. the section hybridized with the negative control showed pink staining only from the application of nuclear fast red. phylogenetic analysis. the sequences of the -bp trsv fragment amplified from the region encoding the capsid protein by the primer pair trsv-f and trsv-r from honeybees, bee pollen, and varroa mites were compared with existing genbank sequences isolated from plants. phylogenetic analysis was conducted in mega ( ) . the sequences were aligned using clustalw, and the sequences that could not be aligned unambiguously at both = and = ends were truncated. a tree was built using the neighbor-joining method ( ) with distances computed using the maximum composite likelihood method ( ) . the reliability of the phylogenies was assessed by bootstrap replication ( replicates) ( ) . node labels correspond to bootstrap support, and values of Ͼ % were regarded as evidence for the phylogenetic grouping. nucleotide sequence accession numbers. the cdna sequence data have been submitted to the genbank sequence database and assigned the accession numbers jq and jq for the helicase and capsid protein coding regions, respectively. the 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interactions with insect and plant hosts genomic rna of an insect virus directs synthesis of infectious virions in plants systemic spread of an rna insect virus in plants expressing plant viral movement protein genes infection with a plant virus modifies vector feeding behavior secoviridae. in virus taxonomy: ninth report of the international committee on taxonomy of viruses tobacco ringspot virus tobacco ringspot virus transmission, movement, and vector relationships of tobacco ringspot virus in soybean transmission of tobacco ringspot virus by xiphinema americanum to a wide range of hosts the grasshopper as a vector of tobacco ringspot virus in soybean effects of tobacco-and tomato ringspot viruses on the reproduction tissues of pelargonium xhortorum transmission and the role of honey bees in field spread of blueberry shock ilarvirus, a pollen-borne virus of highbush blueberry plant pathogens transmitted by pollen bee-mediated transmission of blueberry leaf mottle virus via infected pollen in highbush blueberry the possible role of honey bees in long-distance spread of prunus necrotic ringspot virus from california into washington sweet cherry orchards scientific note on pcr inhibitors in the compound eyes of honey bee molecular approaches to the analysis of deformed wing virus replication and pathogenesis in the honey bee, apis mellifera mutation rates among rna viruses quasispecies theory and the behavior of rna viruses isolation and characterization of viruses related to the sars coronavirus from animals in southern china aids as a zoonosis: scientific and public health implications emergence of influenza a viruses characterization of an avian influenza a (h n ) virus isolated from a child with a fatal respiratory illness population biology of emerging and re-emerging pathogens pollen and seed-transmitted viruses and viroids iruses of plants: descriptions and lists from the vide database introduction to plant viruses, the invisible foe honey bee viruses weight loss and other damage to developing worker honey bees from infestation with varroa jacobsoni varroa jacobsoni oud. in cold climates: population growth, winter mortality and influence on the survival of honey bee colonies life span of apis mellifera carnica pollm. infested by varroa jacobsoni oud. in relation to season and extent of infestation the influence of the mite varroa jacobsoni oud. on the protein concentration and hemolymph volume of the blood of the worker bees and drones of the honey bee, apis mellifera impact of an ectoparasite on the immunity and pathology of an invertebrate: evidence for host immunosuppression and viral amplification global honey bee viral landscape altered by a parasitic mite the transmission of deformed wing virus between honeybees (apis mellifera l.) by the ectoparasitic mite varroa jacobsoni oud molecular evidence for transmission of kashmir bee virus in honey bee colonies by ectoparasitic mite, varroa destructor the role of varroa mites in infections of kashmir bee virus (kbv) and deformed wing virus (dwv) in honey bees varroa destructor is an effective vector of israeli acute paralysis virus in the honeybee, apis mellifera new evidence that deformed wing virus and black queen cell virus are multihost pathogens evolution of cell recognition by viruses: a source of biological novelty with medical implications changes in transcript abundance relating to colony collapse disorder in honey bees (apis mellifera) colony collapse disorder: a descriptive study a historical review of managed honey bee populations in europe and the united states and the factors that may affect them weighing risk factors associated with bee colony collapse disorder by classification and regression tree analysis experimental infection of apis mellifera honeybees with nosema ceranae (microsporidia) how natural infection by nosema ceranae causes honeybee colony collapse detection of multiple viruses in queens of the honey bee apis mellifera l multiple virus infections in the honey bee and genome divergence of honey bee viruses dynamics of persistent and acute deformed wing virus infections in honey bees rt-pcr analysis of deformed wing virus in honeybees (apis mellifera) and mites (varroa destructor) quantitative real-time reverse transcription-pcr analysis of deformed wing virus infection in the honeybee mega : molecular evolutionary genetics analysis (mega) software version . the neighbor-joining method: a new method for reconstructing phylogenetic trees prospects for inferring very large phylogenies by using the neighbor-joining method confidence limits on phylogenies: an approach using the bootstrap we are grateful to gene robinson, nancy a. moran, and john burand for their comments and helpful suggestions on the manuscript.this research was supported in part by a usda-cap grant ( - - ).mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the u.s. department of agriculture. key: cord- -cd cmpoj authors: barzin, amir; schmitz, john l.; rosin, samuel; sirpal, rameet; almond, martha; robinette, carole; wells, samantha; hudgens, michael; olshan, andrew; deen, stephanie; krejci, patrick; quackenbush, eugenia; chronowski, kevin; cornaby, caleb; goins, janette; butler, linda; aucoin, julia; boyer, kim; faulk, janet; alston-johnson, devena; page, cristen; zhou, yijun; fiscus, lynne; damania, blossom; dittmer, dirk p.; peden, david b. title: sars-cov- seroprevalence among a southern u.s. population indicates limited asymptomatic spread under physical distancing measures date: - - journal: mbio doi: . /mbio. - sha: doc_id: cord_uid: cd cmpoj characterizing the asymptomatic spread of sars-cov- is important for understanding the covid- pandemic. this study was aimed at determining asymptomatic spread of sars-cov- in a suburban, southern u.s. population during a period of state restrictions and physical distancing mandates. this is one of the first published seroprevalence studies from north carolina and included multicenter, primary care, and emergency care facilities serving a low-density, suburban and rural population since description of the north carolina state index case introducing the sars-cov- respiratory pathogen to this population. to estimate point seroprevalence of sars-cov- among asymptomatic individuals over time, two cohort studies were examined. the first cohort study, named screennc, was comprised of outpatient clinics, and the second cohort study, named screennc , was comprised of inpatients unrelated to covid- . asymptomatic infection by sars-cov- (with no clinical symptoms) was examined using an emergency use authorization (eua)-approved antibody test (abbott) for the presence of sars-cov- igg. this assay as performed under clia had a reported specificity/sensitivity of %/ . %. screennc identified out of , ( . %) positive individuals among asymptomatic participants accessing health care during april to june , which was increasing over time. a separate cohort, screennc , sampled from march to june , identified out of , ( . %) positive participants. importance this study suggests limited but accelerating asymptomatic spread of sars-cov- . asymptomatic infections, like symptomatic infections, disproportionately affected vulnerable communities in this population, and seroprevalence was higher in african american participants than in white participants. the low, overall prevalence may reflect the success of shelter-in-place mandates at the time this study was performed and of maintaining effective physical distancing practices among suburban populations. under these public health measures and aggressive case finding, outbreak clusters did not spread into the general population. keywords covid- , sars-cov- , antibody, coronavirus, seroprevalence a cute virus infections typically induce a vigorous antibody response. the overwhelming majority of covid- patients also produce antibodies, initially of the igm isotype and shortly thereafter of the igg isotype. seroconversion rates approach % at weeks after symptoms appear and persist for several months ( ) ( ) ( ) ( ) . the composition, quality, median and maximal duration of the igg response are unclear as the earliest cases occurred less than months ago ( ) . both presymptomatic and asymptomatic transmission events of sars-cov- have been reported ( ) ( ) ( ) ( ) ( ) ( ) . it is difficult, however, to define asymptomatic transmission a priori, as the full spectrum of sars-cov- -associated clinical symptoms is still evolving ( , ) . the degree to which asymptomatic transmission contributes to population seroprevalence and eventual herd immunity is unknown and the subject of intense debate. determining the extent to which sars-cov- can be transmitted asymptomatically shapes covid- disease modeling and informs public health interventions. asymptomatic transmission represents a key biological property of this virus. early serology studies for sars-cov- were conducted in densely populated, urban settings, such as wuhan, china; bergamo, italy; or boston, new york, los angeles, or seattle in the united states ( , ) . these studies focused on patients who recovered after severe clinical disease ( ). they were conducted at hot spots of transmission, in urban settings with multiple independent introductory events and extended opportunities for rapid spread, such as multiple modes of public transport and international travel ( ) . north carolina (nc), in contrast, represents a largely suburban and rural population with limited public transport. the first case of covid- was reported on march . as in other locales, symptomatic cases were concentrated among vulnerable populations, such as residents of retirement homes, prison inmates, and workers in meatprocessing plants. "stay-at-home" directives were implemented march to may . statewide, all schools were ordered to close march and did not reopen during the study period. as of june, nc reported , cases and , deaths (nc department of health and human services [dhhs]) for a population of , , people ( census), based primarily on nucleic acid testing (nat) of suspected cases. thus, the fraction of infected persons based on case counts for this area and calendar time using viral nat is . %. this study evaluated two cohorts of asymptomatic patients. screennc enrolled , asymptomatic participants across the university of north carolina health system (unc health), representing different zip codes. these individuals sought medical care unrelated to covid- . individuals with known covid- symptoms, at screening or in their recent past, were excluded. screennc tested , sera from patients in clinical care not related to covid- or recruited to the study. no patient was tested more than once. screennc enrolled april to june , and screennc sampled patients march to june . table shows the demographic composition of the screennc cohort compared to patients accessing unc health sites during the same time period in as well as in , i.e., pre-covid- . table shows the age distribution. there were no differences between overall patient populations accessing unc health in compared to . when comparing the screennc population and patient populations accessing unc health in , there were some notable differences. screennc enrolled fewer ϩyear-old patients who accessed care during the same calendar period ( . % versus . %), slightly more females ( . % versus . %), fewer black or african american participants ( . % versus . %, p Յ . by fisher exact test), and fewer hispanic or latino participants than the comparator unc health population ( . % versus . %, p Յ . by fisher exact test) during the same calendar period. these differences were used in a model of adjusted seroprevalence. sars-cov- igg antibody was detected using the abbott sars-cov- igg assay on the architect platform under emergency use authorization (eua). index values of Ն . were considered positive. this assay has a manufacturer reported analytical sensitivity of % and specificity of . %. independent studies in the u.s. population report sensitivities/specificities of %/ . % ( ) , . %/ . % ( ), . %/ . % ( ) , and %/ . % ( ), respectively. on-site validation (n ϭ ) established a sensitivity of % and a specificity of . % at weeks after symptom onset. intra-assay precision (% coefficient of variation [cv%]) was . %, and interassay cv% was . %. thus, in-house performance was comparable to the manufacturer's specification and to other studies in the united states. screennc identified out of , ( . %) positive participants, and screennc identified out of , ( . %) positive participants. black or african american participants had twice the unadjusted seropositivity rate of whites ( . % versus . %). the analysis of other demographic factors was not possible due to the small number of positive cases. the unadjusted seroprevalence remained constant over time for the screennc population but showed an upward, although imprecise, trend for the larger screennc cohort ( fig. and table ). the counts were adjusted for assay characteristics using the method of rogan and gladen ( ) and cohort characteristics using direct standardization to yield a population this study identifies a very limited seroprevalence of sars-cov- among asymptomatic individuals accessing the unc health system. there was a suggestion of accelerating asymptomatic spread of sars-cov- during the study period and cohort, i.e., the transmission among persons who never subjectively felt ill. the seroprevalence of less than % was lower than estimates from earlier studies, but in line with recent studies using high-accuracy tests ( , ) . the seroprevalence for this low-density community was lower than reported in a large study of convenience samples that focused around "hot spot" cities and did not explicitly exclude persons with past symptomatic infections ( ) . this result may reflect the success of shelter-in-place state mandates and maintaining effective physical distancing among suburban populations during the time frame of our study. it may also reflect the low population density and delayed introduction of the virus compared to population centers and travel nodes. under these circumstances, early outbreak clusters did not expand far into asymptomatic patients, which indicates that continued viral nat and aggressive case finding can curb sars-cov- spread. the data suggest that members of the black community are disproportionately affected by the covid- epidemic, here as demonstrated by asymptomatic acquisition of infection and in other studies as demonstrated by increased incidence of symptomatic cases and deaths. significantly fewer members of the black and latinx communities participated in this study than accessed care in the same calendar time, which may have led to biased enrollment. there are many possible explanations for the variation in population seroprevalence among different preliminary reports and published studies ( , ) . our study populations were derived from individuals accessing health care clinics and may not provide a population-representative study sample. it is very challenging to conduct random population sampling during a public health emergency when "stay-at-home" orders are in place. prevalence of transmission, patterns of behavior, and characteristics of the sample population were time varying during the sample period. logistical constraints, such as lack of personal protective equipment and the primacy of clinical care, limited study access. in nc, only persons with a valid medical reason and "essential workers" were exempted from the "stay-at-home" ordinance, and only those participated in the study. the degree to which underlying health conditions impact analytical assay sensitivity is currently unknown; for instance, screennc included patients with immune suppression and thus likely impairments in their ability to generate high-titer antibodies. defining "asymptomatic" is a challenge and a limitation, as it is based on selfreported clinical symptoms of infection, the exact definition of which continues to evolve. both symptom severity and awareness may differ among different communities. third, there exists a wide variety of antibody tests, each with differing performance characteristics ( , ) . these serological tests are validated on symptomatic, hospitalized patients, not asymptomatic cases. this study employed an eua assay performed in a clia-certified laboratory on a venous blood sample, with demonstrated specificity to detect antibodies only to sars-cov- , not to seasonal coronaviruses. in general, a robust igg response to sars-cov- is correlated with the presence of neutralizing antibodies for coronaviruses, but exactly which level of response and which antigen is a reliable predictor for protection has not been established ( ) . this study only uses the presence of igg as a measure of exposure. the negative predictive value under any of the reported sensitivity/specificity characteristics is Ͼ . % for assumed population seroprevalence of Ͻ %. the positive predictive value is . % for an assumed seroprevalence of % and % for an assumed seroprevalence of %. this implies that perhaps even fewer individuals developed antibodies than reported. alternatively, it is possible that igg responses are lower in asymptomatic than symptomatic patients and decline over time ( ) . in conclusion, based on the data presented in this study, it is unlikely (i) that "herd immunity" can be reached in the foreseeable future and (ii) that asymptomatic spread contributed a substantial number of infections. taken together, these data suggest that early and rigorous shelter-in-place policies and physical distancing measures are important tools to suppress sars-cov- infection in the majority of the population. enrollment. patients enrolled in the study were recruited at nine outpatient clinical sites and two emergency department sites across the state associated with the unc health network. upon arrival for sars-cov- seroprevalence in north carolina ® routine care or scheduled visits for enrollment into the study, patients performed a consent procedure that included reviewing recent covid- clinical history using unc irb-approved questionnaires. once consent was obtained, patients provided ml of venous whole blood. this sample was collected by phlebotomists or nurses at each site and analyzed for the presence of sars-cov- antibody. results were reported in the unc medical record, and patients were notified of negative results via an electronic message. for those with a positive result, a physician was responsible for calling the patients and reviewing the results and answering follow-up questions. the screennc protocol was approved by the unc irb (unc irb - , nct ). screennc samples were sera obtained from unc patients for indications other than covid- . this protocol was also approved by the unc irb, which allowed for waiver of hipaa and consent from these patients (unc irb - ). the comparator cohorts ( and ) were composed of all patients with any procedure seen in the project testing sites. these include two emergency room sites and multiple outpatient locations. canceled appointments are excluded. nineteen departments in the unc health system were identified, as some study sites comprise multiple departments. the cohort covers february to june , and the cohort covers february to june . detection of igg antibody to sars-cov- . igg antibody to sars-cov- was detected with a microparticle chemiluminescence assay (abbott laboratories) on the abbott architect i sr immunoassay analyzer according to the manufacturer's protocol under eua. the abbott sars-cov- igg assay utilizes microparticles coated with sars-cov- nucleocapsid protein. after washing, bound igg was detected via addition of anti-human acridinium-labeled second-step antibody. following a second wash step, pretrigger and trigger solutions were added and a chemiluminescent reaction was detected and reported in relative light units (rlu). the rlu generated is reflective of the amount of antibody bound to the microparticles. the sample rlu was compared to the assay-specific calibrator rlu to generate an index value (s/c). the presence of antibody was reflected by an index value of Ն . . index values of Ͻ . were classified as negative for sars-cov- igg. the abbott sars-cov- igg assay was evaluated in-house to assess sensitivity, specificity, and reproducibility. sensitivity was determined by testing remnant serum samples from patients admitted to unc hospitals and confirmed to be infected by rt-pcr testing of nasopharyngeal swabs. sera collected in the first (n ϭ ), second (n ϭ ), third (n ϭ ), and fourth (n ϭ ) weeks after disease onset were tested. calculated sensitivities were . %, . %, %, and %, respectively. this pattern follows the known natural history of igg antibody development, where titers take to weeks to reach detectable levels after primary infection. specificity was determined by testing sera. two hundred thirty-eight of these were collected prior to january (predating widespread u.s. covid- cases), and were collected after that date. twenty-three of these patients had nasopharyngeal swabs tested by sars-cov- rt-pcr and were negative. additionally, of the samples were from individuals tested for endemic, seasonal coronavirus infection in respiratory secretions and were positive by pcr for the seasonal coronaviruses but not sars-cov- . this group was important to include to establish the specificity of the igg elisa for sars-cov- . the remaining sera were from patients with igm antibodies to cytomegalovirus (cmv) or epstein-barr virus (ebv). this set of sera was not "pristine" sera but reflective of the general population in that it included sera with potentially interfering antibodies to other infectious agents. of the sera collected prior to january , serum samples were from patients containing ige antibodies to common respiratory allergens, were from individuals with positive antinuclear antibodies, and were from individuals with igm antibodies to cmv or ebv. additional sera were obtained from healthy laboratory staff (n ϭ ), pre-solid organ transplant waitlisted patients (n ϭ ), hiv-positive persons (n ϭ ), and a healthy platelet donor cohort (n ϭ ). in total, three serum samples in this set of sera had a positive result. all three were from samples collected prior to january . the estimated specificity of the assay was . %. intra-assay precision (cv%), determined by performing the replicate determinations of a positive sample, was . %. the same sample was tested seven consecutive days and showed an interassay cv of . %. statistical analysis. seroprevalence was estimated using the rogan and gladen estimator to adjust for assay sensitivity and specificity. direct standardization was used to estimate seroprevalence in the unc health population. confidence intervals were obtained using the nonparametric bootstrap percentile method. specifically, within each stratum of age group, gender, and race, sample seroprevalence was estimated and weighted by the stratum's proportion in the population. the rogan-gladen estimate can be negative, so all estimates of seroprevalence were constrained to be within the interval [ , ]. for comparison, demographic data for patients accessing the screening locations during the time period february to june were used. antibody responses to sars-cov- in patients of novel coronavirus disease temporal profiles of viral load in posterior oropharyngeal saliva samples and serum antibody responses during infection by sars-cov- : an observational cohort study profile of igg and igm antibodies against severe acute respiratory syndrome coronavirus (sars-cov- ) antibody responses to sars-cov- in patients with covid- a new coronavirus associated with human respiratory disease in china presymptomatic ttransmission of sars-cov- -singapore potential presymptomatic transmission of sars-cov- asymptomatic cases in a family cluster with sars-cov- infection viral kinetics of sars-cov- in asymptomatic carriers and presymptomatic patients evidence of sars-cov- infection in returning travelers from wuhan, china presymptomatic sars-cov- infections and transmission in a skilled nursing facility viral and host factors related to the clinical outcome of covid- clinical features of cases with coronavirus disease seroprevalence of sars-cov- -specific antibodies among adults in seroprevalence of immunoglobulin m and g antibodies against sars-cov- in china coast-to-coast spread of sars-cov- during the early epidemic in the united states performance characteristics of the abbott architect sars-cov- igg assay and seroprevalence in performance characteristics of four high-throughput immunoassays for detection of igg aantibodies against sars-cov- estimating prevalence from the results of a screening test seroprevalence of antibodies to sars-cov- in sites in the united states severe acute respiratory syndrome coronavirus -specific antibody responses in coronavirus disease patients the receptor binding domain of the viral spike protein is an immunodominant and highly specific target of antibodies in sars-cov- patients clinical and immunological assessment of asymptomatic sars-cov- infections sars-cov- seroprevalence and neutralizing activity in donor and patient blood from the san francisco bay area validation and performance comparison of three sars-cov- antibody assays we thank all the team members for their efforts and the participants of this study for their willingness to participate. we also thank the nurses and physicians who helped study enrollment.this project was funded by the unc school of medicine, unc health, and the university cancer research fund (ucrf). this work was also supported by public health service grants ca , ca , de , and es ; the unc center for aids research (p ai ); the unc lineberger cancer center (p ca ); us epa (cr ); and ul tr from the national center for advancing translational sciences (ncats) and from the national science foundation (grfp dge- ). the content is solely the responsibility of the authors and does not necessarily represent the official views of the national institutes of health.unc health data and further analysis were provided by marshall clark, james champion, kellie walters, and anna jojic at the north carolina translational and clinical sciences institute.the authors do not declare any conflicts of interest. key: cord- -gk jve i authors: beaudoin-bussières, guillaume; laumaea, annemarie; anand, sai priya; prévost, jérémie; gasser, romain; goyette, guillaume; medjahed, halima; perreault, josée; tremblay, tony; lewin, antoine; gokool, laurie; morrisseau, chantal; bégin, philippe; tremblay, cécile; martel-laferrière, valérie; kaufmann, daniel e.; richard, jonathan; bazin, renée; finzi, andrés title: decline of humoral responses against sars-cov- spike in convalescent individuals date: - - journal: mbio doi: . /mbio. - sha: doc_id: cord_uid: gk jve i in the absence of effective vaccines and with limited therapeutic options, convalescent plasma is being collected across the globe for potential transfusion to coronavirus disease (covid- ) patients. the therapy has been deemed safe, and several clinical trials assessing its efficacy are ongoing. while it remains to be formally proven, the presence of neutralizing antibodies is thought to play a positive role in the efficacy of this treatment. indeed, neutralizing titers of ≥ : have been recommended in some convalescent plasma trials for inclusion. here, we performed repeated analyses at -month intervals on convalescent individuals to evaluate how the humoral responses against the severe acute respiratory syndrome coronavirus (sars-cov- ) spike glycoprotein, including neutralization, evolve over time. we observed that the levels of receptor-binding-domain (rbd)-specific igg and iga slightly decreased between and weeks after the onset of symptoms but that rbd-specific igm levels decreased much more abruptly. similarly, we observed a significant decrease in the capacity of convalescent plasma to neutralize pseudoparticles bearing wild-type sars-cov- s or its d g variant. if neutralization activity proves to be an important factor in the clinical efficacy of convalescent plasma transfer, our results suggest that plasma from convalescent donors should be recovered rapidly after resolution of symptoms. u ntil an efficient vaccine to protect against severe acute respiratory syndrome coronavirus (sars-cov- ) infection becomes available, alternative approaches to treat or prevent acute coronavirus disease are urgently needed. a promising approach is the use of convalescent plasma containing anti-sars-cov- antibodies (abs) collected from donors who have recovered from covid- ( ) . convalescent plasma therapy has been successfully used in the treatment of sars, middle east respiratory syndrome (mers), and influenza virus h n pandemics and was previously shown to be associated with improvement of clinical outcomes ( ) ( ) ( ) . experience to date has shown that the passive transfer of convalescent plasma to acute covid- patients is well tolerated and presented some hopeful signs ( ) ( ) ( ) ( ) ( ) . in one study, the convalescent plasma used had high titers of igg to sars-cov- (at least Ն : ), which correlated positively with neutralizing activity ( ) . while it remains to be formally demonstrated, neutralizing activity is considered an important determinant of convalescent plasma efficacy ( ) and regulatory agencies have been recommending specific thresholds for qualifying convalescent plasma prior to its release. while neutralizing function has been associated with protection against reinfection in rhesus macaques ( ) , other antibody functions may be relevant for controlling an acute infection and should be examined to better understand the correlates of convalescent plasma-mediated efficacy ( ) . it was recently reported that the humoral responses against sars-cov- are built rapidly, peaking at week or week after the onset of symptoms but steadily decreasing thereafter ( ) ( ) ( ) . moreover, in a previous cross-sectional study, we reported that the neutralization capacity decreased between the third and the sixth week after the onset of symptoms ( ) . since convalescent patients are generally required to wait for days after recovery to start plasma donations and since they may donate plasma multiple times in the ensuing weeks, most donations are likely to occur even later than this. whether the neutralization capacity of convalescent plasma is stabilized after weeks or decreases further remains unknown. to address this issue, which might have practical implications for the selection of plasma from convalescent donors, we analyzed serological samples from convalescent donors that were collected at and weeks after the onset of symptoms. all of the convalescent donors initially tested positive for sars-cov- by reverse transcriptase pcr (rt-pcr) on nasopharyngeal specimens, with complete resolution of symptoms for at least days before blood sampling. the average age of the donors ( males and females) was years. we collected plasma samples from each individual at two time points: weeks after the onset of symptoms (baseline; median, days) and weeks after ( month; median, days after the onset of symptoms) ( table ) . we first evaluated the presence of receptor-binding-domain (rbd)-specific igg, igm, and iga antibodies by enzyme-linked immunosorbent assay (elisa) as we had recently described ( ) . in agreement with a recent report ( , ) , we observed that all rbd-specific igg, igm, and iga titers significantly decreased between and weeks after the onset of symptoms. we noted that igm and iga titers diminished significantly more abruptly than igg titers (fig. ) . accordingly, the proportions of convalescent individuals presenting detectable titers of igm and iga decreased by ϳ % and ϳ %, respectively, at weeks after the onset of symptoms ( fig. b and c) whereas the percentage of infected individuals presenting detectable titers of igg remained stable (fig. a) . we next used flow cytometry to examine the ability of convalescent plasma to recognize the full-length sars-cov- spike protein expressed at the cell surface. briefly, t cells expressing sars-cov- s glycoproteins were stained with plasma samples, followed by incubation with secondary antibodies recognizing all antibody isotypes. since the sars-cov- strain circulating in europe and north america has the d g mutation ( ), we also evaluated recognition of this variant by flow cytometry. as presented in fig. d , convalescent plasma from . % of donors (all but one) recognized both sars-cov- s variants (wild type [wt] and d g) at baseline. while this percentage was found to have remained stable weeks later, the level of recognition (mean fluorescence intensity [mfi]) was significantly diminished for both wt and d g s-expressing cells, indicating that spike-reactive antibodies were less abundant in convalescent plasma collected at this later time point. interestingly, the mfi values antibodies against cov- s in convalescent individuals ® were almost identical for the cells expressing the wt s and those expressing the d g variant s ( , and , , respectively; fig. d ), suggesting that the mutation did not significantly affect the s conformation. in agreement with recent work, we observed that sars-cov- -elicited antibodies cross-reacted with human sarbecoviruses ( ) (sars-cov; fig. e ) and with another betacoronavirus (oc ) whereas no cross-reactive antibodies to alphacoronavirus (nl and e) s glycoproteins (fig. f) were detected. levels of cross-reactive antibodies recognizing sars-cov and oc s glycoproteins decreased between the two time points, following a trend similar to that shown by the sars-cov- s-reactive antibodies (fig. s ) . we next measured the capacity of plasma samples to neutralize pseudoparticles bearing wt sars-cov- s, its d g variant, or vesicular stomatitis virus g (vsv-g) glycoproteins using t cells stably expressing ace as target cells (fig. ) . previous studies demonstrated that the neutralizing activity of convalescent plasma measured with this method correlates quantitatively with neutralizing activity measured using an authentic sars-cov- neutralization assay ( , ) . neutralizing activity against sars-cov- wt or d g s glycoprotein, as measured by the neutralization half-maximum inhibitory dilution (id ), was detected in % of patients weeks after the onset of symptoms. while we acknowledge that the sensitivity of any given neutralization assay the median of neutralization for baseline (red) or -month (black) plasma samples is shown. (c) neutralization half-maximal inhibitory plasma dilution (id ) values were determined using a normalized nonlinear regression with graphpad prism software. undetectable levels (id Ͻ ) are represented as white symbols. the mean neutralizing titers and the proportions (%) of neutralizers (patients with an id value over ) are shown above the graphs. statistical significance was tested using wilcoxon matched-pair signed-rank tests (ns, not significant; ****, p Ͻ . ). could affect calculations of the percentage of donors with neutralization activity, we note that the percentage of convalescent plasma with undetectable neutralization titers reported here is similar to what was reported in recent studies ( , , ) . sars-cov- neutralization was specific since no neutralization was observed against pseudoparticles expressing vsv-g (fig. ) . neutralizing activity against pseudoparticles bearing the sars-cov s glycoprotein was detected in only % of convalescent plasma and exhibited low potency, as previously reported (fig. ) ( ) . as recently shown, plasma samples from prepandemic sars-cov- -negative and sars-cov-negative individuals showed no neutralization activity against pseudoparticles bearing the sars-cov- or sars-cov spike protein (not shown). of note, while we observed enhanced infectivity for the d g variant compared to its wt sars-cov- s counterpart (see fig. s a in the supplemental material), no major differences in neutralization with convalescent plasma were detected at either time point (fig. s b) , thus suggesting that the d g change does not affect the overall conformation of the spike, in agreement with recent findings ( , ) . the capacity to neutralize sars-cov- s wt-or d g-pseudotyped particles significantly correlated with the presence of rbd-specific igg, igm, iga, and anti-s antibodies (fig. s ) . interestingly, we observed a pronounced ( % to %) decrease in the proportion of convalescent individuals able to neutralize pseudoparticles bearing sars-cov- s glycoprotein between and weeks after the onset of symptoms. moreover, with plasma that still neutralized, the neutralization activity significantly decreased between these two time points (fig. c) . interestingly, rbd-specific igm and neutralizing activity declined more significantly in convalescent plasma over time than rbd-specific igg, iga, and anti-s ab activity ( fig. s a and b) . moreover, while the loss of neutralizing activity on the wt and d g pseudoparticles over time correlated with the loss of anti-rbd igm, iga, and igg antibodies, the correlation was higher for igm than for igg and iga ( fig. s c and d) , suggesting that at least part of the neutralizing activity could be mediated by igm, as recently proposed ( , ) . therefore, if plasma neutralization activity is shown to be required for protection from sars-cov- infection, then our results suggest that this protection could be limited in time and that, in the context of vaccination, multiple boosts might be necessary to mount a durable and effective anti-sars-cov- humoral response. in summary, our results indicate that plasma neutralization activity continues decreasing past the sixth week of symptom onset ( ) . it is currently unknown whether neutralizing activity truly drives the efficacy of convalescent plasma in acute covid- . if this were to be found to be the case, our results suggest that efforts should be made to ensure that convalescent plasma is collected as soon as possible after recovery of the donor from active infection. supplemental material is available online only. text s , docx file, . mb. convalescent plasma as a potential therapy for covid- challenges of convalescent plasma infusion therapy in middle east respiratory coronavirus infection: a single centre experience convalescent plasma treatment reduced mortality in patients with severe pandemic influenza a (h n ) virus infection use of convalescent plasma therapy in sars patients in hong kong deployment of convalescent plasma for the prevention and treatment of covid- a randomized trial of convalescent plasma for covid- -potentially hopeful signals the convalescent sera option for containing covid- early safety indicators of covid- convalescent plasma in , patients effectiveness of convalescent plasma therapy in severe covid- patients effect of convalescent plasma therapy on time to clinical improvement in patients with severe and life-threatening covid- : a randomized clinical trial convergent antibody responses to sars-cov- in convalescent individuals primary exposure to sars-cov- protects against reinfection in rhesus macaques iga dominates the early neutralizing antibody response to sars-cov- cross-sectional evaluation of humoral responses against sars-cov- spike antibody testing for covid- : a report from the national covid scientific advisory panel clinical and immunological assessment of asymptomatic sars-cov- infections increases infectivity of the covid- virus measuring sars-cov- neutralizing antibody activity using pseudotyped and chimeric viruses an mrna vaccine against sars-cov- -preliminary report longitudinal evaluation and decline of antibody responses in sars-cov- infection sars-cov- infections and serologic responses from a sample of u.s. navy service members -uss theodore roosevelt the d g mutation in the sars-cov- spike protein reduces s shedding and increases infectivity waning of sars-cov- rbd antibodies in longitudinal convalescent plasma samples within four months after symptom onset we thank the convalescent plasma donors who participated in this study; the héma-québec team involved in convalescent donor recruitment and plasma collection; the staff members of the crchum bsl and flow cytometry platforms for technical assistance; stefan pöhlmann (georg-august university, germany) for the plasmids key: cord- -hzh tyoo authors: peng, xinxia; gralinski, lisa; ferris, martin t.; frieman, matthew b.; thomas, matthew j.; proll, sean; korth, marcus j.; tisoncik, jennifer r.; heise, mark; luo, shujun; schroth, gary p.; tumpey, terrence m.; li, chengjun; kawaoka, yoshihiro; baric, ralph s.; katze, michael g. title: integrative deep sequencing of the mouse lung transcriptome reveals differential expression of diverse classes of small rnas in response to respiratory virus infection date: - - journal: mbio doi: . /mbio. - sha: doc_id: cord_uid: hzh tyoo we previously reported widespread differential expression of long non-protein-coding rnas (ncrnas) in response to virus infection. here, we expanded the study through small rna transcriptome sequencing analysis of the host response to both severe acute respiratory syndrome coronavirus (sars-cov) and influenza virus infections across four founder mouse strains of the collaborative cross, a recombinant inbred mouse resource for mapping complex traits. we observed differential expression of over small rnas of diverse classes during infection. a majority of identified micrornas (mirnas) showed divergent changes in expression across mouse strains with respect to sars-cov and influenza virus infections and responded differently to a highly pathogenic reconstructed virus compared to a minimally pathogenic seasonal influenza virus isolate. novel insights into mirna expression changes, including the association with pathogenic outcomes and large differences between in vivo and in vitro experimental systems, were further elucidated by a survey of selected mirnas across diverse virus infections. the small rnas identified also included many non-mirna small rnas, such as small nucleolar rnas (snornas), in addition to nonannotated small rnas. an integrative sequencing analysis of both small rnas and long transcripts from the same samples showed that the results revealing differential expression of mirnas during infection were largely due to transcriptional regulation and that the predicted mirna-mrna network could modulate global host responses to virus infection in a combinatorial fashion. these findings represent the first integrated sequencing analysis of the response of host small rnas to virus infection and show that small rnas are an integrated component of complex networks involved in regulating the host response to infection. ferentiate infection by the lethal pandemic influenza virus from nonlethal seasonal influenza virus a/texas/ / infection ( ) . it was also reported that hiv- virus is able to suppress expression of the polycistronic mirna cluster mir- / to enable efficient viral replication ( ) . however, changes in expression of other small rnas during virus infection have not been systematically studied. using next-generation deep-sequencing technology, we recently discovered widespread differential expression of host long ncrnas in response to virus infection ( ) , but the experimental protocol used was not designed to capture small rnas ( ) . in this study, we used deep-sequencing technology to perform a complementary small rna transcriptome analysis of the same severe acute respiratory syndrome coronavirus (sars-cov)-infected lung samples collected from four mouse strains as previously reported. in addition, lung samples collected from the same four influenza virus-infected mouse strains were included in the new analysis. our results show that many known mirnas responded differently to the two virus infections and that many of them were also differentially regulated during lethal influenza virus infection, as shown in a previous study ( ) . we also discovered many non-mirna small rnas and unannotated small rnas that were differentially expressed during infection. the integration of transcriptome sequencing analysis of long transcripts and small rnas showed the intricate interactions of short and long rnas during virus infection. the changes in mirnas positively correlated with the changes in long transcripts cotranscribing from the same locus, indicating that the mirnas were transcriptionally regulated during virus infection. we predicted that differentially expressed mirnas could target the majority of differentially expressed long transcripts during virus infection. to systematically investigate the regulation of small rnas during viral infection, we performed two batches of small rna sequencing analysis using lung samples from virus-infected mice. to follow up our previous study on long ncrnas, we first sequenced the small rnas of the same lung samples collected from four mouse strains infected with sars-cov: s /svimj ( /s ), wsb/eij (wsb), pwk/phj (pwk), and cast/eij (cast) ( ) . for comparisons, we performed small rna sequencing analysis using another set of lung samples collected from additional mice of the same four mouse strains infected with influenza virus. these mouse strains were selected because of their differential range of susceptibility phenotypes as revealed following infection with sars-cov or influenza virus ( ) and because of the opportunity they presented of pursuing downstream quantitative trait locus (qtl) mapping of regulation and function in the collaborative cross, a recombinant inbred mouse resource for mapping complex traits ( ) . directional cdna libraries were constructed using standard illumina protocols for small rna analysis, which targeted small rnas of around to nucleotides (nt). in total, we obtained~ million adaptor-trimmed reads ranging from to nt in length from mouse lung samples (over million reads per sample on average). as expected, the reads exhibited a large peak in abundance in the length range of to nt ( fig. a ; see fig. s in the supplemental material). for each sample, the majority ( % on average) of total short reads were mapped into different classes of abundant small rnas. mirnas composed the most abundant class, accounting for about % of total reads on average ( fig. b ; see also table s in the supplemental material). we observed a wide range of counts of reads (from to an average of , , for the most abundant mirna in a given sample) mapped to individual mirnas in both normal and virus-infected samples, indicating that techniques with dynamic ranges as large as are required for comprehensive profiling of mirna expression. on average, about of , annotated mature mouse mirnas were detected with at least one read in a given sample. the detected mirnas showed very distinctive expression patterns in both normal and virus-infected mouse lung samples. in all samples, the top most abundant mirnas by read count accounted for about % of mirna reads and the top mirnas for % of mirna reads. the most abundant mir-nas and the overall abundance distribution of all detected mir-nas are shown in fig. c and d. differential expression of known host mirnas during virus infection. we first studied annotated mirnas, as host mirnas represented the most abundant class of small rnas observed and the host mirna response to sars-cov infection has not been reported previously. we identified mature mirnas that were differentially expressed during sars-cov or influenza virus infection. thirty were consistently upregulated or consistently downregulated by more than . -fold across three or more mouse strains during sars-cov infection, and were consistently upregulated or consistently downregulated by more than . -fold across three or more mouse strains during influenza virus infection (fig. a ). in addition, of these mirnas were consistently upregulated or consistently downregulated by more than . -fold in two or more mouse strains during both sars-cov and influenza virus infection. additional quantitative pcr (qpcr) analyses of replicate samples with a large subset of differentially expressed mirnas showed good concordance between replicates and statistical congruence (see fig. s in the supplemental material). interestingly, there were mirnas that were upregulated in at least three mouse strains during both sars-cov and influenza virus infection, suggesting that a subset of mirnas such as these commonly responded to different virus infections. however, % ( / ) of the differentially expressed mirnas showed different expression profiles across four mouse strains in sars-cov and influenza virus infections (fig. a) , suggesting that expression of most mirnas was likely both host and virus dependent. although the physiological relevance of small changes in mirna expression remains to be investigated, other studies have indicated that . fold changes in expression can have significant biological impact ( ) . in addition, because we profiled whole lung samples, some of the observed changes in expression could have been due to the infiltration of immune cells into the infected lung. to investigate whether the differential expression patterns of the mirnas were related to viral pathogenesis, we first compared these findings with those from our previous microarray study of host mirnas in mouse lungs infected with the fully reconstructed pandemic influenza virus (r ) ( ). we found that of the differentially expressed mature mirnas identified here were not on the arrays ( mirnas) or not detected ( mirnas) by the previous microarray technology (fig. a) , showing the benefits of deep-sequencing technology, in which the measurement of rnas is independent of prior knowledge of transcript annotations and is achieved with a dynamic range of up to ( ). of those mirnas profiled on the microarray in the previous study, % ( / ) showed at least a . -fold difference between the response to the highly pathogenic r virus infection and the response to minimally pathogenic a/texas/ / virus infection for at least one of three time points studied. these findings suggest that many of these mirnas may be commonly involved in viral pathogenesis. next, we surveyed the changes in expression of differentially expressed mirnas during virus infection across a set of diverse conditions ( fig. b and materials and methods). even with only mirnas surveyed and relatively large variations among replicate samples, we were able to observe distinctive mirna expression changes. these changes were detected under conditions of infection with different viruses and were seen with samples generated in vivo versus in vitro (see fig. s in the supplemental material). first, we observed that of mirnas had very different expression patterns in the highly pathogenic sars-covma versus the minimally pathogenic urbani strain ( fig. b; fig. s ). different expression patterns were also seen with of mirnas in comparisons of the highly pathogenic vn influenza virus to the attenuated vn hemagglutinin (ha) mutant strain. this argues that some of the identified mirnas may be associated with pathogenic outcomes. second, many factors may have contributed to the different expression patterns; however, it appears unlikely that the observed mirna changes were due to pure immune cell infiltration, as mirnas showed strong evidence of differential expression in cultured fibroblasts during infection. third, and not surprisingly, several mirnas showed expression patterns that differed between the in vivo and in vitro models. the differences could be attributed to cell type specificity or to different environmental and/or growth conditions, as two mirnas that did not show obvious differential expression patterns in cultured fibroblasts, mir- in macrophages ( ) and mir- in neutrophils ( ) , are known to be related to different immune cell types. in summary, these results suggest that mirnas are likely involved in viral pathogenesis and that the choice of experimental systems used for mirna studies should be considered a critical and informing component in the study design. differential expression of non-mirna small rnas and novel small rnas during virus infection. as shown in table s in the supplemental material, there were still many (about million in total) "leftover" reads which did not map to annotated or predicted abundant small rnas but were aligned to the mouse reference genome, suggesting that there might be some novel host small rnas relevant to virus infection. therefore, starting by mapping all short reads directly to the mouse reference genome, we carried out a genome-wide search of novel small rnas (see materials and methods). in total, of , , start positions in the genome with at least one uniquely mapped read, we found that about % ( , ) gave at least reads of the same length in a sample, resulting in , nonredundant candidate loci for putative small rnas. about . % ( / , ) of the candidate loci (median length, nt) were differentially expressed during sars-cov and/or influenza virus infection (see table s and fig. s a in the supplemental material); of those candidate loci overlapped with annotated mirna precursors (mirbase version ). we next used a conservative filtering strategy to remove of candidate loci from further analysis because of the possibility of misalignment of short reads originating from other highly expressed loci (see materials and methods). we considered the remaining to represent putative small rna loci, as we reasoned that their showing consistent responses to virus infection suggested that they were more likely to be biologically functional rather than to represent random noise. to validate the analytical approach used to identify these differentially expressed putative small rna loci, we compared the expression ratios calculated for infection versus matched mock infection that we used to identify these putative ( ) and mir- in neutrophils ( ) , are known to be related to immune cells. the other four mirnas came from a small-scale screening performed using qpcr with a subset of randomly selected differentially expressed mirnas in pwk/phj mouse embryonic fibroblasts (mefs) infected with influenza virus (data not shown). also, mir- a* and mir- - p were not measured by previous microarray (see panel a). colors on the heat map indicate the log ratios (the differences between medians of normalized ct values of replicate samples) of expression in virusinfected samples to expression in matched mock-infected samples. red, upregulation; green, downregulation. the "lung ϩ flu" data contrast mirna expression changes in lung samples from mice infected with highly pathogenic influenza virus (wt, vn ) and with minimally pathogenic influenza virus (ha, vn with a mutation in ha protein) at two time points: days and after infections. similarly, the "lung ϩ sars" data contrast mirna expression changes in lung samples from mice infected with highly pathogenic sars-cov (ma, ma ) and minimally pathogenic sars-cov (urb, urbani). the "pwk mefs ϩ flu" data represent temporal mirna expression changes in samples from cultured pwk mefs infected with the mouse-adapted a/pr/ / influenza virus across four time points after infection. a more detailed representation of individual replicate experiments is shown in fig. s . wt, wild type. small rna loci to the corresponding expression ratios that we calculated for the overlapping mirna precursors on the basis of the reads directly mapped to annotated precursor sequences. we obtained very good agreement (analysis of variance [anova] f test, p ϭ . e- ; pearson correlation coefficient, r ϭ . ) between the two estimations. this result confirmed that the analytical approach used here performed well, since we performed an unbiased genome-wide search for all small rnas without considering any known annotations instead of looking only at annotated mirna sequences. to investigate the genomic origins of the putative small rna loci, we compared the loci to those included in a previously compiled nonredundant set of mouse genome annotations ( ). we found that % ( / ) overlapped (sense or antisense) with annotated loci, including coding rnas, ncrnas, and those with unannotated genomic regions ( table ), suggesting that the majority of small rnas originated from genomic regions encoding long transcripts. also, the result suggested that long transcripts from some of these regions have not been well annotated or that some regions formed independent transcriptional units exclusive to small rnas, as % of putative small rna loci did not overlap annotated long transcript-transcribing regions. to understand the classes of putative small rnas produced from the loci, we then compared them to annotated or predicted abundant small rnas. interestingly, many putative small rna loci overlapped non-mirna small rnas such as small nucleolar rnas (snornas) and piwi-associated small rnas (pirnas) ( table ) . snornas guide the chemical modification of ribosomal rnas and other ncrnas and are essential for major biological processes, including protein translation and mrna splicing ( ) . pirnas represent a class of small rnas that are to nt long and have been linked to transcriptional gene silencing in germ line cells ( ) . pirnas have also been identified in various somatic tissues ( , ) . also, we observed length distributions of small rna loci overlapping an-notated small rna loci that were similar to those of small rna loci with no overlap (fig. a) , indicating that our identified loci were indeed producing small rnas. examples of detailed read mapping of loci overlapped with annotated small rnas are shown in fig. b and c; similar results determined using read mapping of putative novel small rna loci are shown in fig. . we observed that putative small rna loci overlapped with annotated snor-nas on the basis of the updated ensembl annotation and that almost all ( of ) of the loci also overlapped with annotated coding or ncrna loci, suggesting that, in addition to mirnas, snornas might represent another large class of small rnas differentially expressed in response to virus infection and originating from long transcript-encoding loci. coupling changes in host short and long rna expression through parallel small rna and whole-transcriptome sequencing. to better understand the host response to virus infection at the system level, we performed an integrative analysis of small rna transcriptome sequencing with our previously reported whole-transcriptome sequencing method using the same set of sars-cov-infected samples ( ) . to the best of our knowledge, studies have profiled host mirna expression changes during virus infection ( , , , ) , but regulation of such mirna expression changes has not been systematically investigated. mirna biogenesis can be regulated at different steps ( ) , including at least three steps of rna processing: (i) the transcription of mirna primary transcripts (pri-mirna) of several thousand base pairs, (ii) the processing of the long pri-mirnas to mirna precursors ( to nt), and (iii) the processing of mirna precursors into mature mirnas (about to nt). as we had previously generated whole-transcriptome data using the set of lung samples infected with sars-cov with which we quantified long transcripts (especially those Ͼ nt in length) ( ), we reasoned that the long pri-mirnas were also quantified. though the transcript structures of pri-mirnas have not been a annotated loci data represent the set of nonredundant annotations of mouse protein-coding loci and ncrnas compiled in reference ( ), combined with the set of unannotated genomic regions identified in the same study. annotated small rnas are as follows: mirna, mirna precursors; snorna, small nucleolar rnas; pirna, piwi-associated small rnas. rna repeat data were downloaded from the ucsc genome browser (http://genome.ucsc.edu/). numbers of putative small rna loci represent the numbers of putative small rna loci overlapped with different classes of annotated loci in terms of genomic coordinates; sense and antisense loci are counted separately. for example, putative small rna loci overlapped with annotated protein-coding loci on the sense strand (column , row ). total sense data represent the total numbers of putative small rna loci that overlapped with annotated loci on the sense strand. total antisense data represent the total numbers of putative small rna loci overlapped with annotated loci on the antisense strand. the putative small rna loci that overlapped with annotated loci were further classified by checking whether they also overlapped with different annotated small loci in terms of genomic coordinates. for example, of small rna loci that overlapped with annotated loci, also overlapped with annotated mirnas (column , row ). completely annotated, it is known that many mature mirnas originate from annotated long (protein-coding or noncoding) transcripts. we reasoned that those annotated loci overlapping mirna precursors would be a good proxy for pri-mirnas, as it has been shown that many mammalian mirnas are transcriptionally linked to expression of the genes from which they origi-nate ( , ) . we then compared the changes in expression of identified mature mirnas obtained during sars-cov infection to the corresponding changes in expression of overlapping loci obtained from the previous whole-transcriptome sequencing analysis ( ) . interestingly, we found that the changes in expression of mature mirnas and the overlapping loci significantly posi- tively correlated (anova p ϭ e- ; pearson correlation coefficient ϭ . ) (fig. a) . further, we took the -kb genomic regions surrounding each annotated mirna precursor ( kb upstream and kb downstream, excluding the precursor region) as another approximation of pri-mirnas and observed a similarly high positive correlation with changes in expression (anova p ϭ . e- ; pearson correlation coefficient ϭ . ) (fig. b) . these results show for the first time that the transcriptional regulation of pri-mirnas could be largely responsible for the differential regulation of mature mirnas during virus infection. to investigate the potential functional impact of differential expression of mature mirnas, we combined the data showing the mrna expression changes measured by the whole-transcriptome sequencing analysis with that from the mirna target predictions. we found that the majority ( %) of mrna loci were predicted as targets of at least of the differentially expressed mature mir-nas identified above (see fig. s a in the supplemental material) and that the differentially expressed mrna loci listed were significantly (p Ͻ . e- [chi-square test]) enriched with predicted targets of those differentially expressed mirnas. the ratio of mirna targets versus nontargets for differentially expressed mrna loci was~ . compared to~ . for those loci that were not differentially expressed during infection, representing an enrichment of about . -fold. these results strongly suggest that collectively differentially expressed mirnas modulate the global host responses to virus infection. notably, about % of those differentially expressed targets were predicted targets of two or more identified mature mirnas, indicating that a single target could be regulated in vivo by multiple mirnas at the same time during virus infection. importantly, % of the targets predicted to be regulated by multiple mir-nas were targeted by both upregulated and downregulated mirnas at the same time, showing that additional studies are necessary to elucidate which specific mirnas, if any, play a regulatory role in the changes of individual targets in vivo (fig. s b) . functional analysis of predicted mirna targets also indicated that many important aspects of the host response (such as innate immunity and cytokine production) were likely modulated by mirnas (see table s in the supplemental material). figure s shows a subnetwork of differentially expressed mirnas and their predicted targets in several antiviral pathways. studies of the host small rna response to virus infection have been focused on mirnas ( , , , ), but there is growing evidence that the mammalian transcriptome comprises a diversity of small rnas in addition to mirnas. for example, human and protozoan snornas have been reported to be processed into mirna-like rnas ( , ) , and the members of a class of novel small rnas have been reported to be derived from many snor-nas ( ) . abundant small rnas are also derived from trnas in a dicer-dependent manner ( ) , and human trna-derived small rnas appear to be involved in the global control of small rna silencing ( ) . to our knowledge, the present study was the first to use comprehensive deep-sequencing technology to characterize virus infection-induced changes in expression of mirnas and other classes of small rnas, including many nonannotated small rnas. as the biological functions of these diverse types of small rnas are largely unknown, virus infection models also offer a unique platform for studying the regulation and biology of these diverse small rnas. for example, the influenza virus ns protein inhibits host pre-mrna splicing through its interaction with snornas ( ) ( ) ( ) . also, it has been shown that sars-cov and other nidoviruses encode a protein with sequence similarity to xendou, a eukaryotic endoribonuclease that is involved in cellular snorna processing ( , ) . though the experimental protocol used here is not optimized for the capture of rna products with =, =-cyclic phosphates as specifically generated by xendou ( ), we did observe that the fold changes of identified small rnas tended to be larger in sars-cov-infected samples than in influenza virus-infected samples (see fig. s b and c in the supplemental material), suggesting that further investigation of the impact of viral proteins on host small rna processing could represent another approach for the study of virus-host interactions. it is known that the characteristics of their secondary structures could be indicative of the types of putative small rnas such as mirnas and therefore of their general functional mechanisms. we therefore investigated whether the putative small rnas identified here tended to have stable secondary rna structures (see materials and methods). in total, ( %) putative small rna loci were predicted by secondary structure analysis; of those loci did not overlap with those of the annotated small rnas (see table s in the supplemental material). interestingly, those that overlapped with annotated small rnas were significantly (chisquare test; p ϭ . e- ) more likely to be predicted by rna secondary structure analysis ( % [ / ]) compared to those which did not overlap any annotated small rnas ( % [ / ]), suggesting that the existing annotation of small rnas might be biased toward those with stable local secondary structures. for example, out of putative small rna loci that overlapped with known mirna precursors, % (one prediction only) to % (all four predictions combined) were predicted with secondary structures, indicating that our automated strategy for structure prediction performed well in terms of finding local rna structures such as hairpins. for those putative loci that overlapped with other classes of small rnas, however, the percentage predicted with secondary structures was much lower, ranging from . % to % for snornas to % for pirnas. a closer look at those loci overlapping snornas showed that % ( / ) of the loci that overlapped with h/aca box family snornas but only about % ( / ) of the loci that overlapped with c/d box family snornas were predicted with secondary structures. since snornas are broadly classified into two families, corresponding to a c/d box with one short (~ nt) hairpin structure and an h/aca box with at least two large hairpin structures ( ) , these results suggest that investigations based solely on a typical rna secondary structurebased approach might miss many small rnas that do not form stable local rna structures per se and that approaches utilizing transcriptome sequencing might be able to identify broader classes of small rnas. compared to the current knowledge regarding noncanonical small rnas, the functional mechanism of mirnas is relatively better understood. however, the regulation of mirna expression changes during virus infection has not been systematically investigated. through integrative analysis of parallel sequencing of both small rnas and long transcripts from the same samples, we were able to infer the regulatory relationships both upstream and downstream of mirnas with respect to the differences in expression. not only did we show that the differential expression of mirnas was likely due to transcriptional regulation, but our data also indicate that those mirnas may collectively play a role in modulating the global host response. since it has been shown that mechanistically mammalian mirnas mainly act to decrease target mrna levels, thus leading to reduced protein output ( ), these results argue that a better understanding of the small rnas, including mirnas, would be required for complete knowledge of the regulation of host responses to virus infections. as evidenced here and in other studies ( ) , multiple mirnas can simultaneously regulate the same targets, arguing that experimental design with the corresponding mirnas measured by whole-transcriptome sequencing analysis using the same samples (x axis). in total, mature mirna loci overlapped annotated loci and were included in the plot. (b) data are presented as described for panel a, with the y axis data showing the log fold changes in expression of the -kb genomic regions surrounding the corresponding mirna precursors measured by whole-transcriptome sequencing analysis of the same samples. in total, mature mirnas were included in the plot, as of genomic regions surrounding differentially expressed mirnas were not subjected to differential expression analysis due to the lack of a sufficient number of reads for quantification in the whole-transcriptome sequencing data. and computational strategies of greater complexity, such as we proposed previously ( ) , are necessary to elucidate the combinatorial nature of mirna-mrna regulatory networks in vivo. our integrated sequencing analysis also offers several additional benefits, such as the investigation of the functions of some long ncrnas. we previously reported that many long ncrnas of unknown function respond to virus infection ( ) , and it is known that long ncrnas can serve as precursors for small rnas ( ) . in this study, we identified a number of small rnas that overlapped with long ncrnas (table ) . for example, mir- , one of the upregulated mirnas identified here (fig. a) , is produced from an exon of the long ncrna bic, which was also observed to be upregulated by whole-transcriptome sequencing analysis ( ) . both mir- and bic were induced in primary murine macrophages stimulated by beta interferon (inf-␤) ( ) . loss-offunction studies showed that mice lacking a functional bic/mir- gene exhibited a defective immune response in vivo and were deficient in cytokine production ( , ) . also, fig. shows two small rnas overlapping snornas encoded in the introns of gas , another long ncrna that was previously identified as an apoptosis regulator ( ) . it is unclear whether snornas from gas play any functional roles in response to virus infection, but it has been hypothesized that the functional effect of gas on t-cell growth may be mediated through its intronic snornas ( ) . these examples suggest that a detailed investigation of the connections between long ncrnas and the coexpressed small rnas might facilitate a better understanding of the regulation of host responses. moreover, even though the study of viral small rnas is beyond the scope of this report, it is worth noting that we also observed many small sequence reads from both viral genomes (see table s in the supplemental material). though isolation of small rnas from sars-cov infections has not been reported, it was shown recently that influenza virus expresses many small rnas ( , ) . in summary, our results convincingly show that, in the future, integrated sequencing analysis of different fractions of the complex transcriptome should facilitate a full understanding of virushost interactions and viral pathogenesis. the small rna transcriptome deepsequencing analysis was performed on lung samples from our previously published study ( ) . briefly, we infected four of the eight founder mouse strains used in generating the collaborative cross, a recombinant inbred mouse resource for mapping complex traits ( ) . these strains included s /svimj ( /s ), wsb/eij (wsb), pwk/phj (pwk), and cast/eij (cast) mice. ten-week-old mice were intranasally infected with phosphate-buffered saline (pbs) alone or with ϫ pfu of mouseadapted severe acute respiratory syndrome coronavirus (sars-cov; rma ), or pfu of influenza a virus strain a/pr/ / (h n ; pr ). to match the previous whole-transcriptome analysis, we performed small rna transcriptome sequencing analysis on the same eight samples from mice with sars-cov infections, including one sars-cov rma infected mouse and one matched mock-infected mouse from each of the four strains at days postinfection (dpi). in addition, we sequenced the small rna transcriptome for samples obtained from influenza virusinfected mice, including two pr -infected mice and one matched mockinfected mouse from each of the four strains at dpi. the additional replicate samples from matched infections were used for evaluation by quantitative reverse transcription-pcr (qrt-pcr). additional virus infections for qpcr assay. twenty-week-old c bl/ mice were infected by intranasal instillation of influenza virus vn ( ϫ pfu), vn ha avirulent (vn with a mutation of the ha protein) ( ϫ pfu), sars-cov rma ( ϫ pfu), or infectious-clone sars (icsars) urbani ( ϫ pfu) in l of pbs or were subjected to mock infection with pbs alone. in this experiment, vn was treated as a highly pathogenic strain and vn ha avirulent as a minimally pathogenic strain of influenza virus, as the % mouse lethal doses were pfu for vn and . pfu for vn ha (unpublished data). similarly, ma was treated as a highly pathogenic strain and urbani as a minimally pathogenic strain of sars-cov based on a previous study ( ) . lung tissues were removed on days and postinfection, and total rna was harvested from an individual lung lobe. there were mice for each time point and animals for the time- rna preparation. the individual lung lobes were homogenized using trizol and a magnalyser system (roche) according to the manufacturer's instructions. rna was further purified using an mirneasy mini kit (qiagen) according to the manufacturer's instructions. rna samples were spectroscopically verified for purity, and the quality of the intact rna was assessed using an agilent bioanalyzer. the assay also confirmed that the rna samples were free of genomic dna contamination. library construction. for sars ma -infected samples, small rna libraries were prepared with a small rna v . sample preparation kit following the manufacturer's instructions (illumina, san diego, ca). total rna was ligated with a = rna adaptor ( =-/ rapp/atctcgtatg ccgtcttctgcttg/ ddc/) specifically modified to target mirnas and other small rnas that have a = hydroxyl group resulting from cleavage by dicer and other rna-processing enzymes and then with a = rna adaptor ( =-guucagaguucuacaguccgacgauc) at the = end of rna with a phosphate group. the = adaptor also included the sequencing primer. rt-pcr amplification was then done using the adaptors as primers, selectively enriching the fragments that had adaptors on both ends. the resulting double-stranded dna libraries were polyacrylamide gel electrophoresis (page) ( % novex tris-borate-edta [tbe] page; invitrogen) purified and size selected to eliminate dimerized adaptors. for influenza virus-infected samples, small rna libraries were prepared using a truseq small rna sample preparation kit (illumina, san diego, ca) following the manufacturer's instructions. this protocol is very similar to that of the version . sample preparation kit but with a change in the = adapter ( = tggaattctcgggtgccaagg) that also targets the = hydroxyl resulting from enzymatic cleavage by dicer. sequencing and read mapping. all libraries were sequenced to nt (sars-cov infection) or nt (influenza virus infection) of read length on individual lanes on a genome analyzer ii system following the protocols of the manufacturer (illumina, san diego, ca). the adaptor sequences were first trimmed from small rna reads. after adaptor trimming, reads Ͻ nt in length were discarded, and reads Ͼ nt were shortened to nt by clipping from the = end. bowtie software ( ) was used to align the trimmed reads to reference sequences, allowing up to two mismatches and with options selected as "-k -m -best -strata," which kept only uniquely mapped reads. for the global classification, the trimmed reads were aligned sequentially to mouse ribosomal sequences (rrna) from genbank, trna sequences (trna) from the genome browser of the university of california, santa cruz (ucsc), (http://genome.ucsc.edu), small cytoplasmic rna (scrna), small nuclear rna (snrna), and small nucleolar rnas (snorna) sequences from ncbi refseq annotation, piwiassociated small rna (pirna) sequences from the functional rna database ( ) , microrna (mirna) precursor sequences from mirbase (http://www.mirbase.org, release ), the mouse reference genome (mm , july , ncbi build , reference strain c bl/ j), and sars-cov (genbank accession no. dq ) or influenza virus (genbank accession no. af to af ) genomic sequences. also, unmapped reads after rrna alignment were mapped directly to the same mouse reference genome to search for nonannotated small rnas. for visualization, bam files were generated using samtools ( ) and displayed using the ucsc genome browser. differential expression analysis of small rnas from annotated loci. to quantify transcript expression, we estimated transcript abundance by counting the total number of reads mapped to each transcript. read sequences that mapped to more than one location were excluded from expression quantification. to compare transcript expression data across different sets of conditions, the transcript abundances, i.e., the raw read counts, were scaled first as the number of reads per million (rpm) mapped for each sample. next, we chose the geometric mean of all samples as a reference. the quantile-quantile (qq) plots of the distribution of differences between the reference and remaining samples in the numbers of log -transformed revolutions per minute were compared. we determined a scaling factor for each sample as the median difference between the corresponding quantile values of the individual sample and the reference. an offset of was added to all normalized values to facilitate comparisons involving one or more values of zero and to reduce the variability of the log ratios for low expression values. for quantification of annotated mirnas, we first removed reads mapped to other abundant small rnas (rrnas, trnas, snornas, snrnas, scrnas, and pirnas) and used the results to map the remaining reads directly to mirna precursors. to accommodate the variability in the rna sequences of individual mature mirnas, i.e., the so-called isomirs ( ) , we counted all reads mapped with start positions located within a -nt window for each annotated mature mirna. only reads of to nt were used for mirna quantification. for other annotated loci, we used the mapping results of the reads to search the mouse reference genome. a nonredundant set of annotations that included annotations of long (Ͼ nt) noncoding rnas was compiled as previously described ( ) . in brief, we clustered the overlapping annotated transcripts into single loci. we found , nonoverlapping ncrna loci ( , of which were larger than nt) in addition to , protein-coding loci. read sequences that mapped to multiple locations on the corresponding reference sequences were excluded from the counts during all differential expression analyses. the repeat information was downloaded as a repeatmasker track using the ucsc genome browser. identification of non-mirna and novel small rnas by a genomewide search. we carried out a genome-wide search of novel small rnas, starting by mapping all short reads directly to the mouse reference genome. we located start positions in the genome mapped with short reads from all samples and counted the number of reads of the same length from the same sample for each start position mapped. we then identified the (top %) most abundant start positions by the read count of all start positions located as described above as candidate loci for putative small rnas. we next merged overlapping candidate loci into single loci to create a set of nonredundant candidate loci for putative small rnas. we counted the number of reads that were to nt in length that mapped to each of nonredundant candidate loci across all samples and estimated the changes in expression of these candidate loci during virus infection by the use of a method similar to that used for mirna differential analysis. the differentially expressed candidate loci were predicted as putative small rna loci. to minimize the possibility that an identification of a non-mirna small rna was due to a partial misalignment of short reads that originated from very abundant rnas sharing high sequence similarities, we located all genomic regions with high sequence similarity to each putative small rna locus. these "homologous" genomic regions were identified as follows: (i) for putative small rna loci that were relatively long (Ͼ nt), we used the standalone blat program with the default setting for dna alignment to align the genomic sequence of the small rna locus to the reference genome sequence ( ); (ii) for small rna locus that were relatively short ( nt or less), we extracted all reads uniquely mapped to the small rna locus as described above. we then used the bowtie program as described above ( ) but with criteria that were less stringent (i.e., with a change from mismatches to mismatches) and with adjustments of the program settings to report all valid alignments (bowtie option "-all") to realign the extracted reads to the reference genomes. for both alignment approaches, we treated the genomic region corresponding to each obtained alignment as a genomic region "homologous" to the small rna locus. to remove redundancies, we merged overlapping homologous genomic regions into single homologous genomic regions. for each identified homologous region, we counted the number of uniquely mapped reads obtained across all samples as described above. we then compared the read counts of these homologous regions to that of the genomic region corresponding to the original small rna locus. we kept a putative small locus for further analysis only when the number of uniquely mapped reads in the genomic region corresponding to the small rna locus was at least twice as large (on average, across all samples) as that of the homologous region with the highest number of uniquely mapped reads. we used rnafold ( ) and rnaz ( ) to perform rna secondary structure predictions for putative small rna loci. conserved rna secondary structures (p Ͼ . ) were predicted using -way multiple alignments downloaded from the ucsc genome browser (http://genome.ucsc .edu) and rnaz ( ) . for predictions performed using rnafold ( ), we extracted three genomic windows of different sizes ( , , and nt) surrounding each putative small rna loci. when a locus was shorter than the desired genomic window, we expanded the locus by first adding neighboring candidate loci within the desired genomic window and then extending the sequence at both ends to achieve the desired width. to evaluate the statistical significance of the secondary structures predicted by rnafold, we generated randomly permutated sequences from the original sequence, with dinucleotide frequencies preserved using ushuffle ( ) , and used rnafold to similarly fold all random sequences. we calculated the percentage of times that a random sequence exhibited a higher minimum free energy level than the original sequence as the p value of the predicted secondary structure for the locus. a prediction with p Ͻ . was considered significant. quantitative real-time pcr (qpcr). quantitative real-time pcr was used to validate expression of selected mirnas on replicate samples. for each mirna qrt-pcr assay, the total rna input used was ng per sample. qpcr (including reverse transcription-and mirna-specific primer sets) was performed using a mircury lna universal rt mi-crorna pcr system (exiqon, woburn, ma). qpcr was performed with an abi real-time pcr system and sybr green chemistry. each assay was run in triplicate with power sybr green pcr master mix (applied biosystems, carlsbad, ca) for mirna detections in a -l total reaction volume. supplemental material for this article may be found at http://mbio.asm.org /lookup/suppl/doi: . /mbio. - /-/dcsupplemental. figure s , pdf file, . mb. figure s , pdf file, . mb. figure s , pdf file, . mb. figure s , pdf file, . mb. figure s , pdf file, . mb. figure s , pdf file, . mb. viral and cellular micrornas as determinants of viral pathogenesis and immunity modulation of hepatitis c virus rna abundance by a 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molecules in somatic tissues microrna profile changes in human immunodeficiency virus type (hiv- ) seropositive individuals computational identification of hepatitis c virus associated microrna-mrna regulatory modules in human livers biogenesis of small rnas in animals microarray profiling of micrornas reveals frequent coexpression with neighboring mirnas and host genes identification of mammalian microrna host genes and transcription units a human snorna with microrna-like functions snorna, a novel precursor of microrna in giardia lamblia small rnas derived from snornas human trna-derived small rnas in the global regulation of rna silencing the influenza virus ns protein: a novel inhibitor of pre-mrna splicing the influenza virus ns protein binds to a specific region in human u snrna and inhibits u -u and u -u snrna interactions during splicing u atac snrna, the highly divergent counterpart of u snrna, is the specific target that mediates inhibition of at-ac splicing by the influenza virus ns protein functional characterization of xendou, the endoribonuclease involved in small nucleolar rna biosynthesis the structure of the endoribonuclease xendou: from small nucleolar rna processing to severe acute respiratory syndrome coronavirus replication capture and sequence analysis of rnas with terminal =, =-cyclic phosphates the expanding snorna world mammalian mi-crornas predominantly act to decrease target mrna levels combinatorial microrna target predictions long noncoding rnas: functional surprises from the rna world requirement of bic/microrna- for normal immune function regulation of the germinal center response by microrna- isolation of genes controlling apoptosis through their effects on cell survival a critical role for non-coding rna gas in growth arrest and rapamycin inhibition in human t-lymphocytes influenza a virus-generated small rnas regulate the switch from transcription to replication influenza a virus expresses high levels of an unusual class of small viral leader rnas in infected cells the collaborative cross, a community resource for the genetic analysis of complex traits a mouse-adapted sars-coronavirus causes disease and mortality in balb/c mice ultrafast and memory-efficient alignment of short dna sequences to the human genome the functional rna database . : databases to support mining and annotation of functional rnas the sequence alignment/map format and samtools application of massively parallel sequencing to microrna profiling and discovery in human embryonic stem cells. genome res blat-the blast-like alignment tool fast folding and comparison of rna secondary structures fast and reliable prediction of noncoding rnas ushuffle: a useful tool for shuffling biological sequences while preserving the k-let counts we thank elizabeth rosenzweig and jean chang (university of washington, seattle) for generating qpcr data. we thank lynn law and janine bryan (university of washington, seattle) for helpful suggestions. key: cord- -hscgmis authors: gralinski, lisa e.; bankhead, armand; jeng, sophia; menachery, vineet d.; proll, sean; belisle, sarah e.; matzke, melissa; webb-robertson, bobbie-jo m.; luna, maria l.; shukla, anil k.; ferris, martin t.; bolles, meagan; chang, jean; aicher, lauri; waters, katrina m.; smith, richard d.; metz, thomas o.; law, g. lynn; katze, michael g.; mcweeney, shannon; baric, ralph s. title: mechanisms of severe acute respiratory syndrome coronavirus-induced acute lung injury date: - - journal: mbio doi: . /mbio. - sha: doc_id: cord_uid: hscgmis systems biology offers considerable promise in uncovering novel pathways by which viruses and other microbial pathogens interact with host signaling and expression networks to mediate disease severity. in this study, we have developed an unbiased modeling approach to identify new pathways and network connections mediating acute lung injury, using severe acute respiratory syndrome coronavirus (sars-cov) as a model pathogen. we utilized a time course of matched virologic, pathological, and transcriptomic data within a novel methodological framework that can detect pathway enrichment among key highly connected network genes. this unbiased approach produced a high-priority list of genes in one pathway out of over , genes that were differentially expressed following sars-cov infection. with these data, we predicted that the urokinase and other wound repair pathways would regulate lethal versus sublethal disease following sars-cov infection in mice. we validated the importance of the urokinase pathway for sars-cov disease severity using genetically defined knockout mice, proteomic correlates of pathway activation, and pathological disease severity. the results of these studies demonstrate that a fine balance exists between host coagulation and fibrinolysin pathways regulating pathological disease outcomes, including diffuse alveolar damage and acute lung injury, following infection with highly pathogenic respiratory viruses, such as sars-cov. revealed the presence of exudative-phase dad along with increased numbers of macrophages in the lung ( , ) . many patients also had lung hemorrhage, noncardiogenic pulmonary edema, and/or hyaline membrane formation in the alveolar spaces. patients with longer-term disease (Ͼ days postinfection [dpi]) manifested with proliferative-or organizing-phase dad that encompassed % to % of the lung ( ) . they also had signs of lung fibrosis in both alveolar and interstitial spaces. sars survivors continue to show lingering effects of their illness with complications including reduced lung elasticity and function ( ) . ali and the development of dad may also occur with other respiratory virus infections, including influenza virus (h n or h n - ) and adult respiratory syncytial virus (rsv) infection, especially in the elderly ( ) ( ) ( ) . much like sars-cov infection, disease progression is first associated with an exudative phase of dad, which may progress into organizing-phase dad ( ) . the presence of exudates in the lung, composed of fibrin and proteinaceous material, blocks normal gas exchange ( ) . without clearance of these exudates, lung pathology progresses toward fibrotic disease with collagen deposition and conversion of the exudates into fibrous tissue. thus, ali and ards represent a common but poorly studied host response to virus-induced severe lung disease. correlation-based gene coexpression network inference approaches are unbiased and powerful tools that can be used to analyze large multidimensional data sets, including expression microarray and proteomics data. specifically, they capture complex relationships between host gene expression patterns and emergent correlative behaviors ( ) ( ) ( ) . these approaches posit that genes and protein products are organized into functional modules according to cellular processes and pathways. in this work, we use weighted gene correlation network analysis (wgcna) methods to interpret the dynamic sars-covmediated transcriptional response. while wgcna has previously been applied to cancer, mouse, and yeast genetics, we use the approach to enhance systems-based analysis of virus infection ( ) ( ) ( ) . key to this approach is the ability to discover systemic gene expression patterns based on underlying correlation structures, which is not biased by existing knowledge of pathways or interactions. wgcna partitions gene expression into groups of transcripts called subnetwork modules with highly correlated behaviors. it has been shown elsewhere that connectivity inferred through wgcna is a strong predictor of related biological function ( ) . a key feature of these subnetwork modules, or eigengenes, is that the most highly connected hub genes within the module provide candidate mediators of disease ( , ) . we further refined these candidates by modifying the tool to identify enriched pathways associated with candidate lists, allowing us to combine connectivity and predicted network structure with biological functions and known interactions within a hypothesistesting framework. pathogenesis in mouse-adapted-sars-cov-infected animals closely mimics the pathologies that were observed in human patients ( ) , including age-dependent disease severity. to model system-wide behaviors following sars-cov infection, we performed a dose-response study that included biological sampling at multiple time points, transcriptional and proteomic systems biology data, and mathematical modeling algorithms to identify signaling networks associated with progression from severe to lethal disease outcomes. these data demonstrate the successful use of highly refined modeling algorithms to identify and validate novel genes and pathways that play critical roles in sars-cov pathogenesis and the development of ali following virus infection in the lung. in a healthy lung, fibrin levels are controlled through the action of the enzyme plasmin and the urokinase pathway through extracellular matrix (ecm) remodeling ( ) . ecm remodeling also involves complex interactions between a number of metalloproteinases and their respective regulatory protein networks ( ) . disruption of the urokinase pathway is associated with fibrotic lung disease or lung hemorrhage, depending on highly directional signaling cascades. tissue plasminogen activator (tpa or plat), negatively regulated by serpine , is given as an anticlotting agent to recent stroke patients to promote cleavage of plasminogen into plasmin and enhance breakdown of fibrin clots ( ) . although ecm and wound healing pathway activation are known to be important in lung disease, these processes are largely unstudied in relation to in vivo respiratory virus pathogenesis. in this work, we demonstrate a critical role for the urokinase pathway in regulating severe end-stage lung disease outcomes following sars-cov infection. to develop an unbiased strategy to explore the molecular mechanisms regulating sars-cov pathogenesis and host responses, we studied virus replication kinetics, clinical disease severity, pathological changes, and variations in host transcriptomics along with targeted proteomics using a dose escalation study. the goal was to identify contrasting outcomes for modeling the role of host responses in disease. groups of week-old c bl /j (b ) mice were infected with to pfu of recombinant mouse-adapted sars-cov (ma ) or mock infected (phosphate-buffered saline [pbs]). clinical changes were noted daily, and biological samples were collected at , , , , and days postinfection (dpi) to allow for profiling of the different phases of infection. mice infected with the lowest dose, pfu, lost little if any weight and showed few clinical signs of disease (fig. a ). mice infected with or pfu experienced transient weight loss and showed minor clinical signs of disease. infection with pfu resulted in continuous weight loss with Ն % weight loss by day ; at late time points, these animals showed significant clinical disease, including hunched posture, ruffled fur, decreased locomotion, labored breathing, and death. virus load in the lung was quantified by plaque assay and was reflective of both the initial infectious dose and the overall replication kinetics. mice infected with pfu of ma had relatively low or undetectable titers (fig. b) . infection with the three higher doses resulted in the highest titers at or dpi, an~ -log drop in titer at dpi, and a decrease of several logs by dpi, regardless of changes in weight loss. virus load was confirmed by quantitative pcr (qpcr) for both virus genome and specific viral genes (see fig. s a in the supplemental material; also data not shown). consistent with a previous report ( ) , minimal to no detectable virus was found in the serum or other organs at dpi (see fig. s b to d), and no detectable virus was present in these other organs or serum at day . pathological findings. lung pathology varied by both infectious dose and time but was not dependent on virus load. full histology scoring is available in table s a in the supplemental material. at dpi, infected mice showed few inflammatory cells and healthy conducting airways, alveoli, and vasculature. by day , mice in all infection groups showed damage to the large and small conducting airways of the lung in a dose-dependent manner, consisting of denuded patches of airway epithelial layers and apoptotic debris, often obstructing the small airways. at dpi, all infected animals had low levels of epithelial cell denudation and airway debris along with mild thickening of the interstitial membranes. low-level perivascular cuffing was also present at this time point, and moderate, sporadic hemorrhage was consistently observed in mice infected with to pfu of ma at dpi. by dpi, lung disease was most pronounced in the parenchyma in mice infected with a -pfu dose and included thickening of the interstitial septum, inflammatory infiltrates including neutrophils, and pink proteinaceous exudates, typical of dad (see fig. s e in the supplemental material). importantly, these mice had clearly visible hyaline membranes, a characteristic of fatal sars-cov infection in humans ( ) . these mice also showed severe and complete lung hemorrhage at dpi, while mice infected with pfu had moderate but notable hemorrhage (see table s a ). mice infected with lower doses had only mild to moderate vascular cuffing and interstitial inflammation along with sporadic and mild hemorrhage but were otherwise absent of severe lung disease. global host expression patterns. lung gene expression patterns were compared between mice with each infection dose and mock-infected mice to gain insight into sars-cov-induced lung disease. consistent with earlier reports ( ) , there was minimal differential expression (de) at the transcript level at dpi (i.e., |log fold change [fc]| of Ͼ . and false discovery rate [fdr] adjusted p value of Ͻ . ), regardless of infectious dose ( fig. a) . however, the number of de genes escalated with increasing dose or number of days postinfection. overlap of de genes was determined between mice infected with pfu and those infected with pfu. the maximum percentage of overlap in de genes was measured at and dpi, despite the low number of de genes. the least overlap between these doses was measured at dpi ( fig. b) , with % of genes showing de at pfu but not at pfu. we hypothesized that these genes with different expression patterns in mice infected with pfu (here referred to as the lethal dose) from those in mice infected with pfu (here referred to as the sublethal dose) could provide insight into the molecular mechanisms and host expression patterns that regulate lethal versus sublethal disease following sars-cov infection. eigengene network analyses following sars-cov infection. gene expression patterns were compared between mice infected with the sublethal dose and lethal dose using the wgcna approach ( ) . in the consensus network analysis, module eigengenes (clusters of genes with similar expression patterns over time) were identified ( with statistically significant connectivity; p value of Ͻ . using permutation test) (fig. a) . many of the eigengenes represented groups of genes with similar expression patterns and dynamics between the lethal and sublethal doses. we chose to focus on one specific module eigengene that displayed distinct dose dynamics, hypothesizing that the differences in transcript expression could explain the differential disease outcome. the eigengene that we pursued, shown in blue and circled in fig. a , had significantly higher expression at , , and dpi in lethally infected mice than in sublethally infected mice ( fig. b ; expression patterns for the other statistically significant eigengenes can be found in fig. s in the supplemental material). this module was prioritizing based on differential expression between doses at day based on overall upregulation in the lethal dose infection and differential module regulation when comparing doses. specifically, ⌬ average log fold change (fc) was calculated by subtracting the average log fc for each dose. using this strategy, we selected the blue module because it is strongly upregulated for the lethal dose ( . average log fc) while the sublethal dosing was only mildly upregulated ( . average log fc) and the resulting ⌬ average log fc was . (an almost -fold difference). this eigengene module was comprised of differentially expressed transcripts. when the unbiased gene list from the blue module was analyzed by the genego metacore knowledge base, the top functional categories were related to cell adhesion, ecm remodeling, and wound healing (see table s in the supplemental material). the same knowledge base was used to visualize the highest-priority ecm remodeling signaling pathways containing proteins en- mice infected with to pfu of sars-cov ma had low levels of transient weight loss, while mice infected with pfu showed increasing weight loss over time. (b) virus titer in the lung was quantitated by plaque assay. the mean value of all samples with detectable virus in each group is shown (three mice at pfu and two each at , , and pfu had detectable virus by plaque assay at day ; bld, below the limit of detection of pfu per lung). coded by the gene set with differential signaling in lethally or sublethally infected mice (see fig. s ). to further refine our candidates, we assessed the connectivity and module membership within known biological pathways. the full statistical analysis workflow is described in materials and methods, and a diagram of the workflow is shown in fig. s . the urokinase pathway was the most significantly enriched for both module membership (p value ϭ . , odds ratio [or] ϭ . ) and transcriptional connec-tivity (p value ϭ . , , permutations), making it the ideal target for validation. while the eigengenes themselves were not priority ranked, the unbiased method that we used to prioritize targets within the blue eigengene model could easily be applied to other modules. the urokinase pathway contributes to the broader ecm remodeling pathway shown in fig. s in the supplemental material, and its members, including serpine , serpine , plat, and plaur, along with other ecm remodeling genes, show significantly higher expression at dpi in the lethal dose than in the sublethal dose (see fig. s a ). while the urokinase pathway has not previously been associated with respiratory virus infection, its involvement in ischemic events and metastatic cancers has been well documented ( ) . furthermore, fibrin turnover and alveolar coagulopathy are known to significantly contribute to the severity of ali following injury or chemical insult ( ) . based on these factors, we explored the impact of the urokinase pathway on sars-cov infection. acute lung injury following acute severe respiratory infection. expanding on the identification of the urokinase pathway, we examined whether conserved rna expression signatures indicated development of a procoagulant or profibrinolytic state after sars-cov infection. tissue factor, factor viia, and serpine are major factors responsible for the development of a procoagulant and antifibrinolytic state in the alveoli ( , ) . changes in alveolar hemostatic balance and intra-alveolar fibrin deposition are also stimulated by cytokine expression after ali. proinflammatory (interleukin- ␤ [il- ␤], tumor necrosis factor alpha [tnf-␣], and il- ) and profibrotic (transforming growth factor ␤ [tgf-␤], connective tissue growth factor [ctgf], and platelet-derived growth factor [pdgf]) cytokine transcripts were significantly elevated, starting by dpi and through the course of the lethal sars-cov ma infection (see fig. s b in the supplemental material). in addition, genes associated with the induction of a procoagulant state (thrombin, factor viia, factor xia, factor xiia, plau, plat, tissue factor f r) ( ) and other fibrinolysin pathway components were altered by infection. both pro-and antifibrinolytic genes in the urokinase pathway showed increased expression in our sars-cov infection model, demonstrating the complicated nature of this signaling cascade. previous reports show that it is rare for one branch of the urokinase pathway to have altered regulation without host feedback loops adjusting the expression of other pathway genes ( ) . in general, the transcripts shown in fig. s b represent a lung with high levels of inflammatory and fibrinolytic gene expression, and this transcriptional picture is supported by the hemorrhage observed in the lungs of sars-cov-infected mice. to increase our understanding of the changes in the lung following acute viral infection, we examined microarray data from previously published respiratory infection models of ards. transcriptional data from the lungs of influenza virus-infected balb/c mice showed similar patterns of differential expression in the genes responsible for coagulation, surfactant production, and fibrinolysis (see fig. s in the supplemental material). furthermore, b mice infected with the h n influenza virus strain ca had high levels of proinflammatory, profibrotic, and urokinase pathway gene expression relative to mock-infected mice along with decreased surfactant expression (doses of to pfu [unpublished data]). overall, these transcription signatures are consistent with reported viral pathological and proteomic findings, strongly supporting the development of altered alveolar hemostatic balance seen during the early exudative phase of dad. urokinase pathway in sars-cov pathogenesis. to further investigate the role of the urokinase and ecm remodeling pathways, we analyzed lung proteomics. proteomics analysis revealed that fibrin beta and gamma chains, factor viii, and cytokeratins, all major components of hyaline membranes ( , ) , have increased expression in the lungs following both lethal and sublethal sars-cov infection compared to mock infection (fig. a) . similarly, surfactant proteins, which typically protect against hyaline membrane formation and lung injury ( ) , have reduced expression in both the lethal and sublethal doses of sars-cov compared to uninfected controls. however, at late time points, several of these same proteins sharply diverge between the lethal and sublethal doses. fibrin beta and gamma chain levels are increased in abundance relative to mock infection only at day in the lethal infection. in contrast, fibrin chains continually decrease after dpi in sublethal infection. a similar pattern is observed with keratin , with increased protein abundance observed in the lethal dose. to further confirm these findings, we used a martius scarlet blue (msb) stain to demonstrate the presence of fibrin deposition in the lungs at day . positive staining was observed in the lungs of lethally infected mice but not in the lungs of mock-infected or sublethally infected animals (fig. b) . together, the proteomics data are consistent with pathophysiological changes resulting in increased fibrin deposition and hyaline membrane formation in the lungs of lethally infected mice. fibrin is cleaved and degraded by plasmin, having been converted from plasminogen by urokinase pathway members ( ) . analysis of plasminogen peptides revealed a distinct and significant increase following a lethal dose of sars-cov (fig. a) . in contrast, the sublethal infection resulted in an initial augmentation but then maintained a low level of plasminogen peptides throughout the course of infection. it is important to note that plasminogen and plasmin are indistinguishable using these proteomics methods. fibronectin, another component downstream of plasmin activation, also demonstrated increased protein expression in the lethal dose compared to the sublethal dose at day . these data are consistent with augmented plau/urokinase activity in the lethal dose despite maintaining similarly elevated levels of serpine , an inhibitor of the urokinase pathway. finally, diseases associated with premature or excessive breakdown of fibrin clots are characterized by hemorrhage and the accumulation of fluid exudates in the alveoli. consistent with the observation of vascular leakage into alveolar spaces and the development of dad, levels of the serum protein albumin found in the lung were significantly elevated in lethally infected mice (fig. a) . targeted knockout mice. to complete the systems biology loop (broad study, model/hypothesis generation, and then testing of the model), we selected a targeted member of the urokinase pathway for validation studies. genes within the blue module were ranked according to their overall connectivity and agreement with the module eigengene expression, and serpine was the highest-ranked urokinase pathway member (correlation with the module eigengene [k me ] of . ). the approximate location of serpine within the blue module is highlighted in fig. a . ser-pine Ϫ/Ϫ mice infected with a sublethal dose of ma continued to lose weight through day postinfection (fig. a ) and showed significantly more weight loss than did b controls at days to postinfection (p value of Ͻ . ). additionally, one serpine Ϫ/Ϫ mouse died, suggesting a necessary and protective role for ser-pine in modulating sars-cov pathogenesis. clinical disease in serpine Ϫ/Ϫ mice mirrored that in lethally infected b animals (fig. a) , with severely decreased locomotion, hunched posture, and labored breathing at late time points. despite differences in clinical symptoms, virus load in the lung showed no significant differences between knockout and wild-type (wt) mice (fig. b ) at or dpi, demonstrating that serpine had no influence on virus replication dynamics. when b and serpine Ϫ/Ϫ mice were infected with the lethal dose of sars-cov, the serpine Ϫ/Ϫ mice succumbed to infection sooner than did the b controls (fig. c ) (p value of Ͻ . ), further indicating a protective role for ser-pine and the urokinase pathway in sars-cov-induced disease. while pfu is lethal in -week-old b mice, wild-type mice rarely succumb before dpi. examining lung sections, we observed cuffing of inflammatory cells around the large airways and vasculature of all infected mice at dpi (see table s b in the supplemental material), which increased by dpi. at dpi, there was mild interstitial membrane thickening and low numbers of inflammatory cells were observed in the alveolar spaces of all infected mice; by dpi, disease in the parenchyma had developed to moderate levels. gross hemorrhage levels of the lung were noted at the time of tissue harvest. ser-pine Ϫ/Ϫ mice trended toward increased hemorrhage in their lung tissue compared to b controls at day , and by dpi, the knockout mice exhibited significantly more lung hemorrhage ). green indicates that expression in serpine -knockout mice is lower than that in b mice, and red indicates that expression in serpine -knockout mice is higher than that in b mice. (p value of Ͻ . by student's t test) (fig. d) . combined, these data confirm our model prediction that serpine and the urokinase pathway play an important role in the pathogenesis of sars-cov infection. examination of gene expression in the lungs of serpine Ϫ/Ϫ mice and b controls showed that sars infection causes greater plat, il- , tissue factor (f ), and serpine expression in the absence of serpine (fig. e ). serpine functions similarly to ser-pine in control of plat and plau activity. il- ␤, tgfb , and plau all had increased expression in serpine Ϫ/Ϫ lungs at day relative to b controls but decreased expression at day , while ptgs and tnf had decreased expression in the serpine Ϫ/Ϫ mice at both time points. these data support the existing evidence of feedback loops within the urokinase pathway as well as confirming the overall dysregulation of the urokinase pathway following sars-cov infection. combined, these transcriptional changes indicate that sars-cov-infected serpine Ϫ/Ϫ mice had increased fibrinolytic activity in their lungs and a decreased inflammatory response relative to b controls. a simplified schematic of the urokinase pathway in the presence and absence of serpine is shown in fig. a and b, illustrating how the lack of serpine could lead to increased plat and plau activity and increased hemorrhage and disease. to further confirm the importance of the urokinase pathway in sars-cov pathogenesis, we infected mice deficient in plat, one of the two plasmin-cleaving proteins that are activated by ser-pine . plat expression was also highly connected to that of the overall blue module with a k me of . . mice deficient in plat should have a complicated phenotype because of the ability of plau to cleave fibrin and compensate in this system. sublethally infected plat Ϫ/Ϫ mice showed a slight, but not statistically significant (p ϭ . ), acceleration in recovery of weight loss compared to b controls (see fig. s a in the supplemental material) and a significant increase in exudates in the lung (p Ͻ . ; see fig. s e ). in contrast, plat Ϫ/Ϫ mice infected with a lethal dose of ma showed notable early mortality compared to b controls, although those knockout mice that survived early infection went on to recover (see fig. s b and c). lethally infected plat Ϫ/Ϫ mice also trended toward less hemorrhage in their lungs than did b controls. b and plat mice infected with equal doses of ma had similar virus loads in the lung at both and days postinfection (see fig. s d ), demonstrating that manipulation of the urokinase pathway did not influence virus replication dynamics but instead impacted the host response in regulating the development and resolution of ali. in this study, we highlight an unbiased modeling approach using systems biology to investigate viral pathogenesis and identify a novel host pathway involved in sars-cov disease progression. while wound healing and extracellular matrix remodeling pathways have previously been associated with lung disease ( ) , this association had not extended into studies of acute respiratory virus infection. our data suggest that dysregulation of the urokinase pathway during sars-cov infection contributes to more severe lung pathology and that serpine plays a protective role following infection. similar changes in the urokinase, coagulation, and fibrinolysin pathway expression signatures are noted following highly pathogenic sars-cov and influenza virus infections (see fig. s b and s in the supplemental material), arguing for a con-served role for these pathways in virus-induced end-stage lung diseases, like ali and ards. the lack of well-defined statistical methods for organizing complex data sets into prioritized modules has hampered the power for systems-based discovery. while gene coexpression networks provide one approach to capture the complex relationships between transcripts, the approach can also be integrated with other types of quantitative data, like infection outcomes, patho- logical findings, and clinical disease severity, resulting in a clearer distinction between principal components (eigengenes) and biological processes. an important goal of these types of integrative analyses is the deconstruction of large data sets into high-priority, constructive gene signatures that provide predictive power for downstream analysis and validation. however, these types of analyses have rarely moved beyond simulations produced from existing data sets. we have modified a wgcna approach to cluster similarly regulated genes into eigengene modules in order to compare and contrast module behavior as a function of virus dose and disease severity. we then developed strategies to prioritize targets for downstream analyses at the level of larger pathway analyses which were followed by analysis of specific genes. this approach was applied to our virus infection data sets containing over , de genes. it resulted in the identification and characterization of four highly prioritized genes in the urokinase pathway whose differential expression was significantly associated with increased disease severity. we note that there are a number of enrichment approaches, such as gene set enrichment analysis (gsea) ( ) , or hypergeometric test-based approaches, such as gostat ( ) , which can identify genes behaving in coordinated ways to aid in interpreting gene candidates. however, these approaches are often focused on differential expression in the context of functional annotation. with our approach, we focus on de novo-inferred connectivity and the distribution of the most highly connected module members within canonical pathways. this allows for a much more focused refinement of the candidate list and enables the generation of direct hypotheses for perturbation testing among the candidates. our unbiased transcriptomics analysis approach identified eigengenes with different expression patterns (fig. a) . the eigengene network analysis and prioritization also identified several highly ranked innate immune signaling components, including myd , stat , and ptgs , as having high connectivity with the severity of sars-cov disease outcome. earlier studies have independently demonstrated that myd -and stat -knockout mice are highly susceptible to sars-cov pathogenesis and that blocking prostaglandin function protects old mice from sars-cov pathogenesis ( ) ( ) ( ) . stat signaling is known to affect cell cycle and apoptotic processes, and stat -knockout mice are particularly sensitive to bleomycin-induced lung fibrosis with increased collagen levels and enhanced fibroblast proliferation ( ) . together, these previous reports further confirm the efficacy of our modeling approach. to further validate our model, we analyzed genes from one module eigengene with connections to wound healing and ecm remodeling pathways, including the urokinase pathway, which displayed dose-dependent expression changes. both histology and proteomic data demonstrated that urokinase activity increased in a dose-dependent manner following sars-cov infection. to complete a validation loop, we characterized disease outcomes in serpine Ϫ/Ϫ and plat Ϫ/Ϫ mice. the serpine Ϫ/Ϫ mice, deficient in a major protein in the urokinase pathway, were significantly more susceptible to sars-cov infection than were wt controls in terms of both weight loss and lung hemorrhage (fig. a and d) , while the plat Ϫ/Ϫ mice showed a mixed pathogenic phenotype that suggested improved outcome but significant impairment of lung function (see fig. s a and e in the supplemental material). these data confirm predictions that the urokinase pathway and ecm remodeling are important for regulating sars-cov pathogenic outcomes and demonstrate the delicate balance between development of hemorrhage and fibrosis following ali. while previous studies of sars-cov pathogenesis showed the presence of fibrin in the lung following infection, the importance of ser-pine and the urokinase pathway in development of disease was unknown. furthermore, the use of unbiased methods to identify the urokinase pathway as a regulator of sars-cov disease represents an important advance in analysis of microarray data. ali and its more severe form, ards, are devastating end-stage lung diseases that can arise following a variety of acute insults to the lung epithelium and occur frequently following infection with sars-cov and h n and h n influenza viruses in humans ( , , ) . despite significant advances in treatment options, the overall mortality rates remain substantial and range between and %, resulting in~ , deaths in the united states and over a million deaths globally each year ( ) . while the underlying molecular mechanisms governing virus-induced ali remain to be elucidated, our transcriptomics, proteomics, modeling, and validation approaches using targeted knockout mice have coalesced into a model of altered hemostatic balance defined by the expression of procoagulative and antifibrinolytic factors resulting in the induction of an exudative phase of dad after infection. the urokinase pathway regulates fibrinolytic and procoagulative responses designed to prevent vascular permeability and hemorrhage ( , ) . fibrin levels dramatically increased following lethal sars-cov ma infection ( fig. a and b) . excess fibrin was likely mediated by serpine -driven inhibition of urokinase and tissue type plasminogen activators (plau and plat) and by blockade of plasmin activity by ␣ -plasmin inhibitor, whose transcripts are elevated following sars-cov infection. we suggest that lethal sars-cov infection overwhelms the normally protective, profibrinolytic signaling of the urokinase pathway, leading to overall dysregulation, including increased serpine expression, and severe lung disease. while fibrin is required for normal wound healing, persistent and excessive intra-alveolar fibrin levels can contribute to acute inflammatory and chronic interstitial lung disease. fibrin stimulates the production of profibrotic growth factors ( ) , and many profibrotic cytokine transcripts, like tgf-␤, ctgf, and pdgf, are elevated following sars-cov or influenza virus infection in mice (see fig. s and s in the supplemental material) ( , ) . pulmonary surfactant protein and transcript signatures are also reduced following acute viral infection (fig. a ) (see also fig. s ) ( ) , potentially leading to collapse or closure of alveoli and loss of lung compliance (changes in lung volumes) ( ) . in combination, a high-fibrin/low-surfactant intra-alveolar environment provides an ideal environment for fibroblast adherence and growth, resulting in collagen deposition and development of lung fibrosis ( ) . finally, fibrin and fibrin breakdown products increase vascular permeability, stimulate migration and proliferation of inflammatory cells, and promote recruitment of neutrophils to the lung ( , ) . although we cannot absolutely ascribe this phenotype to elevation in the levels of fibrin and fibrin breakdown products, fluorescence-activated cell sorting (facs) analysis shows significant increases in lung neutrophil counts, as a function of increasing sars-cov dose (data not shown). more recent studies have also shown that serpine inhibits neutrophil apoptosis ( ) , suggesting that neutrophil recruitment and effector function likely contribute to more severe disease outcomes following sars-cov ma infection. similar findings have been reported with highly pathogenic influenza viruses, including h n and influenza viruses ( ) . fibrin accumulation in the lung is a hallmark of ali and ards, and a reduced capacity to cleave and remove fibrin deposits corresponds with a poor clinical patient outcome ( ) . notably, increased serpine levels were measured in the blood of sars-cov-infected patients during the - epidemic, and increased serpine expression has been measured in the lungs of sars-cov-infected macaques ( , ) . necropsy findings from h n influenza patients revealed accumulations of fibrin in the lung along with pulmonary edema and other signs of dad and hemorrhage ( ) . furthermore, and h n influenza virus-infected mice showed increased levels of urokinase pathway transcripts (see fig. s in the supplemental material; also data not shown). the host response to streptococcus pneumoniae also includes upregulation of serpine expression to protect against hemorrhage and ali. the deubiquitinase cyld provides critical control of serpine expression ( ) , and in the absence of cyld, infected mice develop lung fibrosis. experiments using the mouse model for pneumonic plague have shown that deletion of the bacterial plasminogen activator, which cleaves plasminogen and also serpine , changes the course of disease and protects mice from lethal lung injury ( ) . combined, these data demonstrate the critical balance of coagulative and fibrinolytic signaling in the lung following injury and suggest a common modality for development of pathogen-induced ali and ards after microbial infection. the systems biology approach promises to aid in our understanding of complex biological processes through the highthroughput, unbiased analysis of broad data sets. previous work has demonstrated the ability to identify distinct transcriptomic signatures predictive of the immune response to the yellow fever and influenza vaccines ( , ) . our approach highlights the ability to maintain statistical rigor and analytical prioritization that is both driven by and focused on biological relevance and experimental validation. using this approach, mathematical models and their predictions, as well as the subsequent perturbations and refinement, allowed us to characterize the molecular mechanisms that lead to the initiation and progression of infectious disease pathogenesis. a comparative approach with multiple viruses, such as sars virus and highly pathogenic influenza virus, can identify common pathways that lead to severe end-stage lung disease and provide high-value targets for therapeutic approaches that address multiple pathogens. these data demonstrate the successful use of systems biology approaches to identify and then validate novel genes and pathways that play critical roles in sars-cov pathogenesis in the lung and suggest targeting urokinase and coagulopathy-related signaling pathways as a therapeutic approach to treat virus-induced ali. recombinant mouse-adapted sars-cov (ma ) was propagated on vero e cells, and its titer was determined. for virus titration, half of the right lung was used to give pfu per lung using vero e cells with a detection limit of pfu ( ) . all experiments were performed in a class ii biological safety cabinet in a certified biosafety level laboratory containing redundant exhaust fans by workers wearing personnel protective equipment, including tyvek suits, hoods, and highefficiency particulate air (hepa)-filtered powered air-purifying respirators (paprs). animals. c bl/ j (stock no. ), serpine Ϫ/Ϫ (stock no. ), and plat Ϫ/Ϫ (stock no. ) mice were obtained from the jackson laboratory (bar harbor, me). mice were anesthetized with a mixture of ketamine and xylazine and intranasally infected with either l of phosphate-buffered saline (pbs) alone or ma (e.g., to pfu /animal). animals were maintained in hepa-filtered sealsafe cages (techniplast, buguggiate, italy). all animal housing and care were conducted in accordance with all university of north carolina (unc)-chapel hill institutional animal care and use committee guidelines. lung tissues from a total of infected mice were harvested at each dose on days , , , and postinfection in the dose-response study. lung tissues from a total of infected mice were harvested at days and postinfection in the knockout mouse studies. mock-infected animals were age matched and harvested at each time point. weight loss significance was determined by student's t test (microsoft excel), and significance in survival data was determined by the mantel-cox test (graphpad). qpcr assays were used to confirm the infection status of all mice. all mouse studies were performed at the university of north carolina (animal welfare assurance #a - ) using protocols approved by the unc institutional animal care and use committee (iacuc). histological analysis and hemorrhage. gross hemorrhage of lung tissue was observed immediately after euthanasia and scored on a scale of (no hemorrhage in any lobe) to (extreme and complete hemorrhage in all lobes of the lung). lung tissues for histological analysis were fixed in % formalin (fisher) for at least days, tissues were embedded in paraffin, and -m sections were prepared by the unc histopathology core facility. to determine the extent of inflammation, sections were stained with hematoxylin and eosin (h&e) and scored in a blinded manner that is detailed in text s in the supplemental material. slides containing adjacent sections of lung tissue were stained with martius scarlet blue (msb) to visualize fibrin by the unc histology core. images were captured using an olympus bx microscope with an olympus dp camera. rna isolation, microarrays, quantitation of viral rna species, full details of statistical analysis of gene expression data, and data dissemination are all discussed in text s in the supplemental material. to identify groups of host transcripts that showed coordinated regulation in response to infection with sars-cov, we applied wgcna ( , ) . the wgcna method detects signaling subnetworks or modules consisting of groups of genes that are highly connected according to a neighborhood proximity metric called the topological overlap (to). to quantifies the degree of shared network neighbors. modules are represented as eigengenes by taking the first principal component of each set of module transcripts, which describe most of the variance in the module gene expression. we have adapted wgcna into a workflow with three key steps: (i) de novo network construction, (ii) consensus network analysis, and (iii) network enrichment (see fig. s in the supplemental material). the de novo network construction and the derivation of signaling modules are described in detail in text s in the supplemental material. in addition to analysis of the individual doses, we utilized a consensus approach to identify modules present across both networks (see fig. s ). two nodes should be connected in a consensus network only if all of the input networks agree on that connection. we define the consensus network similarity between two nodes as the minimum of the input network similarities. this allows identification of regulatory differences between two networks, even when the resulting topology/modules are conserved. novel prioritization within network modules. we prioritized the statistically validated host response modules to identify signaling events that differed between doses at day based on overall upregulation of the -pfu-dose treatment and differential module regulation when comparing treatments. average log fcs were calculated across modules for each dose. modules were labeled as upregulated at day if the average log fc was greater than zero. a ⌬ average log fc was calculated by subtracting the average log fc for each dose at day . network enrichment was then performed in two stages: functional enrichment using gene ontology (see text s in the supplemental material for details) and connectivity enrichment. to aid in the prioritization of the candidates to be validated within the modules, we examined the connectivity distribution relative to known canonical pathway membership within each module. we first identified canonical pathways that intersected with a given wgcna module and then took the average to connectivity between intersecting members. ten thousand random samplings of sets of genes of the same size were used to derive a null distribution and determine if a given pathway was enriched for to connectivity. this provides information regarding possible biological relevance to our putative candidates that had been ranked within the module based on their similarity to the eigengene. proteomics samples. for proteomics analysis, sars-cov-or mockinfected lung samples were collected and washed with mm ammonium bicarbonate buffer, homogenized in ml of m urea in mm ammonium bicarbonate buffer with glass beads for s at , rpm, and then incubated for h at room temperature. samples were then centrifuged at , rpm to remove debris and immediately stored at Ϫ °c. at each time point, analysis of individual mouse lungs provided intensity values for proteins derived from multiple peptides. the mean intensity (abundance) for each protein was then graphed as an average (n ϭ for infection, n ϭ for mock infection) for each group at each time point. missing or absent values were not scored; however, if no value was observed in any of the samples at a time point, the sample was registered with a single , representing "not detected." full details of the proteomics sample preparation methods are in the supplemental material. supplemental material for this article may be found at http://mbio.asm.org /lookup/suppl/doi: . /mbio. - /-/dcsupplemental. text s , docx file, . mb. figure s , tif file, . mb. figure s , tif file, . mb. figure s , tif file, . mb. figure s , tif file, . mb. figure s , tif file, . mb. figure s , tif file, . mb. figure s , tif file, . mb. table s , docx file, . mb. table s , docx file, . mb. a novel coronavirus associated with severe acute respiratory syndrome severe acute respiratory syndrome coronavirus-like virus in chinese horseshoe bats severe acute respiratory syndrome isolation of a novel coronavirus from a man with pneumonia in saudi arabia lung pathology of severe acute respiratory syndrome (sars): a study of autopsy cases from singapore pulmonary pathology of severe acute respiratory syndrome in toronto lung pathology of fatal severe acute respiratory syndrome sars: clinical virology and 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resulting in diminished t cell responses upon respiratory virus infection in mice susceptibility of signal transducer and activator of transcription- -deficient mice to pulmonary fibrogenesis clinical features and outcomes of severe acute respiratory syndrome and predictive factors for acute respiratory distress syndrome clinical characteristics of human cases of highly pathogenic avian influenza a (h n ) virus infection in china acute respiratory distress syndrome: estimated incidence and mortality rate in a million-person population base response network analysis of differential gene expression in human epithelial lung cells during avian influenza infections highly pathogenic avian influenza virus h n infects alveolar macrophages without virus production or excessive tnf-alpha induction respiratory syncytial virus infection alters surfactant protein a expression in human pulmonary epithelial cells by reducing translation efficiency epithelial and surfactant changes in influenzal pulmonary lesions alveolitis and collapse in the pathogenesis of pulmonary fibrosis disorganization of cultured vascular endothelial cell monolayers by fibrinogen fragment d the role of fibrin degradation products in neutrophil recruitment to the lung inhibition of neutrophil apoptosis by pai- h n and pandemic influenza virus infection results in early and excessive infiltration of macrophages and neutrophils in the lungs of mice functional genomics highlights differential induction of antiviral pathways in the lungs of sars-cov-infected macaques analysis of thrombotic factors in severe acute respiratory syndrome (sars) patients retrospective analysis of necropsy findings in patients of h n and their correlation to clinical features cyld negatively regulates transforming growth factor-betasignalling via deubiquitinating akt a plasminogenactivating protease specifically controls the development of primary pneumonic plague systems biology of vaccination for seasonal influenza in humans systems biology approach predicts immunogenicity of the yellow fever vaccine in humans vaccine efficacy in senescent mice challenged with recombinant sars-cov bearing epidemic and zoonotic spike variants the control of the false discovery rate in multiple testing under dependency key: cord- - qfhsj n authors: alagaili, abdulaziz n.; briese, thomas; mishra, nischay; kapoor, vishal; sameroff, stephen c.; de wit, emmie; munster, vincent j.; hensley, lisa e.; zalmout, iyad s.; kapoor, amit; epstein, jonathan h.; karesh, william b.; daszak, peter; mohammed, osama b.; lipkin, w. ian title: middle east respiratory syndrome coronavirus infection in dromedary camels in saudi arabia date: - - journal: mbio doi: . /mbio. - sha: doc_id: cord_uid: qfhsj n the middle east respiratory syndrome (mers) is proposed to be a zoonotic disease; however, the reservoir and mechanism for transmission of the causative agent, the mers coronavirus, are unknown. dromedary camels have been implicated through reports that some victims have been exposed to camels, camels in areas where the disease has emerged have antibodies to the virus, and viral sequences have been recovered from camels in association with outbreaks of the disease among humans. nonetheless, whether camels mediate transmission to humans is unresolved. here we provide evidence from a geographic and temporal survey of camels in the kingdom of saudi arabia that mers coronaviruses have been circulating in camels since at least , are distributed countrywide, and can be phylogenetically classified into clades that correlate with outbreaks of the disease among humans. we found no evidence of infection in domestic sheep or domestic goats. clusters of human infection indicate that human-to-human mers-cov transmission can occur ( , ) . however, the origin of the infection in most cases remains unknown. analysis of human mers-cov sequences by cotten et al. has revealed the presence of at least three circulating genotypes within the ksa alone ( ) . phylogenetic analyses of complete and partial genome sequences enabled estimates of the timing and geographic origins of individual viral clades. the authors proposed that mers-cov emerged in humans in and noted that sequence divergence between clades is consistent with several sporadic introductions of the virus into the human population, presumably from an animal reservoir. efforts to identify an animal reservoir have focused on bats and camels. bats harbor a wide range of betacoronaviruses ( ) ; furthermore, bat cell lines display the mers-cov receptor, dipeptidyl peptidase ( ) , and can be experimentally infected. a short sequence fragment consistent with mers-cov was reported in a bat in bisha, ksa, collected in close proximity to the home and workplace of the index case patient from whom the initial virus isolate was obtained ( ) . that same patient owned four pet dromedary camels (dc). serological analysis of those dc revealed the presence of antibodies reactive with mers-cov; however, no mers-cov sequences were found by pcr analysis of nasal or rectal swabs or serum. additional human cases have been associated with exposure to dc, and in some instances, investigators have described both serologic and genetic evidence of mers-cov infection in dc. memish and coworkers reported pcr detection of mers-cov sequences in a dc with respiratory illness owned by an individual with mers-cov who had no history of contact with other infected humans ( ). haagmans et al. investigated an outbreak of the disease among humans on a qatari farm and found mers-cov sequences in nasal swabs from of seropos-itive dc. analysis of open reading frame a (orf a) and fragments representing orf b, spike, and orf b revealed similarity but not identity to sequences obtained from the mers-covinfected humans at the same farm. the authors provide evidence that mers-cov can infect dc but cautiously conclude that data are insufficient to determine whether the infection spread from dc to humans, from humans to dc, or via another host to both species ( ) . several groups have reported serological reactivity with mers-cov or a closely related virus in dc in the middle east ( ) ( ) ( ) ( ) ( ) . in two regions of the ksa, hemida and colleagues detected antibodies to mers-cov in % of dc but not in sheep, goats, cattle, or chickens. the seroprevalence was lower in dc Ͻ year of age ( % versus %), suggesting widespread infection in early life ( ) . to determine the prevalence of mers-cov infection in dc throughout the ksa, we undertook a nationwide survey by using both serological and molecular methods. serum, whole blood, and rectal and nasal swabs were freshly collected from dc, sheep, and goats in november and december of in the southwestern (gizan), western (taif), northwestern (tabuk), eastern (hofuf), and central (unizah, riyadh) regions of the ksa (table ) . we also collected archived serum samples obtained from dc in through (table ). sera were initially tested for the presence of antibodies reactive with mers-cov by using a cell enzyme-linked immunosorbent assay (elisa) based on vero cells infected with mers-cov. subsets of sera positive by elisa were tested in western blot assays that used extracts of vero cells infected with mers-cov and a luciferase immunoprecipitation system (lips) assay based on recombinant mers-cov nucleoprotein. potential serologic cross-reactivity with bovine coronavirus (bo-cov) was addressed by testing for reactivity with bo-cov nucleocapsid protein by lips assay. the presence of viral nucleic acids in rectal and nasal swabs and a subset of serum and whole blood samples was assayed by reverse transcriptionquantitative pcr (rt-qpcr) with primers targeting the upe and orf a genome regions of mers-cov ( , ) . one hundred fifty ( %) of dc sampled countrywide in were found to have antibodies to mers-cov by elisa. the prevalence of seropositivity was higher in adult dc (Ͼ years of age; / , %) than in juvenile dc (Յ years of age; / , %) (p Ͻ . , test). the lowest prevalence of seropositive juveniles was found in the southwestern region, in proximity to the city of gizan ( / , %) (fig. a) . a higher prevalence of antibodies to mers-cov in older animals was also seen in samples obtained in years prior to . in , the seroprevalence was % ( / ) in juveniles versus % ( / ) in adults in the central region (riyadh, kharj). in , the seroprevalence was % ( / ) in juveniles versus % ( / ) in adults (riyadh, rumah) ( table ) . seroreactivity was also found in samples collected from dc as early as the s: / ( %) in , / ( %) in , / ( %) in , / ( %) in , and / ( %) in . analysis of selected seropositive dc sera by western immunoblot assay indicated two distinct reaction patterns that showed reactivity either to mers-cov spike glycoprotein alone or to spike glycoprotein and nucleocapsid protein. these results were concordant with results of mers-cov nucleocapsid lips assays ( fig. ; see table s in the supplemental material). potential serologic cross-reactivity to bo-cov was addressed by analyzing bo-cov nucleocapsid reactivity, which has been shown to be subgroup specific in covs ( ) . overall, % ( / ) of dc were positive for bo-cov, ranging from % in the southwest (gizan) to % in the east (hofuf) and % in adult animals versus % in juvenile animals; animals (juveniles in taif) were seropositive for bo-cov exclusively, while the remaining % ( / ) were reactive to bo-cov and mers-cov, and % ( / ) were reactive to mers-cov alone (see table s in the supplemental material). rectal and nasal swabs collected in parallel with serum samples from the same animals were assayed for mers-cov nucleic acids by rt-qpcr. nucleic acids were most frequently detected in nasal swabs; rectal swabs were found positive in only three cases; in two of them, the nasal sample was also positive. the regional distribution of pcr-positive animals is shown in fig. b . in contrast to serology, where mers-cov-reactive antibodies were more prevalent in adults than in juveniles ( % versus %), mers-cov nucleic acids were found more frequently in juveniles ( / , %) than in adults ( / , %) (p ϭ . , test). the five samples with Ͼ copies were all from juveniles, four of them from seronegative animals. the prevalence of pcr-positive dc ranged from % in taif in the west to % in gizan in the southwest. pcr analysis of a random selection of serum and whole blood samples collected from nasal or rectal swab pcr-positive, seropositive, and seronegative dc revealed no evidence of viremia (see table s in the supplemental material). these included adults and juveniles phlebotomized in , adults and juveniles phlebotomized in , and adults and juveniles phlebotomized in . these animals included the five juveniles with the highest viral genome sequence load in nasal swabs. serum samples collected from goats (n ϭ ) and sheep (n ϭ ) in in the central region (unizah, riyadh) were not immunoreactive with mers-cov but were immunoreactive with bo-cov ( % of goats, n ϭ ; % of sheep, n ϭ ). nasal swabs from goats and sheep were negative in rt-qpcr assays for mers-cov upe. to test the validity of rt-qpcr results and determine phylogenetic relationships of viral sequences found in dc in the ksa to previously reported sequences, we amplified and sequenced longer regions of the spike, orf ab, and nucleocapsid genes from rt-qpcr-positive samples (for the sequences of the primers used, see table s in the supplemental material). eleven of swab samples with Ͼ copies in upe rt-qpcr yielded products for sequencing. no suitable products were obtained from samples with lighter viral sequence loads (Ͻ copies) (see table s in the supplemental material). phylogenetic analysis of a , nucleotide (nt) region of the spike gene and a , -nt region of the orf ab gene indicated Ͻ % divergence from previously published mers-cov sequences (fig. ) (genbank accession no. kj to kj ). the nucleocapsid sequence was identical to the previously reported mers-cov sequences. mers-cov is posited to be a zoonosis. however, the evolutionary history of mers-cov and the reservoirs and vectors for human infection remain obscure. early anecdotal reports that some mers-cov victims had exposure to dc led to serologic investigation of dc in spain and oman ( ) , jordan ( ), egypt ( ) , and the ksa ( ) that revealed antibodies to mers-cov. definitive evidence that dc can be infected with mers-cov was obtained when viral sequences were detected in nasal swabs from dc sampled in close proximity to outbreaks of the disease among humans in qatar ( ) and jeddah, ksa ( ) . nonetheless, as noted by nishiura and colleagues, current data do not fulfill the two criteria required to implicate dc as a significant reservoir species in the epidemiology of mers-cov ( ) , i.e., (i) that dc are sufficient to maintain mers-cov and (ii) that the presence of dc is essential to the continuous transmission of infection. results presented here do not establish the latter; however, they do provide evidence for the former. our study is the first comprehensive countrywide survey of dc from the ksa, the country with the most recorded mers-cov cases, to use both serological and molecular diagnostic methods. analysis of specimens from western regions of the country, from tabuk in the northwest, taif in the west, and gizan in the southwest, revealed regional differences. although the seroprevalence was high in adults throughout the country at Ͼ %, in juveniles, it ranged from % in the east to % in the southwest. the sero-prevalence in dc Յ years of age was lower than that in older animals, confirming the results of hemida et al. ( ) . molecular analysis of nasal and rectal swab specimens indicated the highest prevalence of mers-cov sequences in dc in the west and northwest. nasal swabs with heavy sequence loads (Ͼ copies) also clustered in the taif region. a second sample collection in the west (taif) separated from the first by an interval of months confirmed the presence of heavy viral sequence loads in nasal swabs collected from juvenile animals sampled in this area (data not shown). these findings suggest that continuous, longer-term surveillance is necessary to determine the dynamics of virus circulation in dc populations. lower prevalence rates of both mers-cov and bo-cov were evident in samples from the southwest. this may relate in part to the enforcement of restrictions of livestock movement in and out of gizan province implemented after the rift valley fever outbreak in but also to the generally lower dc population density in this region than in other regions of the ksa. viral nucleic acids were more commonly detected in nasal swabs than in rectal specimens and were more frequent in juvenile than in adult animals. these findings, together with the absence of viremia and the known respiratory tract tropism of several other coronaviruses, suggest that airborne transmission is the most likely mode of mers-cov transmission. although nucleic acid copy numbers were commonly highest in juvenile animals that were seronegative or had low antibody titers, positive findings were also obtained with specimens from highly seropositive and adult animals. our findings in archived dc specimens, although restricted to serology, strongly suggest that mers-cov or a closely related virus has been circulating in dc in the ksa for at least decades. complete genomic sequences of mers-cov found in contemporary dc in the ksa are identical to sequences of viruses recovered from human mers-cov victims (unpublished data). although we speculate that dc are potential reservoirs for human transmission, we cannot prove this relationship from the current data. rigorous epidemiological investigation of the potential for exposure to dc in sporadic cases of mers-cov (those where there is no opportunity for human-to-human transmission) is required to test this model. if dc can be implicated, other questions will arise. did mers-cov truly emerge as a human pathogen in , or were cases of cryptic infection not appreciated because of a lack of suitable diagnostic tests? we may be able to address this conundrum by using archived human materials. if evidence of human mers-cov infections cannot be detected prior to , we must entertain the possibility that mutation facilitated cross-species transmission. however, we see no path to address this possibility absent access to historical dc respiratory tract specimens. the only archived dc specimens we have been able to locate are dc sera; our efforts to recover mers-cov sequences from camel blood have been unsuccessful. what are the roles of bats, if any, as reservoirs of mers-cov? these limitations notwithstanding, the most urgent public health concern, raised in work we and others have reported that focuses on dc infection, is to determine the role of these animals in sporadic human infection. the evidence is clearly sufficient to support targeted investigation of direct or indirect exposure to dc in the disease among humans. sample collection. samples included dc, sheep, and goat sera; whole blood and nasal and rectal swabs freshly collected in ; and archived serum samples from , , , , , , and . two rectal and two nasal swabs were obtained from each animal. one rectal swab and one nasal swab were placed into rnalater (life technologies, carlsbad, ca), and one rectal swab and one nasal swab were placed into viral transport medium (becton dickinson, franklin lakes, nj). all were stored at Ϫ ºc. after h in fixative, plates were rinsed three times with phosphate-buffered saline (pbs) and placed in pbs for storage at °c. plates were loaded with infected and noninfected cells in alternating rows to generate a differential reading for each serum tested. positivity was defined as an infected-cell optical density of Ͼ . and Ͼ ϫ the noninfected-cell optical density. test sera were diluted : , in pbs- . % tween - % bovine serum albumin; secondary antibodies were rabbit anti-goat igg (hϩl)-horseradish peroxidase conjugate ( : , ; bio-rad, hercules, ca), rabbit anti-sheep igg (hϩl)-horseradish peroxidase conjugate ( : , ; bio-rad), and anti-llama igg-horseradish peroxidase conjugate ( : , ; bethyl laboratories, montgomery, tx). western blot. extracts of noninfected vero cells or vero cells infected with mers-cov strain emc were generated at rocky mountain laboratories, loaded onto discontinuous and . % sds gels (bio-rad), and transferred onto nitrocellulose with iblot transfer stacks (invitrogen iblot; life technologies). lanes were loaded with alternating infected and noninfected extract samples, and a pair were cut for incubation with dc sera ( : in blocking solution) after blocking of the membrane in pbs- . % tween - % dry milk blocking solution for h. membranes were washed three times with pbs- . % tween after a -h incubation with serum and then incubated for another . h with secondary antibody ( : , in blocking solution; anti-llama igg-horseradish peroxidase conjugate; bethyl laboratories). following three more washes, the membranes were developed with westernsure premium chemiluminescent substrate (li-cor, lincoln, ne) and read on a c-digit blot scanner (li-cor). lips assay. the nucleocapsid proteins of bo-cov and mers-cov were pcr amplified with primers introducing appropriate restriction sites for cloning into vector pren- fused to the c terminus of the renilla luciferase reporter ( ) . sequence-confirmed construct dna was purified from escherichia coli cultures (qiagen, hilden, germany), and transfected into cos- cells (african green monkey kidney, atcc crl- ; g; lipofectamine; invitrogen, life technologies). cells were harvested at h posttransfection in lysis buffer ( mm tris [ph . ], mm nacl, mm mgcl , % triton x- , % glycerol, protease inhibitors), and the protein extract was clarified by two rounds of centrifugation at , ϫ g. the target concentration was determined in relative light units (rlu), and approximately ϫ rlu were incubated with test serum ( : ) in a final volume of l buffer a ( mm tris [ph . ], mm nacl, mm mgcl , % triton x- ) per well in -well plates (catalog no. ; thermo/nunc, waltham, ma). after h of incubation at room temperature, protein a/g beads (catalog no. ; pierce, junction city, or) were added and the mixtures were transferred to -well filter plates (catalog no. msbvn b ; millipore, billerica, ma) for another hour of incubation at room temperature with shaking. bead-bound anti-gen was washed eight times with buffer a and three times with pbs (tecan hydroflex, maennedorf, switzerland) and then read with coelenterazine substrate (renilla luciferase assay system; promega, madison, wi) on a centro lb luminometer (berthold, bad wildbad, germany). nucleic acid extraction and pcr. total nucleic acids were extracted from nasal swabs, serum, and whole blood on a qiacube with cador reagent kits (qiagen) or rneasy reagent kits for extraction of rna from rectal swabs. real-time qpcr used a onestep real-time qpcr buffer (invitrogen, life technologies) and primer/probes upe and orf a ( , ) . products for sequencing were generated by rt-pcr. cdna was reverse transcribed with superscript iii and random hexamer primers. pcr was performed with amplitaq gold (life technologies) and primers designed to amplify a , -nt region of the spike gene (heminested pcr), a -nt region of the n gene (nested pcr) or a , -nt region of the orf ab region (heminested pcr). for the sequences of the primers used, see table s in the supplemental material. products were purified by agarose gel electrophoresis and with qiaquick gel extraction kits (qiagen) and subsequently sequenced on both strands by the dideoxynucleotide chain termination method (genewiz, south plainfield, nj). supplemental material for this article may be found at http://mbio.asm.org /lookup/suppl/doi: . /mbio. - /-/dcsupplemental. middle east respiratory syndrome coronavirus (mers-cov)-update. world health organization isolation of a novel coronavirus from a man with pneumonia in saudi arabia state of knowledge and data gaps of middle east respiratory syndrome coronavirus (mers-cov) in humans family cluster of middle east respiratory syndrome coronavirus infections hospital outbreak of middle east respiratory syndrome coronavirus transmission and evolution of the middle east respiratory syndrome coronavirus in saudi arabia: a descriptive genomic study human betacoronavirus c emc/ -related viruses in bats, ghana and europe dipeptidyl peptidase is a functional receptor for the emerging human coronavirus-emc middle east respiratory syndrome coronavirus in bats, saudi arabia mers-cov-eastern mediterranean ( ): animal reservoir, camel, suspected, official. promed-mail middle east respiratory syndrome coronavirus in dromedary camels: an outbreak investigation middle east respiratory syndrome coronavirus (mers-cov) serology in major livestock species in an affected region in jordan seroepidemiology for mers coronavirus using microneutralisation and pseudoparticle virus neutralisation assays reveal a high prevalence of antibody in dromedary camels in egypt middle east respiratory syndrome coronavirus neutralising serum antibodies in dromedary camels: a comparative serological study middle east respiratory syndrome (mers) coronavirus seroprevalence in domestic livestock in saudi arabia detection of a novel human coronavirus by real-time reverse-transcription polymerase chain reaction assays for laboratory confirmation of novel human coronavirus (hcov-emc) infections evaluation of serologic and antigenic relationships between middle eastern respiratory syndrome coronavirus and other coronaviruses to develop vaccine platforms for the rapid response to emerging coronaviruses missing information in animal surveillance of mers-cov antibody-profiling technologies for studying humoral responses to infectious agents we are grateful to his highness, prince bandar bin saud al saud, president, saudi wildlife authority, for his unlimited support and encouragement; to devon welsh and kawthar muhammad for mobile laboratory coordination; to andrew schultz for statistical analysis; to ellie kahn for editorial contributions; to aaloki shah, parisa zolfaghari, mia r. key: cord- -p enjxiq authors: hattori, shin-ichiro; higshi-kuwata, nobuyo; raghavaiah, jakka; das, debananda; bulut, haydar; davis, david a.; takamatsu, yuki; matsuda, kouki; takamune, nobutoki; kishimoto, naoki; okamura, tadashi; misumi, shogo; yarchoan, robert; maeda, kenji; ghosh, arun k.; mitsuya, hiroaki title: grl- , an indole chloropyridinyl ester, completely blocks sars-cov- infection date: - - journal: mbio doi: . /mbio. - sha: doc_id: cord_uid: p enjxiq we assessed various newly generated compounds that target the main protease (m(pro)) of severe acute respiratory syndrome coronavirus (sars-cov- ) and various previously known compounds reportedly active against sars-cov- , employing rna quantitative pcr (rna-qpcr), cytopathicity assays, and immunocytochemistry. here, we show that two indole-chloropyridinyl-ester derivatives, grl- and grl- , exerted potent activity against sars-cov- in cell-based assays performed using veroe cells and tmprss -overexpressing veroe cells. while grl- and the nucleotide analog remdesivir blocked sars-cov- infection, viral breakthrough occurred. no significant anti-sars-cov- activity was found for several compounds reportedly active against sars-cov- such as lopinavir, nelfinavir, nitazoxanide, favipiravir, and hydroxychroloquine. in contrast, grl- exerted potent activity against sars-cov- ( % effective concentration [ec( )] = . μm) and dramatically reduced the infectivity, replication, and cytopathic effect of sars-cov- without significant toxicity as examined with immunocytochemistry. structural modeling shows that indole and chloropyridinyl of the derivatives interact with two catalytic dyad residues of m(pro), cys and his , resulting in covalent bonding, which was verified using high-performance liquid chromatography–mass spectrometry (hplc/ms), suggesting that the indole moiety is critical for the anti-sars-cov- activity of the derivatives. grl- might serve as a potential therapeutic for coronavirus disease (covid- ) and might be optimized to generate more-potent anti-sars-cov- compounds. jpn/ty/wk- (sars-cov- wk- ), employing a quantitative rna-qpcr assay with cell culture supernatants, cytotoxicity assays, and immunocytochemistry. grl- and grl- exert potent activity against sars-cov- . we first tested the antiviral activity of the compounds against sars-cov- wk- by employing a series of compounds that had previously been reported to be active against sars-cov ( ) ( ) ( ) and identified grl- and a newly synthesized compound, grl- , as potent inhibitors of sars-cov- (table ) . when veroe cells were exposed to sars-cov- wk- at a multiplicity of infection (moi) of . and cultured in the presence of various concentrations of the two indole chloropyridinyl esters grl- and grl- , the compounds were found to be highly potent against sars-cov- wk- with % effective concentration (ec ) values of Ϯ and . Ϯ . m, respectively, using rna-qpcr (table ) . we further examined the antiviral activity of these two compounds together with other selected compounds that had previously been reported to be active against sars-cov and/or sars-cov- , including remdesivir, lopinavir, nelfinavir, favipiravir, hydroxychloroquine, nitazoxanide, and nafamostat ( , ( ) ( ) ( ) . to compare the antiviral activities of the compounds as accurately as possible, the compounds were examined at the same time in one assay. the data shown in fig. (see also table s in the supplemental material) confirmed that grl- and grl- were active against sars-cov- wk- with ec values of Ϯ and . Ϯ . m, respectively. remdesivir was also found to be active against sars-cov- wk- (ec ϭ . Ϯ . m). the ec values for lopinavir, nelfinavir, and nitazoxanide were Ϯ , . ϩ . , and Ϯ m, respectively; however, their specificity index values were only . , , and . , respectively (table s ) . thus, such ec values were thought possibly to be affected by their cytotoxicity (see below). while favipiravir and nafamostat were apparently nontoxic at all concentrations tested, the ec values of both were Ͼ m, suggesting that neither of the compounds was effective against the virus ( fig. ; see also table s ). hydroxychloroquine appeared to be nontoxic at up to m, and its ec turned out to be . Ϯ . m; however, its cytotoxicity was suspected to have contributed to its apparent antiviral activity (see below). immunocytochemistry features of veroe and veroe tmprss cells exposed to sars-cov- . we first examined the immunocytochemistry features of veroe cells under conditions of exposure of the cells to sars-cov- wk- . when veroe cells were cultured alone and stained with texas red-x dye conjugated phalloidin, the cytoskeleton filamentous actin (f-actin) was well visualized as a mesh-like structure (see top left his are indicated in stick mode. for clarity, only a monomer structure is shown and the covalent protease inhibitor n was omitted. the picture was generated using ucsf chimera. activity of grl- and grl- against the infectivity and replication of sars-cov- in veroe cells a grl- nd Ϯ grl- . Ϯ . . Ϯ . a the data shown represent mean ec values Ϯ standard deviations (sd). the assays were conducted on at least different occasions, and the data show representative results. all assays were performed in duplicate. nd, not done. inset in fig. s in the supplemental material), suggesting that the cells were healthy and replicating. in contrast, when the cells were exposed to sars-cov- wk- , the f-actin was rearranged, disrupted, and destroyed upon infection by sars-cov- wk- . superimposition of -color images (red, blue, and green, indicating f-actin, nuclei, and sars-cov- antigens, respectively) showed that no nonspecific staining with the antibody against sars-cov- (an igg fraction from the serum of a covid- convalescent patient [convigg] was used as the primary antibody for immunofluorescence staining) had occurred in sars-cov- wk- -unexposed veroe cells (top right inset in fig. s ). however, most of the sars-cov- wk- -exposed veroe cells stained in green, indicating most of those cells were infected and producing viral components (bottom right inset in fig. s ). we also examined the cell destruction effects of the virus by the use of immunocytochemistry performed with two cell lines, veroe and veroe tmprss cells. the veroe cells were exposed to sars-cov- wk- . the viral copy numbers in the culture supernatants (left axis, open bars) and percent inhibition (right axis, red lines) of each compound in veroe cells were determined using rt-qpcr. each compound was tested on at least different occasions. for details of the ec and cc values, see table s . the data illustrate representative ones and are shown as means Ϯ standard deviations (sd). all compounds were tested and compared in one assay performed in duplicate. images in fig. s show that robust actin filaments were observed when the veroe and veroe tmprss cells were cultured alone (insets a and d), while actin filaments were all gone (inset b) and most of those cells were stained in green when convigg was used as the primary antibody in veroe cells exposed to sars-cov- wk- and cultured in the absence of test compounds. when the highly sars-cov- -susceptible veroe tmprss cells ( ) were exposed to sars-cov- wk- , most of the cells were infected, their actin filaments were totally destroyed, and the cells were killed by the virus detached from the bottom of microculture titer plate wells, resulting in the loss of the cells (inset e). we also employed a murine anti-spike monoclonal antibody as the primary antibody in our immunostaining (insets c and f in fig. s ). convigg detected much higher levels of sars-cov- -infected cells than were seen when the murine anti-spike monoclonal antibody was used as the primary antibody (compare insets b and c). thus, we chose the igg fraction from the convalescent covid- individual for use as the primary antibody for further study. it is known that the infectivity and replication of sars-cov- significantly vary depending on the types of target cells used. therefore, we quantitatively examined the infectivity and replication of sars-cov- wk- in two cell lines, veroe cells and veroe tmprss cells plus sars-cov- wk- . upon exposure of veroe cells to sars-cov- wk- , the viability of the veroe cells was reduced to close to % by h following the viral exposure and the viability was found to have further reduced to ϳ % by h in culture (fig. s a ). in contrast, the viability of veroe tmprss cells exposed to sars-cov- wk- went down quickly to ϳ % by h in culture and further down to ϳ % to ϳ % by h in culture. we also determined the numbers of sars-cov- wk- rna copies in the supernatants in the same experiment. in the and h following the viral exposure, veroe tmprss cells produced greater numbers of sars-cov- wk- rna copies than veroe cells, while at h and after, the veroe cells and veroe tmprss cells produced comparable numbers of rna copies (fig. s b) . images of the cultures captured under light microscopy and immunocytochemistry results confirmed that the veroe tmprss cells were more susceptible to the infectivity and cytopathicity of sars-cov- wk- (fig. s c) . thus, the assay performed using veroe cells and sars-cov- wk- is referred to as a "low-stringency assay" in the present report, while that performed using veroe tmprss cells and sars-cov- wk- is referred to as a "high-stringency assay." association of apparent antiviral activity with the cytostatic/cytotoxic nature of test compounds. we carefully investigated whether the two compounds grl- and remdesivir exerted antiviral activity at , , and m (fig. a ) without significant cytostatic or cytotoxic effects. we first examined light microscopic images of veroe cells exposed to sars-cov- wk- and cultured in the presence of grl- or remdesivir (fig. a) . veroe cells were exposed to sars-cov- wk- for h, the virus was washed off, and the cells were cultured in the presence of various concentrations of test compound for days. veroe cells cultured alone (left top inset, fig. a ) appear to be robust; however, many of the cells exposed to sars-cov- wk- and cultured in the absence of test compounds showed granular patterns (bottom inset at left in fig. a ). all of the veroe cells exposed to the virus and cultured with grl- or remdesivir (at m) appeared to be protected (top row of insets at right in fig. a) . in order to corroborate the data representing the antiviral activity of grl- and remdesivir described above, we further examined the inhibitory activity of those compounds using detailed immunocytochemistry. when veroe tmprss cells, which are known to be highly susceptible to the infectivity of sars-cov- ( ), were cultured alone, robust cellular cytoskeleton filamentous actin (f-actin) was seen in the form of mesh-like structures in red and a number of nuclei (in blue) were identified, signifying that those cells were healthy and replicating (top panel at extreme left in fig. b ). however, when veroe tmprss cells were exposed to sars-cov- wk- and cultured in the absence of test compounds, the results showed that most of the cells had been infected and destroyed by the virus, were detached from the bottom of the microtiter culture plates, and had been lost during the staining procedure as seen with fluores-cence microscopy (bottom, extreme left in fig. b ). most of the residual cells stained in green, indicating that those cells were infected and producing viral proteins and that the cellular f-actin was almost totally lost due to rearrangements and destruction caused by the virus ( ) . when veroe tmprss cells were exposed to sars-cov- wk- and cultured in the presence of various concentrations of lopinavir and nelfinavir, many virus-infected cells were seen at and m and stained in green, indicating that these two compounds had no detectable antiviral activity in the assay. at m, most of the cells were lost ( fig. b ; see also fig. s a ), presumably due to both the infection by the virus and the toxicity of the agents. these data strongly suggest that the apparent antiviral effect of nelfinavir and lopinavir stemmed from their cytotoxicity, producing "false" antiviral effects as assessed using rna-qpcr (fig. ) . in contrast, when sars-cov- wk- -exposed veroe tmprss cells were cultured in the presence of m grl- or remdesivir, there were essentially no infected cells seen. the cellular f-actin was almost completely preserved, indicating that the sars-cov- wk- exposed veroe tmprss cells remained healthy and replicating (fig. b ). of note, the level of antiviral activity of grl- was moderate and a substantial number of cells stained in green, indicating that viral breakthrough had occurred even with m grl- (fig. b ). grl- completely blocks the infectivity of sars-cov- , while various compounds reportedly active against sars-cov- fail to block infectivity. veroe tmprss cells are highly susceptible to the infectivity of sars-cov- wk- (insets b and e in fig. s ). in order to ensure detection of even moderate antiviral activity of test compounds against sars-cov- , we subsequently used the low-stringency combination of veroe cells and sars-cov- wk- to further examine selected compounds, including nitazoxanide, nafamostat, chloroquine, and hydroxychloroquine. as shown in fig. c (see also fig. s a ), nitazoxanide did not exert activity against the virus at and m and only a few nuclei remained at m, strongly suggesting that nitazoxanide had no antiviral activity and that its cytotoxicity resulted in a false antiviral effect as assessed using rna-qpcr and shown in fig. . nafamostat was not significantly cytotoxic but was inactive against the virus at all the concentrations tested ( in this regard, chloroquine reportedly blocks the catabolism and/or degradation of cellular proteins through increasing endosomal ph, possibly resulting in the maintenance of actin filaments despite of sars-cov- wk- infection ( ) . chloroquine also reportedly interferes with glycosylation of the ace receptor, one of the cellular entry points of sars-cov, possibly resulting in moderating the infection by sars-cov- ( , ) . however, almost all veroe cells were lost when the cells were cultured in the presence of m chloroquine because of its toxicity ( ). the effects of hydroxychloroquine were almost the same as those of chloroquine. of note, when veroe cells were exposed to sars-cov- wk- and cultured in the presence of m chloroquine or hydroxychloroquine, a number of the cells were infected by the virus; nevertheless, the actin filaments were moderately conserved (fig. s b ). in this regard, the wst- assay used in the present study ( ) evaluated the mitochondrial nad(p)h-dependent cellular oxidoreductase enzyme activity in living cells that results in reduction of wst- to formazan dyes. therefore, the data from wst- assays, obtained under defined conditions, reflect the number of viable cells. sars-cov- wk- , cultured in the presence of grl- or remdesivir, and examined under a microscope (magnification, ϫ ). (b) grl- , grl- , and remdesivir potently blocked the infectivity and cytopathic effect of sars-cov- wk- in veroe tmprss cells, while lopinavir, nelfinavir, and favipiravir failed to block the infection. (c) grl- at m completely blocked sars-cov- wk- infection in veroe cells, while nafamostat and hydroxychloroquine did not exert significant antiviral activity. nafamostat did not exert significant toxicity to the cells, but it totally failed to block the infectivity of the virus. for immunocytochemistry, the igg fraction of serum from a convalescent covid- individual was employed as the primary antibody. the sars-cov- antigens, f-actin, and nuclei are shown in green, red, and blue, respectively. the image in each inset in panels b and c represents the merged image. chloroquine reportedly does not target mitochondria but damages other cellular organelles involved in protein synthesis and metabolism such as lysosome, endoplasmic reticulum, and golgi apparatus, resulting in compromised cellular growth and functions ( ) . thus, the results of wst- assays did not reflect cellular robustness or functionality, especially with regard to chloroquine and hydroxychloroquine. as shown in fig. s b , in the veroe cells exposed to relatively low concentrations ( and m) of chloroquine and hydroxychloroquine, following infection, the virus did not replicate well because the protein synthesis in the veroe cells was compromised by the compounds, moderately maintaining actin filament structures. however, the damaging effects of chloroquine and hydroxychloroquine at m were significant and virtually no cells were left in the wells of microtiter culture plates (fig. c) (fig. s b ; see also fig. s ). grl- reproducibly and completely blocked the infectivity and cytopathic effect of sars-cov- wk- as examined with the low-stringency assay using veroe cells ( fig. c ; see also fig. s c ). when veroe cells were exposed to sars-cov- wk- and cultured in the presence of remdesivir or grl- at . , . , , , and m, there was further suppression of viral replication at m and either nearly complete (remdesivir) or complete (grl- ) suppression at m as assessed using either the convigg or the murine anti-spike monoclonal antibody as the primary antibody (fig. s ). examining the activity of m remdesivir and grl- against sars-cov- wk- with veroe cells, the appearance of actin filaments proved to be virtually the same as seen in the high-stringency assay performed using veroe tmprss cells (fig. b) . these data strongly suggest that the veroe cells protected by remdesivir and grl- were viable and that the integrity of the cells was likely maintained. grl- and grl- covalently bind to m pro of sars-cov- . finally, we built molecular models to understand the structural interactions of grl- - and grl- with m pro of sars-cov- . we started with the recently published crystal structure of sars-cov- m pro (rcsb pdb identifier [id] y f) ( ) . grl- represents an indole chloropyridinyl ester. grl- has the same moiety as grl- but with an additional propene substituent on the indole nitrogen. when m pro cys attacked grl- and formed an initial complex in the active site (fig. a) , the hydroxyl oxygen (or the oxyanion) formed hydrogen bond interactions with the backbone amide nitrogens of gly , ser , and cys . the indole moiety is appropriately positioned to form pi-pi interactions with his and his . the pyridinyl nitrogen is positioned to form polar interactions with gly , and the chlorine atom forms halogen bond interactions with the backbone amide nitrogen of thr . these interactions of different moieties of grl- , including the chloropyridinyl group, may help stabilize the initial reaction intermediate. following acylation, the chloropyridinyl group departs, and the carbonyl indole is bound to cys with a covalent bond. the presence of covalently bound carbonyl indole was verified by high-performance liquid chromatography-mass spectrometry (hplc/ms). newly generated recombinant sars-cov- -m pro was incubated with dimethyl sulfoxide (dmso), grl- , or grl- for min. dmso-treated m pro gave a molecular weight of , . consistent with full-length m pro (expected molecular weight, , . ) (fig. s a) . grl- treatment and grl- treatment increased the mass by . and . amu ( fig. s b and c) , respectively. the increase in the observed mass of m pro is consistent with acylation of m pro by two compounds (expected increases of . and . ). the modeled complex of m pro with grl- was further minimized, and the interactions are shown in fig. b . the carbonyl carbon has hydrogen bond interactions with the backbone amine nitrogens of gly , ser , and cys . slight rotations of the rings make favorable pi-pi interactions of both his and his with the indole moiety. we also examined the interactions of grl- with m pro . the initial and final complexes are shown in fig. c and d, respectively. the presence of the propene substituent seems to have drastically reduced the binding interactions of grl- in comparison to grl- . while the chloropyridyl group has polar interactions, there was only one polar interaction of the hydroxyl (or oxyanion) moiety with the backbone of cys . the polar interactions with gly and ser seen for grl- were lost. these reductions in interactions are most likely due to the change in conformation with the additional substituent and to the shallow binding site of m pro . the grl- complex with sars-cov- m pro seen following completion of the acylation reaction is shown in fig. d . this complex also had a lower number of polar interactions with m pro than the grl- complex. overall, the difference in these interactions may at least in part explain the greater potency of grl- than grl- . thermal stability of m pro in the absence or presence of grl- . we also examined the thermal stability of m pro in the presence of grl- using differential scanning fluorimetry (dsf) and cypro orange ( , ) . as illustrated in fig. , the melting temperature (t m ) of m pro ( m) alone in experiment was . °c, while the t m value decreased to . , . , and . °c in the presence of , , and m grl- , respectively. the shift of t m to lower temperatures is reportedly due to the destabilization of the protein by covalent binding compounds ( ) . thus, the present data corroborate that grl- forms a covalent bond with m pro . as a control, we determined the t m value for hiv- protease in the presence of an hiv- protease inhibitor, grl- ( ) . the t m value determined for hiv- protease ( m) alone in experiment was . °c, while when the same hiv- protease was made to interact with grl- , the t m value significantly shifted to the right, giving a t m value of . °c and signifying that grl- had very effective noncovalent interactions. therefore, these thermal stability data corroborate our nanolc-ms results indicating that grl- forms covalent interactions with m pro and that grl- exerts its antiviral activity against sars-cov- by performing covalent binding with m pro . while grl- and remdesivir significantly blocked the infectivity and replication of sars-cov- , they still permitted viral breakthrough ( fig. b ; see also fig. s in the supplemental material). however, grl- completely blocked the infectivity, replication, and cytopathic effect of sars-cov- in both the high-stringency and lowstringency assays. in our study, the ec value of nitazoxanide was Ϯ m; however, its specificity index was only . (see table s in the supplemental material). thus, such an ec value was thought to be affected by its inherent cytotoxicity. in fact, nitazoxanide did not block the infectivity of sars-cov- wk- as examined with immunocytochemistry (fig. c) . with regard to another compound, hoffman et al. recently reported that nafamostat, a serine protease inhibitor, blocked sars-cov- infection in calu- cells exposed to sars-cov- with an ec value of as low as nm ( ) . note, however, that calu- cells are not very susceptible to sars-cov- infection and do not support viral replication. thus, in the assays performed using calu- cells and in an alternative assay that was performed without using live infectious sars-cov- , instead using "pseudotype entry" as an endpoint, it was found that the ec values can be excessively low (i.e., nm) ( , ) compared to the ec values obtained in the assays using other cell lines such as veroe cells. thus, we refer to the cell-based assay systems using calu- cells as a "very-low-stringency" assays, where moderately or only slightly active compounds tend to get very low ec values. using both veroe cells and veroe tmprss cells with sars-cov- wk- in the present study, nafamostat failed to show significant antiviral activity (ec values of Ͼ m). thus, we conclude that neither nitazoxanide nor nafamostat exerted significant activity against sars-cov- wk- in the present study. as described above, cytostatic and cytotoxic effects of test compounds in cell-based assays are often mistakenly interpreted as representative of apparent antiviral activity since the production of the relevant virus is reduced by the inherent cytostatic and cytotoxic effects of the test compounds ( , ) . indeed, none of such toxic agents (i.e., daunorubicin and adriamycin) have proven to be of clinical utility as antiviral agents. of note, we seriously attempted to sever the data representing reduction of virus copy grl- completely blocks sars-cov- infection ® numbers due to cytostatic/cytotoxic effects from the data representing the virusspecific inhibitory activity of the test compounds. we believe that our detailed immunocytochemistry results clearly segregated the effects of virus-specific antiviral activity from the cytostatic/cytotoxic effects of the test compounds. we conclude that in the present study, no detectable anti-sars-cov- activity was present in compounds such as hiv- protease inhibitors (nelfinavir and lopinavir), favipriavir, hydroxychloquine, and others reportedly active against sars-cov- and that grl- potently blocked the infectivity and cytopathicity of sars-cov- . indeed, the results from the present study show that the previously reported activity of nelfinavir, lopinavir, nitazoxanide, chroloquine, and hydroxychroloquine against sars-cov- ( , ( ) ( ) ( ) was incorrectly judged to represent specific activity against sars-cov- and that the reduction of viral production observed was due to inherent cytostatic and/or cytotoxic effects of those compounds. favipiravir and nafamostat were not very toxic but did not show detectable antiviral activity in the present study. our modeling studies strongly suggest that grl- and grl- interact with m pro and exert their activity against sars-cov- . the results of our m pro molecular weight analysis performed with hplc/ms (fig. s ) show that both compounds covalently bond with m pro . furthermore, the data representing thermal stability of m pro in the presence of grl- showed a relatively odd feature, i.e., a shift of the stability curve to a lower temperature, corroborating the idea that grl- forms covalent bonds with m pro . in regard with the covalent bonding, it is concerning that compounds forming irreversible covalent interactions may cause permanent injuries to critical cellular components by binding covalently to produce serious adverse effects. however, the duration of administration of a therapeutic(s) to patients with covid- could be as short as days or weeks at the longest. thus, certain adverse effects due to such remedies might be acceptable if lives are expected to be saved. also, it is possible that grl- -m pro covalently linking ketal may convert to a carbonyl with noncovalent binding with m pro in a reversible manner. therefore, the covalent interactions of grl- and its analogs may not pose serious toxicity. gc- , reportedly one of the most potent sars-cov- m pro inhibitors, has an ec value of . m as examined with regard to the inhibition of cytopathic effect of sars-cov- using veroe cells ( ) . the ec value representing the potency of gc- is comparable to the ec value ( . Ϯ . m) determined for grl- ( fig. ; see also table s ); however, for an accurate comparison, both compounds have to be tested within the same assay using the same cell type and the same virus strain. grl- might thus serve as a potential therapeutic agent against covid- , and optimization of grl- based on the present data is essential to develop more-potent anti-sars-cov- compounds for treating covid- . however, the potency of grl- is moderate, and optimization is essential. it is noteworthy that a series of sars-cov pl pro inhibitors have been reported ( ) ( ) ( ) ) . combination therapy consisting of a potent sars-cov- m pro inhibitor and a potent sars-cov- pl pro inhibitor could benefit infected individuals significantly more than monotherapy performed with a sars-cov- protease inhibitor. moreover, if the combination of a potent novel m pro inhibitor and a potent sars-cov- rna polymerase inhibitor proves to be significantly more effective against the virus than each class alone, such combined therapy could be more effective in controlling sars-cov- infection in a manner comparable to that witnessed in the area of the therapy against hiv- infection and aids ( , ) . if the combination of grl- and remdesivir proves to be significantly more potent than administration of grl- or remdesivir alone, combined therapy might be more effective in controlling sars-cov- infection. cells, viruses, and antiviral compounds. veroe cells and tmprss -overexpressing veroe (veroe tmprss ) cells were obtained from the japanese collection of research bioresources (jcrb) cell bank (osaka, japan). veroe cells were maintained in dulbecco's modified eagle's medium (d-mem) supplemented with % fetal bovine serum (fcs), g/ml of penicillin, and g/ml of streptomycin. veroe tmprss cells were maintained in d-mem as mentioned above in the presence of mg/ml of g . sars-cov- strain jpn/ty/wk- (sars-cov- wk- ) was obtained from the national institute of infectious diseases (tokyo, japan). the antiviral agents lopinavir (sigma-aldrich, st. louis, mo); nelfinavir, nafamostat, hydroxychloroquine, and nitazoxanide (tokyo chemical industry, tokyo, japan); favipiravir (medchemexpress, monmouth junction, nj); and chloroquine (selleck, sylvanfield drive, houston, tx) were purchased. remdesivir was obtained from clifford lane, national institute of allergy and infectious diseases, national institutes of health, bethesda, md. grl- and grl- were synthesized by a. k. ghosh. each compound except remdesivir was dissolved in dmso at mm, and remdesivir was prepared with saline solution at mm concentrations as stock solutions. antiviral activity, cytotoxicity, cytopathicity, and virus replication assays. for antiviral assay, cells were seeded in a -well plate ( ϫ cells/well) and incubated. after day, virus was inoculated into cells at multiplicity of infection (moi) of . . after an additional days, cell culture supernatants were harvested and viral rna was extracted using a qiaamp viral rna minikit (qiagen, hilden, germany), and quantitative rt-pcr (rt-qpcr) was then performed using one step primescript iii rt-qpcr mix (takara bio, shiga, japan) following the instructions of the manufacturers. the primers and probe used for detecting sars-cov- envelope ( ) for cytopathicity and virus replication assay, cells ( cells/well in a -well plate) were exposed to sars-cov- wk- ( % tissue culture infective doses [tcid ]) for h, washed, and cultured in fresh culture medium. at , , , , or h postinfection (hpi), viral rna copy numbers were determined using rna-qpcr as mentioned above, and the cytopathicity of sars-cov- wk- was determined using the wst- assay. the percentage of cell viability was calculated using the following formula: percent cell viability ϭ [od ( nm) value of cells Ϫ mean od value of veroe tmprss cells at hpi]/[mean od value of uninfected cells Ϫ mean od value of veroe tmprss cells at hpi] ϫ (where "od" represents optical density). in this formula, the mean od value of veroe tmprss cells at hpi was the same as in the wells without cells, indicating that the virus-exposed veroe tmprss cells had been totally destroyed by the virus as examined at hpi. immunocytochemistry. cells in a -well microtiter culture plate were fixed with % paraformaldehyde-phosphate-buffered saline (pbs) for min, washed with pbs ( l/well) three times for min each time, and then blocked with a blocking buffer ( % goat serum, % bovine serum albumin [bsa], . % triton x- , pbs ϫ) for h. after removal of the blocking buffer, the cells were immediately stained with the primary antibody mouse monoclonal anti-sars-cov/sars-cov- (covid- ) spike antibody ( a ) (genetex, alton pkwy irvine, ca, usa) or a convalescent igg fraction, which was isolated from serum of a convalescent covid- individual using a spin column-based antibody purification kit (cosmo bio, tokyo, japan) overnight at °c. the stained cells were washed with pbs ( l/well) three times for min each time, and the cells were incubated with secondary antibody goat polyclonal anti-mouse igg-alexa fluor antibody (thermo fisher scientific, waltham, ma, usa) or goat polyclonal anti-human igg-alexa fluor fab fragment antibody (jackson immunoresearch laboratories, inc., west grove, pa, usa), together with texas red-x dye-conjugated phalloidin (thermo fisher scientific) for f-actin visualization for h. after washing of the cells with pbs ( l/well) three times for min each time, dapi ( =, -diamidino- -phenylindole) solution (thermo fisher scientific)-pbs ( l/well) was added to stain nuclei. signals were acquired with a cytation cell imaging multimode reader (biotek, winooski, vt, usa). molecular modeling of the interaction of grl- and grl- with sars-cov- protease. we started with a crystal structure of sars-cov- main protease (m pro ) with rcsb pdb id y f. we deleted the dimethyl sulfoxide and added hydrogens to the protein atoms, water oxygens, and the crystalized inhibitor. the protonation states of asparagines, glutamines, and histidines were determined, and the orientations of all hydrogen atoms, including those attached to crystal waters, were optimized to improve hydrogen bonding interactions. using the opls force field, restrained minimization was performed (with a cutoff value of . Å for the root mean square differences of heavy atoms from the crystal structure coordinates). the assay steps described above were performed with the protein preparation wizard present in maestro. the structure thus obtained was used for molecular docking. the inhibitor molecules grl- and grl- were built in maestro, and minimized conformations were generated using the ligprep module. the covalent docking submodule of glide was used, and a docking grid encompassing the volume occupied by the inhibitor from the crystal structure was generated. a nucleophilic attack by cys- of sars-cov m pro on the ligand ester carbonyl was chosen as the mode of reaction. this choice was made because the literature on sars-cov demonstrates that the active site cysteine residue undergoes acylation reaction with ester groups, with formation of a covalent bond with the carbonyl carbon followed by the departure of a part of the ligand. recently determined crystal structures also demonstrate that cys- of sars-cov- protease forms covalent bond with inhibitors. an initial mode of interaction that involves the whole ligand molecule was thus generated. subsequently, grl- and grl- were manually cleaved from these docked complexes to generate the appropriate thiocarbonyl complexes. these complexes were further minimized using the opls force field. all simulations were done using software versions/modules present in maestro version . . (schrödinger llc, new york, ny). expression and purification of m pro . the sars-cov m pro -encoding sequence was cloned into pgex- t vector (genscript, piscataway, nj). the plasmid construct was transformed into bl star (de ) cells (thermo fisher scientific). the culture was grown in terrific broth media supplemented with ampicillin. protein expression was induced by adding mm isopropyl ␤-d-thiogalactopyranoside at an optical density at nm of . . protein expression continued at °c overnight. sars-cov- m pro was purified first by affinity chromatography using glutathione s-transferase (gst) sepharose b (ge healthcare, piscataway, nj). the gst tag was cleaved off by the use of thrombin and separated from m pro via gst affinity chromatography, providing the intact m pro with an additional n-terminal glycine residue. the cleaved m pro was further purified by size exclusion chromatography using a hiload superdex -pg column (ge healthcare) in a reaction mixture containing mm tris (ph . ), mm nacl, and mm dithiothreitol. the protease was confirmed to be Ͼ % pure based on sds-gel electrophoresis and hplc/ms chromatography as shown in fig. s in the supplemental material. m pro molecular weight analysis with hplc/ms. the newly generated sars-cov- m pro (see above) was diluted to approximately m ( g/ml) in mm tris buffer (ph . ) with mm sodium chloride and mm dithiothreitol, and . l of m pro was treated either with dmso ( . l)- mm grl- ( . l) or with dmso ( . l)- mm grl- ( . l) at a final concentration of m for each compound. the preparation was incubated at °c for min and then diluted -fold ( l) with high-performance liquid chromatography-mass spectrometry (hplc/ms) running buffer a (water with . % formic acid and . % trifluoroacetic acid). to detect the molecular weight of the protease, analysis was done using a quadrupole time of flight (qtof) mass spectrometer (agilent ) in positive mode with liquid chromatography (agilent ) (agilent, santa clara, ca). a sample ( l) was separated on a zorbax extend c column ( . by mm, . -m pore size) (agilent) over min using a % acetonitrile gradient. separations started with % buffer a and % buffer b (acetonitrile with . % formic acid and . % trifluoroacetic acid) to % buffer b and then ramped to % in the following min and then returned to starting conditions min later. the intact and modified forms of the protease eluted at approximately min. the tof settings were as follows: gas temperature, °c; drying gas rate, liters/min; nebulizer, pounds per square inch gauge (psig); sheath gas temperature, °c; fragmenter, v; skimmer, v. molecular weights were determined by protein deconvolution using agilent mass hunter software (agilent). thermal stability analysis of m pro complexed with grl- using differential scanning fluorimetry. thermal stability was examined using differential scanning fluorimetry. an m pro preparation ( m dissolved in mm tris [ph . ]) that included mm edta was mixed with various amounts of a test compound and incubated at °c for h. subsequently, l of the solution was gradually heated from °c to °c, and the changes of fluorescence intensity were documented using a real-time pcr system (applied biosystems). the % t m ( % melting temperature) values were determined as the temperature at which the relative fluorescent intensity level reached %. supplemental material is available online only. china novel coronavirus investigating research team. . a novel coronavirus from patients with pneumonia in china a pneumonia outbreak associated with a new coronavirus of probable bat origin feng z. . early transmission dynamics in sustaining containment of covid- : global sharing for pandemic response world health organization. . coronavirus disease (covid- ) situation report clinical features of patients infected with novel coronavirus in wuhan remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus ( -ncov) in vitro from sars to mers: crystallographic studies on coronaviral proteases enable antiviral drug design autoprocessing mechanism of severe acute respiratory syndrome coronavirus c-like protease (sars-cov clpro) from its polyproteins maturation mechanism of severe acute respiratory syndrome (sars) coronavirus c-like proteinase the papain-like protease from the severe acute respiratory syndrome coronavirus is a deubiquitinating enzyme the papain-like protease of severe acute respiratory syndrome coronavirus has deubiquitinating activity discovery, synthesis, and structure-based optimization of a series of n-(tert-butyl)- -(narylamido)- -(pyridin- -yl) acetamides (ml ) as potent noncovalent small molecule inhibitors of the severe acute respiratory syndrome coronavirus (sars-cov) cl protease structures of two coronavirus main proteases: implications for substrate binding and antiviral drug design design, synthesis, and evaluation of inhibitors for severe acute respiratory syndrome c-like protease based on phthalhydrazide ketones or heteroaromatic esters synthesis and evaluation of keto-glutamine analogues as potent inhibitors of severe acute respiratory syndrome clpro inhibitor recognition specificity of mers-cov papain-like protease may differ from that of sars-cov synergistic inhibitor binding to the papain-like protease of human sars coronavirus: mechanistic and inhibitor design implications structure-based design, synthesis, and biological evaluation of a series of novel and reversible inhibitors for the severe acute respiratory syndrome-coronavirus papain-like protease design, synthesis and antiviral efficacy of a series of potent chloropyridyl ester-derived sars-cov clpro inhibitors progress in anti-sars coronavirus chemistry, biology and chemotherapy structure-based design, synthesis, and biological evaluation of peptidomimetic sars-cov clpro inhibitors design and synthesis of peptidomimetic severe acute respiratory syndrome chymotrypsin-like protease inhibitors inhibition of papain-like protease plpro blocks sars-cov- spread and promotes anti-viral immunity a noncovalent class of papain-like protease/deubiquitinase inhib-grl- completely blocks sars-cov- infection ® itors blocks sars virus replication the species severe acute respiratory syndromerelated coronavirus: classifying -ncov and naming it sars-cov- hydroxychloroquine, a less toxic derivative of chloroquine, is effective in inhibiting sars-cov- infection in vitro sars-cov- cell entry depends on ace and tm-prss and is blocked by a clinically proven protease inhibitor hiv protease inhibitor nelfinavir inhibits replication of sarsassociated coronavirus enhanced isolation of sars-cov- by tmprss -expressing cells early events during human coronavirus oc entry to the cell the effects of basic substances and acidic ionophores on the digestion of exogenous and endogenous proteins in mouse peritoneal macrophages chloroquine is a potent inhibitor of sars coronavirus infection and spread effects of weakly basic amines on proteolytic processing and terminal glycosylation of secretory proteins in cultured rat hepatocytes crystal structure of sars-cov- main protease provides a basis for design of improved alpha-ketoamide inhibitors a novel central nervous systempenetrating protease inhibitor overcomes human immunodeficiency virus resistance with unprecedented am to pm potency recent developments in the use of differential scanning fluorometry in protein and small molecule discovery and characterization nafamostat mesylate blocks activation of sars-cov- : new treatment option for covid- inhibition of hiv- replication by daunorubicin selective inhibition of hiv replication by adriamycin in macrophages but not in lymphocytes boceprevir, gc- , and calpain inhibitors ii, xii inhibit sars-cov- viral replication by targeting the viral main protease trends and causes of mortality in a population-based cohort of hiv-infected adults in spain: comparison with the general population the effect of combination antiretroviral therapy use among hiv positive children on the hazard of aids using calendar year as an instrumental variable we thank clifford lane (national institute of allergy and infectious diseases, national institutes of health) for kindly providing remdesivir, thomas misteli (national cancer institute) for his critical reading and editing of the manuscript, and asuka fujiwara (national center for global health and medicine) for technical help. this study utilized the high-performance computational capabilities of the biowulf linux cluster at the national institutes of health, bethesda, md (https://hpc.nih.gov).the present work was supported in part by a grant for development of novel drugs for treating covid- (h. mitsuya, a ) from the intramural research program of national center for global health and medicine, in part by the intramural research our contributions were as follows: conceptualization, s.-i. hattori, a. k. ghosh, and key: cord- -mio przi authors: mcaloose, denise; laverack, melissa; wang, leyi; killian, mary lea; caserta, leonardo c.; yuan, fangfeng; mitchell, patrick k.; queen, krista; mauldin, matthew r.; cronk, brittany d.; bartlett, susan l.; sykes, john m.; zec, stephanie; stokol, tracy; ingerman, karen; delaney, martha a.; fredrickson, richard; ivančić, marina; jenkins-moore, melinda; mozingo, katie; franzen, kerrie; bergeson, nichole hines; goodman, laura; wang, haibin; fang, ying; olmstead, colleen; mccann, colleen; thomas, patrick; goodrich, erin; elvinger, françois; smith, david c.; tong, suxiang; slavinski, sally; calle, paul p.; terio, karen; torchetti, mia kim; diel, diego g. title: from people to panthera: natural sars-cov- infection in tigers and lions at the bronx zoo date: - - journal: mbio doi: . /mbio. - sha: doc_id: cord_uid: mio przi despite numerous barriers to transmission, zoonoses are the major cause of emerging infectious diseases in humans. among these, severe acute respiratory syndrome (sars), middle east respiratory syndrome (mers), and ebolaviruses have killed thousands; the human immunodeficiency virus (hiv) has killed millions. zoonoses and human-to-animal cross-species transmission are driven by human actions and have important management, conservation, and public health implications. the current sars-cov- pandemic, which presumably originated from an animal reservoir, has killed more than half a million people around the world and cases continue to rise. in march , new york city was a global epicenter for sars-cov- infections. during this time, four tigers and three lions at the bronx zoo, ny, developed mild, abnormal respiratory signs. we detected sars-cov- rna in respiratory secretions and/or feces from all seven animals, live virus in three, and colocalized viral rna with cellular damage in one. we produced nine whole sars-cov- genomes from the animals and keepers and identified different sars-cov- genotypes in the tigers and lions. epidemiologic and genomic data indicated human-to-tiger transmission. these were the first confirmed cases of natural sars-cov- animal infections in the united states and the first in nondomestic species in the world. we highlight disease transmission at a nontraditional interface and provide information that contributes to understanding sars-cov- transmission across species. and provide epidemiological and genetic evidence for human-to-animal transmission of the virus. our data show that tigers and lions were infected with different genotypes of sars-cov- , indicating two independent transmission events to the animals. importantly, infected animals shed infectious virus in respiratory secretions and feces. a better understanding of the susceptibility of animal species to sars-cov- may help to elucidate transmission mechanisms and identify potential reservoirs and sources of infection that are important in both animal and human health. keywords one health, panthera leo, panthera tigris, sars-cov- , in situ hybridization, lion, rrt-pcr, tiger, virus isolation, whole-genome sequencing, zoo, zoonotic infection c oronaviruses are a recognized cause of disease in humans and animals ( ) . among them are the viruses that cause colds in humans, multisystemic disease in domestic cats, and gastrointestinal and respiratory diseases in pigs and poultry. coronavirus disease (covid- ) caused by severe acute respiratory syndrome-related coronavirus (sars-cov- ) ( ) was first reported in wuhan, hubei province, china at the end of december ( ) . within weeks the virus spread globally, and by july , over million people were infected and more than , had died (https://www.who.int/ emergencies/diseases/novel-coronavirus- ; accessed july ). genome sequence analysis has shown sars-cov- to be most closely related to a bat coronavirus (ratg - ), and horseshoe bats are currently considered the likely source of the ancestral virus from which the currently circulating sars-cov- virus was derived ( , ) . subsequent genetic adaptation of the currently circulating virus in an intermediate animal host(s) or after human transmission has been proposed ( , ) . the exact details of how long the virus had been circulating in animals prior to transmission to people is unknown. however, a recent study suggests the virus may have been circulating in bats for several decades ( ) , and an early cluster of human covid- cases had an epidemiological link to the huanan seafood wholesale market in wuhan where a variety of live wild animals were sold ( ) . the current sars-cov- pandemic and outbreaks of severe acute respiratory syndrome (sars) and middle east respiratory syndrome (mers) before it raise awareness and concerns about zoonotic (animal-tohuman) diseases and cross-species transmission of coronaviruses ( ) ( ) ( ) ( ) . given the suspected zoonotic origin of sars-cov- , identifying susceptible animal species, reservoirs, and transmission routes between species is a topic of global scientific and public interest. natural sars-cov- infections in animals have been reported in dogs, cats, and farmed mink in hong kong, europe, china, and the united states ( ) ( ) ( ) . infection in most of these cases has been linked to households or settings in which human owners or caretakers have tested positive for sars-cov- and infection from humans to animals has been presumed. experimental inoculation studies have shown that sars-cov- infects and replicates with high efficiency in domestic cats, ferrets, and fruit bats and poorly in dogs; pigs, chickens, and ducks do not seem to support productive sars-cov- infection ( , ) . importantly, virus shedding and horizontal transmission have been shown in cats and ferrets ( ) ( ) ( ) following experimental inoculation. in this study, we report natural infection of tigers (panthera tigris) and lions (panthera leo) with sars-cov- at the wildlife conservation society's (wcs's) bronx zoo, new york, ny, and provide a detailed genomic characterization of viruses obtained from infected animals and keepers who had close contact with the sars-cov- -positive animals. these were the first confirmed animal infections in the united states and occurred in march , when, due to widespread community transmission ( ) , new york was a global sars-cov- epicenter. audible wheezing despite remaining eupneic. by april, an additional malayan tiger (tiger ) and two amur tigers (panthera tigris altaica) (tigers and ) housed in the same building as tiger but in different enclosures and three african lions (panthera leo krugeri) (lions , , and ) housed in a separate building developed similar respiratory signs. all animals otherwise exhibited normal behavior and activity. clinical respiratory signs resolved in less than days ( to april ) in all animals except tiger , whose clinical signs lasted days (resolution of clinical signs on april ). an additional amur tiger (tiger ) in the same building as tigers to did not develop clinical respiratory disease. detection of sars-cov- in affected animals. a broad diagnostic investigation was performed in tiger on april, days after the onset of clinical signs. this included physical examination, thoracic and abdominal radiography and ultrasonography, and collection of respiratory (nasal swab, oropharyngeal swab, and tracheal wash) and blood samples. thoracic radiography and ultrasonography revealed small, multifocal regions of peribronchial consolidation. cytologic examination of tracheal wash fluid identified necrotic epithelial and inflammatory cells consistent with tracheitis ( fig. a fig tracheal wash cytology (a and b) and in situ hybridization (ish) (c and d). tiger . (a) flocculent material from the trachea consists of stringy mucus with enmeshed degenerate cells characterized by condensed nuclei and loss of distinct cellular features (arrows). (b) few intact cells (short arrow) and degenerate epithelial cells (long arrow) are admixed with abundant round to amorphous cellular debris and granular degenerate mucus (arrowheads). inset: degenerate epithelial cell (upper right) with nuclear fragmentation (karyolysis) and an adjacent intact neutrophil (lower left). modified wright's stain. (c and d) incubation with sars-cov- -specific probe is positive (red puncta) throughout the mucinous material, in the cytoplasm of intact and degenerate epithelial and inflammatory cells, and in cellular debris. red chromogenic assay; hematoxylin counterstain. (note: a software or equipment malfunction produced a faint horizontal line that may be visible in panels a and b.) and b). in situ hybridization (ish) colocalized sars-cov- rna within necrotic epithelial and inflammatory cells in the fluid ( fig. c and d and see also fig. s ). all respiratory samples (nasal, oropharyngeal, and tracheal wash) were negative on virus isolation for feline herpesvirus and feline calicivirus and on targeted pcr testing and metagenomic analysis for common feline pathogens (table s , bioproject accession no. prjna ); all were positive for sars-cov- by real-time reverse transcription-pcr (rrt-pcr) using primers and probes targeting portions of the nucleocapsid (n , n , and n ) and envelope (e) genes (table s ). minion and sanger-based sequencing of the full sars-cov- spike (s) gene, an internal region of the n gene, and the rna-dependent rna polymerase (rdrp) confirmed the virus in the respiratory samples (table s and data set [see "data availability" in materials and methods for definitions and locations of all data sets]). fecal samples collected opportunistically from each animal (symptomatic tigers to , asymptomatic tiger , and symptomatic lions to ) tested positive for sars-cov- by rrt-pcr (table s ). results were confirmed by amplicon sequencing (table s and data set ). infectious sars-cov- detected in respiratory and fecal samples from affected animals. virus isolation was performed on all respiratory and fecal samples. cytopathic effect (cpe) was observed in vero cells inoculated with tracheal wash fluid from tiger ( fig. a and b ) and fecal samples from tiger and lion (table s ). results were confirmed by rrt-pcr (cdc n assay) and/or ish and immunofluorescence assays ( fig. c and d) . additionally, a neutralizing antibody titer of detected in tiger confirmed active sars-cov- infection in this animal (table s ) . epidemiologic and diagnostic investigation of zoo staff. subsequent to confirmation of sars-cov- infection in the animals, an epidemiologic investigation of zoo staff identified zoo keepers and two managers who provided care for and had close (Յ . -m) but not direct contact with the tigers or lions between march (the date on which the zoo was closed to the public due to the pandemic) and march to april (timeline of disease onset in the animals). four staff ( tiger and lion keepers) reported mild respiratory symptoms (including fever, cough, chills, myalgia, and fatigue) between and march . nasopharyngeal samples and blood were collected from these staff members on april , and rrt-pcr and a microsphere immunoassay (mia; to detect igg antibodies) were performed; staff who did not report symptoms were not tested. all tested keepers had evidence of current or prior sars-cov comparative genomics and phylogenetic and haplotype network analysis. nine complete sars-cov- genome sequences (from four tigers, three lions, and two keepers) and eight full-length s gene sequences (from seven symptomatic animals and one asymptomatic animal) were generated directly from respiratory and/or fecal samples (data sets and ). compared to the wuhan-hu- sequence (genbank accession number nc_ ), all tiger and keeper sequences contained six single-nucleotide polymorphisms (snps) with nine additional ambiguous sites ( fig. s and table s ). a total of sites differed between the three lion sequences and wuhan-hu- ( fig. s and table s ). viral sequences in the tigers, lions, and keepers clustered into common sars-cov- clades (fig. a) . those from tigers and tiger keepers clustered with clade g (defined by the d g substitution in the spike protein); the lion sequences clustered with clade v (defined by the g v substitution in orf a) (fig. a ). median-joining haplotype network analysis of the viral sequences corroborated results of phylogenetic analyses ( fig. b and data set ). nucleotide sequence and amino acid analysis of the spike protein of sars-cov- in tigers and lions was performed. compared with the wuhan-hu- strain, the tiger and lion sars-cov- s gene sequences had to nucleotide differences that resulted in several nonsynonymous substitutions (fig. ). of five substitutions in the tiger strains, only one (g d) was found in available human sars-cov- strains. these changes were not observed in the viral sequences from the lions (fig. ). our results document susceptibility and natural sars-cov- infection in tigers and lions. these were the first confirmed animal infections in the united states and the first to be described in a nondomestic species in the world. genomic and epidemiological data support a close evolutionary relationship between the viral strains in the tigers and those in the tiger keepers. notably, the genetic differences and the distant phylogenetic relationship between sequences recovered from the tigers/tiger keepers and lions and the relationship of these strains in the context of global sequences indicate that tigers and lions were infected by two different sars-cov- genotypes. these data suggest that at least two independent sars-cov- introductions occurred, one in tigers and another in lions. importantly, the sars-cov- genome sequence from tiger was identical to the viral sequence from keeper (a tiger keeper) and to other human sars-cov- strains detected in new york (ny-cdc- [mt ] and ny-qdx- [mt . ]). these observations, temporal overlap in animal and human infections, and a lack of new animal introductions to the collection support the conclusion of transmission from an infected keeper(s) to the tigers. whether this was direct or indirect (e.g., fomite, food handling/preparation, or shared enrichment items) and whether subsequent tiger-to-tiger transmission (aerosol, respiratory droplet, etc.) occurred were not determined. a clear association and transmission source were not identified for the lions. the lion sars-cov- sequences were more divergent than those in the tigers and keepers. interestingly, nine of the snps (relative to the wuhan-hu- reference strain) shared by all three lion viruses were also found in a human strain (closest strain, gisaid accession no. epi_isl_ ) detected in connecticut, usa. two lion keepers were serologically positive for sars-cov- , but viral rna was not detected, and sars-cov- could not be confirmed in their respiratory samples. however, given close contact between keepers and animals and the serological evidence indicating infection of two of the lion keepers, it is possible that while asymptomatic, they or other asymptomatic staff may have transmitted virus to the lions. the host range of sars-cov- and other coronaviruses is determined primarily by the interaction of the virus s glycoprotein, specifically the spike subunit (s ), and the cellular receptor, angiotensin-converting enzyme ii (ace ) ( ) . in silico predictions have shown high binding potential between the s receptor binding domain (rbd) and domestic cat ace receptor and conservation of three of five amino acid residues that are critical for interaction with the sars-cov- s glycoprotein in human and domestic cat ace ( ) . these observations are supported by reports describing natural and experimental infection of domestic cats with sars-cov- ( , ) and the data here that show a high degree of conservation between ace in humans and domestic and wild felids. further work is needed to determine if these changes affect sars-cov- receptor binding and pathogenicity in felids and humans. infections in the tigers and lions occurred at a time before sars-cov- testing was widely available in the united states and when there was limited evidence of pre-or asymptomatic viral shedding ( ) . additionally, at that time, keepers caring for the tigers and lions did not generally wear personal protective equipment (ppe) given the (historical) low risk of infectious respiratory disease transmission between humans and domestic or nondomestic felid species. results of this investigation prompted the immediate development of new protocols for ppe use in the enclosures of nondomestic felids and other known or susceptible species including mustelids, viverrids, and chiroptera (ppe was already in place for work with nonhuman primates) at the bronx zoo. they also contributed to the development of similar recommendations by other zoo and wildlife organizations (https://www.aza.org/aza-news-releases/posts/aza-and-aazv-statement-on -covid- -positive-tiger-in-new-york). the role of domestic and wild animal species in the epidemiology of sars-cov- is not completely understood. to date, the reported number of cases of sars-cov- infection in domestic and wild animal species is low, and to our knowledge, no other zoos worldwide have confirmed cases in their animals. this is notable when considered in the context of the large number of human cases and close interactions between people, their pets, and wild animals in their care. however, the fact that companion animals, farmed mink, and zoo animals are susceptible to sars-cov- infection and shed infectious virus in respiratory secretions and/or feces ( - ) makes the humananimal-environment interface an important area for further one health-based studies ( ) . in general, a better understanding of sars-cov- susceptibility across a wide range of animal species will help to elucidate transmission mechanisms and identify potential reservoirs and sources of infection important in both animal and human health. in the last decades, at least three major coronavirus epidemics (sars, mers, and covid- ) have occurred. a feature shared by these and other novel viruses of humans including ebolaviruses and human immunodeficiency virus (hiv) is an origin in a wild sars-cov- in tigers, lions, and keepers at a zoo ® animal host. despite a traditionally held perception of low risk, scientists and conservationists around the world have long recognized and shared concerns related to human activities that increase human-wildlife interactions and zoonotic disease transmission risk ( ) ( ) ( ) . as long as anthropogenic development and population growth bring humans and wildlife into increasing proximity, legal and illegal harvesting persists, and consumption of wildlife and wildlife products exists, there will be continued and significant risk of pandemic viral emergence with devastating global impact on human and animal health, economies, food security, and biodiversity. personal protective equipment (ppe), including n or surgical masks, face shields or goggles, and disposable gloves, were not generally worn when working with the tigers and lions prior to the sars-cov- pandemic but were worn during all animal handling and sample collection subsequent to the development of clinical signs in tiger . diagnostic specimens were submitted to the university of illinois veterinary diagnostic laboratory (uiuc-vdl) and animal health diagnostic center at cornell university (cornell ahdc) (both part of the national animal health laboratory network) for broad diagnostic investigation (respiratory samples-tiger ) and specific sars-cov- testing (fecal samplesall animals). epidemiologic investigation. subsequent to the development of clinical signs and positive test results in the tigers and lions, an epidemiologic investigation into possible human infections in staff working with these animals was conducted by the new york city (nyc) and new york state (nys) public health laboratories in conjunction with the u.s. centers for disease control and prevention (cdc). the investigation focused on the time period between march (date on which the zoo was closed to the public due to the pandemic) and march to april (the timeline of disease onset in the animals). twelve staff members ( keepers and managers) were identified who had responsibilities that offered opportunities for close (Յ . -m) but not direct contact with the animals during this time period. this included moving animals between enclosures and exhibits, feeding, training sessions, enrichment activities, greetings, and social interactions (e.g., chuffing, a form of vocalization that tigers performed that involves air exhalation and which keepers also use to greet tigers). additionally, lion keepers worked at a desk that was located less than . m from metal-mesh-fronted lion enclosures. four keepers reported being mildly symptomatic (including fever, cough, chills, myalgia, and fatigue) with signs beginning in each prior to or concurrently with illness in animals on , , , and march . nasopharyngeal swab and blood samples were collected on april from the symptomatic keepers, and sars-cov- -specific rrt-pcr and a microsphere immunoassay (to detect igg antibodies) were performed; sampling and testing were not performed in the eight additional staff who did not report symptoms. all four of the tested keepers had evidence of sars-cov- infection (one rrt-pcr-positive tiger keeper [keeper ], one rrt-pcr-and serology-positive tiger keeper [tiger ], and two serology-positive lion keepers [keeper and keeper ]). none of the keepers reported being sick at work. all stayed home for at least days from the onset of illness, and none returned to work prior to a minimum of symptom-free days and fever-free hours in compliance with organizational covid- policies and cdc and ny department of health (doh) guidelines. rrt-pcr-positive specimens were forwarded to cdc for whole-genome sequencing (wgs) and haplotype network analysis to characterize the human samples and further compare the human and animal viral genome sequences. interviews with the tiger and lion keepers suggested that up to two additional keepers may have had signs or symptoms suggestive of mild and transient covid- ; however, they did not self-report being sick, may not have recognized their symptoms as being consistent with covid- , and were not tested. non-sars-cov- respiratory pathogen testing. nucleic acid extracted from the respiratory specimens (nasal and oropharyngeal swabs and tracheal wash fluid) from tiger was tested by real-time pcr (rpcr) or rrt-pcr for several common feline respiratory pathogens (cornell ahdc) including bordetella bronchiseptica ( ) , influenza a virus (cdc universal assay) ( ) , mycoplasma cynos ( ) , mycoplasma felis ( ) , pneumovirus ( ) with probe modification ( -carboxyfluorescein [fam]-cttcatcacttttggcctgg cccag-bhq ), and streptococcus equi subsp. zooepidemicus ( ) . additionally, samples were tested for chlamydia psittaci, chlamydia felis, and chlamydia abortus using a conventional pcr assay ( ) , and virus isolation was performed using inoculated feline pulmonary cells to test for feline herpesvirus and feline calicivirus. all assays have been adapted and optimized and are used routinely in feline infectious respiratory disease diagnostic testing. tracheal wash fluid cytology. direct smears of buoyant, flocculent material in the tracheal wash fluid and cytocentrifuge smears of the remaining fluid were prepared and stained with a romanoski stain (modified wright's stain) using an automated stainer (hema-tek ; siemens). the stained slides were examined using standard bright-field microscopy by a board-certified veterinary clinical pathologist (cornell ahdc). in situ hybridization for sars-cov- rna. unstained cytologic smears of tracheal wash fluid from tiger were fixed in ice-cold % methanol for min and stored at Ϫ °c until shipped to uiuc college of veterinary medicine zoological pathology program (zpp) on cold packs. upon arrival, the slides were submerged for an additional min in % neutral buffered formalin at °c, air dried, and then placed in % ethyl alcohol for min at room temperature. an in situ hybridization (ish) chromogenic manual assay was performed using the rnascope . hd detection kit red and a -pair oligonucleotide probe targeting the sars-cov- s gene of the wuhan hu- complete genome (nc_ . ; advanced cell diagnostics catalog no. ) according to the manufacturer's directions (advanced cell diagnostics, inc., newark, ca). positive-control slides consisted of vero cells infected with sars-cov- isolated from tiger . a control probe targeting the dapb gene from the bacillus subtilis strain smy (advanced cell diagnostics catalog no. ) was used as a negative control on all cytology sections in parallel with the sars-cov- target probe (see fig. s ). additional negative controls to rule out cross-reactivity with tiger rna and the felid alphacoronavirus included a cytocentrifuge preparation of cell cultures infected with the alphacoronavirus feline enteric coronavirus (fecov) (kindly provided by gary whittaker, cornell university); formalin-fixed, paraffin-embedded (ffpe) unstained sections of normal malayan tiger trachea, lung, and oropharyngeal tissue; and lung, lymph node, and intestine from a fecov-positive domestic cat (fig. s ) . samples from the control tiger and domestic cat were collected opportunistically in and , respectively, and archived as part of routine necropsy procedures (wildlife conservation society's [wcs's] bronx zoo and uiuc-zpp, respectively). virus isolation. virus isolation on respiratory and fecal samples was performed in vero (atcc ccl- ), vero e , and vero cells under biosafety level conditions at the cornell ahdc and the national veterinary services laboratory (nvsl). cells were cultured in minimum essential medium eagle (mem-e; gibco, gaithersburg, md) supplemented with . to % fetal bovine serum (fbs; gibco), iu/ml penicillin, and g/ml streptomycin (growth medium). cells were seeded in -well culture plates or t flasks and cultured at °c with % co for to h. before inoculation, respiratory swabs and tracheal wash fluid samples were diluted at : , : , and : , in serum-free mem-e containing ui/ml penicillin, g/ml streptomycin, and . g/ml amphotericin b (all from gibco); swabs from fecal samples were placed in ml of sterile pbs supplemented with . % bovine serum albumin (bsa) (sigma-aldrich, st. louis, mo) containing ui/ml penicillin, g/ml streptomycin, and . g/ml amphotericin b (all from gibco). cells were rinsed with mem-e and inoculated with l of each respiratory sample dilution in individual wells of a -well plate or . ml of the diluted fecal sample in a t flask and adsorbed for h at °c with % co for h. mock-inoculated cells were used as negative controls. after adsorption, replacement medium was added, and cells were incubated at °c with % co and monitored daily for cytopathic effect (cpe) for days. cell cultures with no cpe were frozen, thawed, and subjected to three blind passages with inoculation of fresh vero cell cultures with the lysates as described above. sars-cov- infection in cpe-positive cultures was confirmed with sars-cov- specific rrt-pcr using the cdc n primer and probe set (sequences available upon request), an immunofluorescence assay using a mouse monoclonal antibody against the sars-cov n protein ( , ) , and rnascope in situ hybridization as described above. virus neutralization assay. seroconversion of tiger to sars-cov- was assessed by a virus neutralization assay (vn; cornell ahdc). twofold serial dilutions ( : to : , ) of a serum sample collected on april ( days after the onset of clinical signs) were incubated with % tissue culture infective doses (tcid ) of sars-cov- on vero cells for h at °c. following incubation of serum and virus, l of a cell suspension of vero ccl- cells was added to each well of a -well plate and incubated for h at °c with % co . virus cytopathic effect (cpe) was used as an indicator of virus infection/replication. neutralizing antibody titers were expressed as the reciprocal of the highest dilution of serum that completely inhibited cpe. archived frozen sera from another tiger (archived frozen at cornell ahdc) and positive human control sera (deidentified convalescent human sera provided by cayuga medical center, irb protocol ep) were included, and all samples were tested in triplicate with results averaged. a cell culture control was included in the assays, and the virus working dilution was back-titrated. sars-cov- rrt-pcr. nucleic acid was extracted from nasal and oropharyngeal swabs and tracheal wash fluid (tiger ) using the magmax isolation kit (thermo fisher scientific, waltham, ma) and from fecal samples (tigers to and lions to ) using the magmax core nucleic acid purification kit (thermo fisher scientific, waltham, ma) and an automated nucleic acid extractor (king fisher flex purification system; thermo fisher scientific, waltham, ma) according to the manufacturer's instructions. analysis for sars-cov- rna was performed with real-time reverse transcriptase (rrt) pcr and either the -ncov cdc qpcr probe assay targeting three regions of the nucleocapsid (n) gene (n , n , and/or n ; integrated dna technologies [idt], inc., coralville, ia) (uiuc-vdl, cornell ahdc) or the cdc qpcr probe assay, and in-house primers for the envelope (e) gene with the agpath-id one-step rt-pcr kit (applied biosystems, foster city, ca) (uiuc-vdl) (primer and probe sequences are available upon request). thermal cycler conditions consisted of reverse transcription and enzyme activation at to °c for min and °c for min, respectively, followed by to cycles of °c for to s and to °c for s. positive ( -ncov_n_positive control; idt, coralville, ia, and a synthesized plasmid [genscript] e gene control) and negative (distilled or nuclease-free water) controls, plus internal amplification controls (xeno or beta-actin; thermo fisher scientific, waltham, ma), were included as separate reaction mixtures. following initial rrt-pcr testing at both institutions, samples were submitted to the nvsl for confirmatory testing, using the -ncov cdc qpcr probe assay and n and n primers (sequences available upon request). amplicon sequencing. minion and sanger amplicon sequencing was used to confirm rrt-pcr results at cornell ahdc and nvsl, respectively (primer sequences are available upon request). for minion-based sequencing, targets were amplified directly from tracheal wash or fecal samples using the superscript iv one-step rt-pcr system (thermo fisher scientific, waltham, ma). primers targeted the complete spike (s) gene ( , bp) and an internal region of the n gene ( bp). universal oxford nanopore-compatible adapter sequences were added to the = end of each primer sequence to allow pcr-based barcoding. amplicons were purified (ampure xp beads [beckman coulter, brea, ca]; . : volumetric bead-to-dna ratio), and dna quantification was performed on a qubit fluorometer . (double-stranded dna [dsdna] high-sensitivity assay kit; thermo fisher scientific, waltham, ma). samples were subsequently diluted to . nm in a total of l and used as the input for the library preparation following the d pcr barcoding ( ) genomic dna (sqk-lsk ) protocol (oxford nanopore technologies, oxford, uk). final dna libraries were loaded in a flo-min r . flow cell to start the sequencing runs. sanger sequencing was performed using primers targeting partial regions of s, n, and rnadependent rna polymerase (rdrp) genes (primer sequences available upon request). amplicons were generated directly from nasal and oropharyngeal swabs and tracheal wash fluid using the superscript iii one-step rt-pcr system (thermo fisher scientific, waltham, ma). reaction mixtures were purified using the qiagen pcr purification kit (qiagen, germantown, md). dna was amplified for sanger sequencing using the bigdye terminator v . cycle sequencing kit (thermo fisher scientific, waltham, ma) and sequenced on the applied biosystems xl genetic analyzer (thermo fisher scientific, waltham, ma). whole-genome sequencing and phylogenetic analysis. whole-genome sequencing (wgs) was performed on tracheal wash fluid and fecal specimens from all individual tigers and lions as previously described ( ) . individual fecal samples from tigers to and lions to and cell culture viral isolates were subjected to sequencing with either minion-based amplicon sequencing using overlapping primers covering the full viral genome (amplicons with an average size of ϳ , bp; primer sequences are available upon request) or the ion ampliseq kit for chef dl and ion ampliseq sars-cov- research panel (thermo fisher scientific, waltham, ma) (data sets and ). minion libraries were prepared as previously described ( ) using the native barcode kit, exp-nbd , ligation sequencing kit, sqk-sqk (oxford nanopore technologies), and sequenced on an r . flow cell for h. ion targeted libraries were sequenced using an ion chip on the ion s system using the ion -ion -ion kit (thermo fisher scientific, waltham, ma). viral isolates were sequenced with the minion or ion ampliseq approach as described above. whole-genome sequencing on the rrt-pcr-positive specimens from the two sars-cov- -positive keepers was performed as previously described using an amplicon sequencing approach and sanger sequencing ( ) (data set ). all genomes for each animal that were assembled using data generated from different sequencing platforms (illumina, minion, and/or ion torrent) were combined into a single consensus sequence for each animal (data set ). the assemblies for a given animal were aligned with the wuhan-hu- reference sequence (nc_ . ) using mafft v. . ( ) . the reference sequence was then removed from the alignment, and a consensus sequence for the virus sequence recovered from each animal was generated using the consambig program in emboss v. . . . ( ) . when a single assembly shifted alignment due to a single base insertion or repeat nucleotide, the alignment was rerun after removal of the offending nucleotides. to compare the outbreak genomes to others isolated from humans in the same geographic region, all available sars-cov- genomes from new york were downloaded from ncbi on april . these were clustered at . % identity using vsearch v. . . ( ) , and the consensus sequences from each cluster were aligned along with wuhan-hu- reference sequence, using mafft v. . ( ) . a phylogenetic tree was constructed using the consensus sequence from each animal, keepers, sequences from new york, and the wuhan-hu- reference sequence (nc_ . ) using the gtr-gamma model in raxml v. . . ( ) . haplotype network analysis. haplotype network analyses were conducted with two overlapping data sets using popart software ( ) using the median joining algorithm ( ) . data set contained nine genomes generated from the bronx zoo cases: four tigers, three lions, and two tiger keepers. data set contained the nine genomes from data set , as well as additional genomes (including the sars-cov- reference wuhan-hu genome) generated from a total of countries to better understand genetic relatedness of bronx zoo cases in the context of the global pandemic. the top blast results for the lion sequences were included in data set . for both data sets, the entire genome alignment was examined visually for accuracy and evidence of large-scale rearrangements to rule out the likelihood of multiple single-nucleotide polymorphisms (snps) being the result of a single evolutionary event. subsequently, whole-genome alignments were converted into an snp matrix by removing columns containing identical bases, gaps, and ambiguous bases. the lengths of final snp matrices were nucleotides (nt) (data set ) and nt (data set ). data availability. primer and probe sequence information for rrt-pcr and amplicon and wholegenome sequencing is available upon request. all remaining data are available in the main text or the supplemental material or as follows: data set , metagenomics data obtained from respiratory specimens from tiger have been deposited in srr under prjna ; data set , whole-genome consensus sequences obtained from sars-cov- detected in fecal samples from tigers to and lions to have been deposited in genbank under accession numbers mt , mt , mt , mt , mt , and mt , respectively; data set , whole-genome sequences obtained from sars-cov- tiger and lion isolates (tgr /ny/ , tgr /ny/ , and ln /ny/ ) have been deposited in genbank under accession numbers mt , mt , and mt , respectively; data set , whole-genome sequences obtained from sars-cov- strains in keepers and have been deposited in genbank under accession numbers mt and mt , respectively. supplemental material is available online only. the emergence of sars, mers and novel sars- coronaviruses in the st century the species severe acute respiratory syndrome-related coronavirus: classifying -ncov and naming it sars-cov- china novel coronavirus investigating and research team. . a novel coronavirus from patients with pneumonia in china a pneumonia outbreak associated with a new coronavirus of probable bat origin the proximal origin of sars-cov- full-genome evolutionary analysis of the novel corona virus ( -ncov) rejects the hypothesis of emergence as a result of a recent recombination event evolutionary origins of the sars-cov- sarbecovirus lineage responsible for the covid- pandemic pathways to zoonotic spillover host range and emerging and reemerging pathogens risk factors for human disease emergence reverse zoonotic disease transmission (zooanthroponosis): a systematic review of seldomdocumented human 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production and characterization of monoclonal antibodies against the nucleocapsid protein of sars-cov complete genome sequence of sars-cov- in a tiger from a u ncov- sequencing protocol rapid, sensitive, full-genome sequencing of severe acute respiratory syndrome virus coronavirus mafft multiple sequence alignment software version : improvements in performance and usability emboss: the european molecular biology open software suite vsearch: a versatile open source tool for metagenomics raxml version : a tool for phylogenetic analysis and post-analysis of large phylogenies popart: full-feature software for haplotype network construction median-joining networks for inferring intraspecific phylogenies we declare no competing interests. this manuscript represents the opinions of the authors and does not necessarily reflect the position of the u.s. centers for disease control and prevention. key: cord- -ssen q authors: albrecht, randy a.; liu, wen-chun; sant, andrea j.; tompkins, s. mark; pekosz, andrew; meliopoulos, victoria; cherry, sean; thomas, paul g.; schultz-cherry, stacey title: moving forward: recent developments for the ferret biomedical research model date: - - journal: mbio doi: . /mbio. - sha: doc_id: cord_uid: ssen q since the initial report in , the domestic ferret has become an invaluable biomedical research model. while widely recognized for its utility in influenza virus research, ferrets are used for a variety of infectious and noninfectious disease models due to the anatomical, metabolic, and physiological features they share with humans and their susceptibility to many human pathogens. however, there are limitations to the model that must be overcome for maximal utility for the scientific community. here, we describe important recent advances that will accelerate biomedical research with this animal model. populations. it is likely that new models and transgenic animals will be developed in the near future. the sequencing of the ferret genome ( ) was instrumental in advancing functional genomic analysis. numerous groups developed reagents to monitor gene-specific mrna expression levels via taqman-based or sybr green-based real-time reverse transcription-pcr assays for a plethora of targets. many of these primers are available free of charge through the national institute of allergy and infectious diseases (niaid) established bei resources (https://www.beiresources.org/home.aspx). bruder et al. described the development of an expression microarray platform that included the identification of genes with consistent baseline transcription profiles across tissues that could be used as housekeeping genes ( ) . our group developed and is validating a fluidigm panel with distinct immune response and lung injury and repair genes. beyond transcription, tisoncik-go et al. described an integrated omics analysis that profiles lipids, metabolites, and proteins in the respiratory compartments of influenza virus-infected ferrets ( ) . combined, these tools provide powerful resources to the research community. despite its relevance for biomedical research, there are limitations of the ferret model for immunologic studies due to the dearth of reagents. screening of commercially available antibodies for cross-reactivity with markers on innate and adaptive cell subsets and cytokines in ferrets has yielded limited success ( table ) . to resolve this, a group of researchers from around the world are working together to develop validated reagents and assays to improve our understanding of the innate and adaptive immune responses in the ferret. to date, recombinant proteins representing a range of intrinsic, innate, and adaptive immune markers are under development, and some are already available from commercial sources ( , ) . these include type i and iii interferons (ifns), rig-i and toll-like receptors, cytokines, and chemokines, as well as cell surface markers for immune and nonimmune cells. in terms of adaptive immune responses, kirchenbaum and ross recently developed a monoclonal antibody against the ferret b cell receptor light chain that is useful in distinguishing kappa versus lambda b cell responses ( , ) . enzymelinked immunosorbent spot (elispot) and flow cytometric assays have been developed to quantify the isotypes of antibody-secreting cells (igg or iga) ( ) , pan-b cells (cd ϩ , cd ␣ ϩ ), and ig ϩ b cells ( , ) . t cell phenotyping has been limited to quantification of overall cd ϩ t cells, including cd ϩ and cd ϩ subsets, by flow cytometric assays and identification of antigen-specific effector responses by detecting ifn-␥ secretion in flow-based intracellular cytokine secretion assays or elispot assays ( ) . an in vivo depletion of cd t cells using a cross-reactive human monoclonal antibody has been shown to delay influenza virus clearance ( ) . to increase our toolbox, the centers for excellence in influenza research and surveillance (ceirs) network has undertaken a large project to rapidly produce monoclonal antibodies and develop assays to support the universal influenza vaccine initiative ( ) . antibodies in production include b cell markers (cd , cd , cd , cd , cd , cd , cd , cd , cd , cxcr , and fcr), t cell markers (cd , ccr , cd e, cd , cd l, cd , cd l, cd , cd , pd- , cxcr , interleukin- receptor [il- r], and il- ra) and others (cxcr , cd , il- , il- , and il- ). these much-needed reagents will facilitate efforts to establish immunologic assays to interrogate the innate and adaptive immune responses to infection and vaccination at the level of detail that is routinely applied to studies of mouse or human immunology. importantly, the ferret model will allow correlates of protection to be established after vaccination and infection in conjunction with transmission studies, which are not available in the mouse models. additionally, the longer life span of the ferret relative to the mouse will allow analysis of the evolution of the immune response to sequential infection and/or vaccination ( ) , permitting more accurate modeling of the immune response in humans. while there has been exciting progress, much work remains to move the ferret model forward. toward this goal, the ceirs group has produced fibroblasts and primary nasal and tracheal epithelial cells and cell lines, established a repository of defined tissues and cell types (table ) , and are working with the j. craig venter institute to define the ferret major histocompatibility complex (mhc). an exciting achievement is the completion of the pacbio sequencing of the ferret mhc (granger sutton, personal communication). while these are important steps, the ultimate goal is to provide the biomedical research community with validated reagents and protocols they can trust to ensure the rigor and reproducibility in experiments utilizing the ferret model. in support of this goal, many of the reagents created through the ceirs network will be made publicly available through the ceirs data processing and coordinating center (dpcc) website (http://www.niaidceirs.org/resources/ceirs-reagents/). we thank everyone involved in team ferret, whose names we will not list for fear we might miss someone, as well as others producing reagents for the ferret model. we also thank diane post (niaid) and the members of the ceirs network for feedback, advice, and constructive criticism. studies in the embryology of the ferret anatomy of the ferret heart: an animal model for cardiac research a model of spinal cord injury drug effects on after discharge and seizure threshold in lissencephalic ferrets: an epilepsy model for drug evaluation a ferret model of copdrelated chronic bronchitis airway disease phenotypes in animal models of cystic fibrosis development of ferret as a human lung cancer model by injecting -(n-methyl-n-nitrosamino)- -( -pyridyl)- -butanone (nnk) building the ferretome cloned ferrets produced by somatic cell nuclear transfer adeno-associated virus-targeted disruption of the cftr gene in cloned ferrets crispr/cas -mediated genome engineering of the ferret live attenuated influenza vaccine is safe and immunogenic in immunocompromised ferrets influenza transmission in the mother-infant dyad leads to severe disease, mammary gland infection, and pathogenesis by regulating host responses impaired heterologous immunity in aged ferrets during sequential influenza a h n infection the draft genome sequence of the ferret (mustela putorius furo) facilitates study of human respiratory disease transcriptome sequencing and development of an expression microarray platform for the domestic ferret integrated omics analysis of pathogenic host responses during pandemic h n influenza virus infection: the crucial role of lipid metabolism flow cytometric and cytokine elispot approaches to characterize the cell-mediated immune response in ferrets following influenza virus infection screening monoclonal antibodies for cross-reactivity in the ferret model of influenza infection generation of monoclonal antibodies against immunoglobulin proteins of the domestic ferret (mustela putorius furo) infection of ferrets with influenza virus elicits a light chain-biased antibody response against hemagglutinin vaccine-specific antibody secreting cells are a robust early marker of laiv-induced b-cell response in ferrets contemporary seasonal influenza a (h n ) virus infection primes for a more robust response to split inactivated pandemic influenza a (h n ) virus vaccination in ferrets a universal influenza vaccine: the strategic plan for the national institute of allergy and infectious diseases immune history shapes specificity of pandemic h n influenza antibody responses a virus obtained from influenza patients the pathogenesis of respiratory syncytial virus infection in infant ferrets ferrets as a novel animal model for studying human respiratory syncytial virus infections in immunocompetent and immunocompromised hosts identification of small-animal and primate models for evaluation of vaccine candidates for human metapneumovirus (hmpv) and implications for hmpv vaccine design comparison of wild-type and subacute sclerosing panencephalitis strains of measles virus. neurovirulence in ferrets and biological properties in cell cultures assessment of the ferret as an in vivo model for mumps virus infection infection of mice, ferrets, and rhesus macaques with a clinical mumps virus isolate further studies on the neonatal ferret model of infection and immunity to and attenuation of human parainfluenza viruses effect of upper respiratory infection on hearing in the ferret model virology: sars virus infection of cats and ferrets a neutralizing human monoclonal antibody protects against lethal disease in a new ferret model of acute nipah virus infection the domestic ferret (mustela putorius furo) as a lethal infection model for species of ebolavirus rift valley fever: a report of three cases of laboratory infection and the experimental transmission of the disease to ferrets influenza enhances susceptibility to natural acquisition of and disease due to streptococcus pneumoniae in ferrets in vivo localization of staphylococcus aureus in nasal tissues of healthy and influenza a virus-infected ferrets helicobacter infections in laboratory animals: a model for gastric neoplasias a new experimental infection model in ferrets based on aerosolised mycobacterium bovis animal models of pneumocystosis immune system cells in healthy ferrets: an immunohistochemical study cellular immune response in the presence of protective antibody levels correlates with protection against influenza in ferrets evaluation of the humoral and cellular immune responses elicited by the live attenuated and inactivated influenza vaccines and their roles in heterologous protection in ferrets key: cord- - m i h authors: pope, welkin h.; jacobs-sera, deborah; russell, daniel a.; rubin, daniel h. f.; kajee, afsana; msibi, zama n. p.; larsen, michelle h.; jacobs, william r.; lawrence, jeffrey g.; hendrix, roger w.; hatfull, graham f. title: genomics and proteomics of mycobacteriophage patience, an accidental tourist in the mycobacterium neighborhood date: - - journal: mbio doi: . /mbio. - sha: doc_id: cord_uid: m i h newly emerging human viruses such as ebola virus, severe acute respiratory syndrome (sars) virus, and hiv likely originate within an extant population of viruses in nonhuman hosts and acquire the ability to infect and cause disease in humans. although several mechanisms preventing viral infection of particular hosts have been described, the mechanisms and constraints on viral host expansion are ill defined. we describe here mycobacteriophage patience, a newly isolated phage recovered using mycobacterium smegmatis mc( ) as a host. patience has genomic features distinct from its m. smegmatis host, including a much lower gc content ( . % versus . %) and an abundance of codons that are rarely used in m. smegmatis. nonetheless, it propagates well in m. smegmatis, and we demonstrate the use of mass spectrometry to show expression of over % of the predicted proteins, to identify new genes, to refine the genome annotation, and to estimate protein abundance. we propose that patience evolved primarily among lower-gc hosts and that the disparities between its genomic profile and that of m. smegmatis presented only a minimal barrier to host expansion. rapid adaptions to its new host include recent acquisition of higher-gc genes, expression of out-of-frame proteins within predicted genes, and codon selection among highly expressed genes toward the translational apparatus of its new host. phages (average, . % gc). the genome diversity and percent gc range of the mycobacteriophages support a model for phage genome evolution in which phages migrate much more rapidly across a diverse bacterial landscape than their genomes ameliorate toward that of any one host ( ) . thus, mycobacteriophages at the lower end of the percent gc spectrum are predicted to have infected lower-gc hosts in their recent evolutionary pasts ( ) . we note that there are other phage-host systems with mismatched gc contents, such as t and the right arm of phage lambda ( . % and . %, respectively), relative to their escherichia coli host ( . %). there is also substantial variation in trna content of the mycobacteriophages. many have no trna genes, and some have only one or a small number, while others-such as the members of clusters c and m-have more than ( , ) . the rationale for carriage of these is unclear, and there is no obvious correlation between trna content and percent gc that might reflect trna acquisition to augment gene expression in newly acquired hosts. it has been noted that there is no close correlation between the trna specificities encoded by d and infrequently used codons ( ) or between bxz and phage codon preferences of putative highexpression genes ( ) . analysis of the codon usage of mycobacteriophage genomes showed that there is variation in codon usage preferences ( ) . in some phage genomes, the trnas may counteract host measures to protect themselves from infection by trna destruction ( ) . here, we describe mycobacteriophage patience, a newly isolated phage of m. smegmatis mc that is a singleton with no close relatives and has a gc content of . %, representing the extreme low end of the percent gc spectrum for mycobacteriophages. of the predicted patience protein-coding genes, % are "orphams" with no close mycobacteriophage relatives in the phamerator_ database, and most of the genes with homologues in other mycobacteriophages are most closely related to those in clusters h, r, and d, which also have below-average gc contents. however, patience has a distinctly different codon usage profile from both its host and other mycobacteriophages and an abundance of codons that are rarely used in the host. proteomic analysis using mass spectrometry provides evidence for expression of at least patience proteins, two of which are from cryptic open reading frames (orfs) embedded within annotated genes. we propose that patience is a relatively recent visitor to the mycobacterium neighborhood, having evolved primarily in lower-gc hosts within the actinomycetales, and is in the process of adapting to growth in its new high-gc genetic environment. isolation and genome sequencing of mycobacteriophage patience. mycobacteriophage patience was isolated by direct plating of an environmental sample taken from near the nelson mandela school of medicine at the university of kwazulu-natal (ukzn), durban, south africa, using m. smegmatis mc as a host ( ) . isolation and purification of patience were components of a -week workshop on mycobacterial genetics offered in july ; the genome was sequenced at the university of pittsburgh and annotated in a second -week workshop at ukzn in july . patience forms normal-size hazy (~ -mm-diameter) plaques on m. smegmatis mc at °c under standard conditions, although we have been unsuccessful in recovering stable lysogens. it can easily be propagated on solid media to titers greater than pfu/ml. the genome is , bp long, circularly permuted, and presumably terminally redundant. for linear presentation, coordinate is designated the beginning of an open reading frame upstream of the large terminase subunit consistent with the organization of the cluster h phages that contain homologues of the first open reading frame at their left ends ( ) . the genbank submission (jn ) has been reported previously ( ) . genome annotation identified putative open reading frames (orfs) and one trna gene (table ) ; one additional orf was annotated using mass spectrometry analysis (see below). virion morphology and virion page analysis. mycobacteriophage patience is morphologically a member of the siphoviridae with an isometric head and a long flexible tail (fig. a) . the diameter of the heads averages nm, and the tails average nm in length, among the longest of the mycobacteriophages described to date, similar to those of the cluster m phages ( nm [ ] ); other phages with notably long tails are those in cluster h (approximately nm) and cluster r (approximately nm) ( ) . sds-page analysis of patience particles shows three abundant proteins and at least other lower-abundance proteins between the sizes of and kda (fig. b) . relationship of patience to other mycobacteriophages. the patience genome is not closely related to other phage genomes, although dot plot analysis shows weak similarity to cluster h phages (fig. ) . the related segments span only %, %, and % of genome lengths with barnyard, predator, and konstantine genomes, respectively (fig. ) , and the closest matching segment of patience and barnyard is a , -bp region ( % nucleotide identity) corresponding to the capsid subunit genes. alignment of the genome maps of patience, barnyard, konstantine, and predator in phamerator shows their architectural relationships (fig. ) , and although the nucleotide sequence similarity is minimal, % of patience genes have homologues in cluster h phages (fig. ) . however, more than half of the patience orfs are orphams (genes with no close mycobacteriophage relatives [ fig. ; table ]). the overall gc content of the patience genome is . % and is maintained almost throughout the genome (fig. a ). there are two notable departures suggesting recent acquisitions by horizontal exchange. one is within patience gene , where the = end has % nucleotide identity to rosebush gene (subcluster b ) and the elevated percent gc reflects that of the b phages (fig. b ). patience gp is related to tail fibers of many other mycobacteriophages that are implicated in host range determination ( ). the second example is gene ( % gc [ fig. c ]), although it is an orpham with no close mycobacteriophage relatives and is of unknown function. patience genome organization. genome annotation of patience indicates that all open reading frames and one trna gene are transcribed in the same direction ( fig. ; table ). the overall coding capacity is . %, and there are no intergenic spaces greater than bp. the recognizable virion structure and assembly functions lie within the gene to region, and the siphoviral syntenic arrangement of terminase, portal, protease, capsid, headtail connector proteins, major tail subunit, tail assembly chaperones, tape measure, and minor tail protein genes is observed, spanning kbp of the genome ( ) . this is atypically long not only because of the long tape measure gene corresponding to the long phage tail but because of a dozen genes of mostly unknown function located between the terminase large subunit (gene ) and portal (gene ) genes, between the portal (gene ) and protease (gene ) genes, and between the muf-like gene (gene ) and the putative scaffolding gene (gene ) (fig. ) ; most of these are orphams (i.e., they do not have a close mycobacteriophage rela-tive), with the exceptions of genes and (fig. ). gene encodes a putative endo vii protein and has weak similarity (Ͻ % identity) to genes in phages konstantine and predator table s in the supplemental material. (subcluster h ), as well as dori (singleton). the gene upstream of the terminase large subunit gene encodes a protein with similarities to putative hnh homing nucleases ( fig. ) but may function as part of the dna packaging machinery as described for hk gp ( ) . the four leftmost genes (genes to ) are of unknown function. the lysis cassette is located downstream of the virion structural genes and contains an endolysin gene (gene , lysin a) and a putative mycolylarabinogalactan esterase (gene , lysin b) ( , ) . genes and code for proteins containing two and four predicted transmembrane domains, respectively, that may act as holins or lysis chaperones ( ) . the lysis cassette is not closely related to that of the cluster h phages, and lysin a is most closely related to the corresponding genes of cluster m phage pegleg gp and bongo gp ( % identity), with an org-u domain organization ( ) . in contrast, lysin b is most closely related to phages in subcluster b ( % identity to gadget gp ), illustrating the mosaic nature of the lysis cassette ( ) . patience gp has no homologues, but gp has distant relatives in the cluster d phages (Ͻ % identity), where the genes are located between lysin a and lysin b. of the other patience orfs, only seven can be assigned putative functions, most of which are involved in dna metabolism (helicases, reca, dna polymerase iii [pol iii] alpha subunit, the three most abundant proteins likely correspond to the major tail subunit (gp ), the capsid subunit (gp ), and gp , with predicted molecular masses of . kda, . kda (after processing), and kda, respectively. the major tail subunit may migrate slower than its predicted mass, as observed in some other phages ( ) . numbers at right are molecular masses in kilodaltons. table ]). these all have homologues in a variety of mycobacteriophages as well as in phages of gordonia and corynebacterium. one (gene ) encodes a mazg-like protein, a putative nucleoside triphosphate pyro-phosphohydrolase that is common in a variety of phage genomes ( ) . patience has a single trna gene, and the trna is predicted to be charged with glutamine and has the anticodon =-uug [i.e., trna gln (uug)]. it is unclear how the patience genes are tran- ( ) and the database mycobacteriophage_ , containing complete genome sequences, and aligned by the left ends of the tape measure gene. maps are displayed in three tiers with pairwise nucleotide sequence similarities displayed in spectrum coloring, with violet being the most similar and red the least similar (minimal blastn cutoff e value is Ϫ ). genes are shown as colored boxes, with colors reflecting pham assignments for each gene (gene members of a phamily have the same color). genes shown in white are orphams and have no mycobacteriophage relatives with greater than . % amino acid identity or a blastp e value lower than Ϫ . phamily assignments with the number of phamily members in parentheses are above each gene. scribed, and there are no strongly predicted siga-like promoters, similar to other mycobacteriophages such as giles ( ), even though siga-like promoters are active in other mycobacteriophages ( ) ( ) ( ) . table s in the supplemental material). using stringent criteria for peptide identification, we identified of the previously annotated predicted gene products ( different gene products, including previously unannotated genes) present in at least one of the samples, including of the putative virion structure and assembly proteins (genes to ), of which are particle associated (fig. ) . although gp was not detected, particle-associated peptides corresponding to a previously unannotated -codon gene between genes and were identified, which we designate gene (fig. ) . particles contain four additional products, gp , gp , gp , and gp , although gp is abundant in lytic growth and the few particle-associated peptides could be contaminants in the phage preparation (table s ). the capsid subunit (gp ) appears to be proteolytically cleaved between residues and to generate a . -kda mature protein product; that and the gp major tail subunit ( . kda) and gp ( kda) likely correspond to the three major species observed by sds-page (fig. b) . it is noteworthy that-with the exception of -residue gp -all of the genes corresponding to insertions within the otherwise canonically syntenic virion structure and assembly operon are present in patience particles, although only spectra of gp were identified and could represent contaminants from the lysate (fig. ; table s ). of the total of patience proteins identified in infected cells, only one-gp -was not identified in the late-infected ( . -hpostinfection) sample. patience gp was identified in the earlyinfected sample and is presumably not expressed late in infection but also is turned over rapidly ( fig. ; see also table s in the supplemental material). many of the virion proteins (genes to ) are expressed at higher levels (using peptide spectral count as a surrogate for expression) in the late-infected sample, although there is also substantial expression of many of these at the early time. of the annotated proteins not identified in any of the samples, are predicted to be smaller than kda and may have escaped detection if they are not expressed at high levels. twelve are predicted to be membrane or wall associated and are likely excluded from the soluble fractions used for analysis. peptide abundance in infected cells is expected to generally correlate with expression, although other factors such as protein size and trypsin cleavage efficiencies influence peptide detection. first, we note that peptides at or near the n terminus confirm the previously annotated translation start site for proteins but also identify seven (gp , gp , gp , gp , gp , gp , and gp ) for which revisions of the annotated start sites are supported. for proteins, the peptide coverage is insufficient to be informative about start site usage. interestingly, seven proteins, all of them particle associated (gp , gp , gp , gp , gp , gp , and gp ), are acetylated at their n terminus (following methionine loss), although the functional significance-if any-is not known. six are acetylated at an n-terminal threonine following methionine removal (the seventh is a serine acetylation), although not all proteins with n-terminal threonine residues are acetylated. gp is unusual in that the n-terminal-most peptides start at residue of the annotated product following a glycine in the Ϫ position, indicating that they were not generated either by tryptic digestion or by translation initiation. other processes such as posttranslational processing or intron splicing prior to translation may be involved. we also note that gp is present in the particles but that the peptide coverage ( total spectra) is restricted to the n-terminal % of the protein, whereas in infected cells, the spectra reflect % coverage of the protein ( total spectra). this could be explained by assembly-associated protein processing. surprisingly, we identified several peptides corresponding to translation of regions wholly embedded within annotated genes. two of these were identified only with peptides for which we have lower confidence and will not be considered further. the evidence supporting translation of the other two is, however, quite strong. the first of these is a -bp open reading frame transcribed on the same strand, in a different reading frame, within gene , the dna polymerase iii catalytic subunit (see fig. s a and s a in the supplemental material). a total of instances of seven different peptides were identified, and the spectra and fragmentation tables strongly support the peptide assignments (see text s in the supplemental material). moreover, peptides correspond to the extreme n terminus with the methionine present, and there is a strong ribosome binding site upstream (fig. s ) . although the protein is seemingly expressed, the functional relevance is unclear. the predicted product has no close database relatives, and the evidence for conservation is ambiguous (fig. s a ). barnyard contains a similar open reading frame within its polymerase, and the products share % amino acid identity, whereas the corresponding segments of the polymerases share only % identity. this region of the polymerase is more distantly related in konstantine ( % identity), and the second open reading frame is not conserved (fig. s a) . a similar scenario is seen within patience gene , where two peptides corresponding to a -bp open reading frame on the same strand were identified with high confidence (see fig. s b in the supplemental material). peptides corresponding to the predicted n terminus were not identified, although a start codon with a strong ribosome binding site is present (fig. s b) . the corresponding segment of patience gp is conserved in barnyard gp and konstantine gp , but the second open reading frame is not. presumably, either expression of embedded out-of-frame genes is more common than anticipated or these reflect expression artifacts resulting from the use of noncognate host transcription and translation systems. codon selection and adaptation for mycobacterial growth. because m. smegmatis has a relatively high gc content ( . %), it is not surprising that these mutational biases (reflected in base composition) result in frequent usage of gc base pairs in thirdcodon positions (fig. a ; see also fig. s and s in the supplemental material). each of the most commonly used of the synonymous codons has gc in the third position, and m. smegmatis mc carries trnas with anticodons corresponding to all codons with gc in the third position, except for cgc (fig. s ) . all of the nnu codons are present infrequently and (with the exception of cgu) are decoded by wobble pairing in the codon third position (fig. s and s ) ; m. tuberculosis h rv has an almost identical trna profile. mycobacteriophages such as twister ( % gc) and kayacho ( % gc), with nucleotide compositions similar to that of m. smegmatis, have similar synonymous codon distributions (see fig. s and s in the supplemental material). in contrast, patience codon usage patterns are distinctly different, with notably different distributions of codons with respect to their third-position content relative to m. smegmatis ( fig. a ; see also fig. s and s ). there are a total of nine switches to a different most commonly used synonymous codon from m. smegmatis, seven of which are nnu codons that are rarely used in the host (five with normalized fig. s ] ). in total, nnu codons (except cgu, for which there is a cognate host trna) represent over % of all patience codons; in contrast, these are only % of m. smegmatis codons. this represents an extreme end of a trend between the usage of nnu codons and the overall gc content of the phage, where usage of nnu increases as percent gc decreases (fig. s ). although codon usage shows that patience spent a significant portion of its evolutionary past in hosts with moderate gc contents, it is not clear what its current host range is. if patience currently exploits more gc-rich hosts, then it would experience not only mutational biases, which would increase its gc content, but selective pressure on its most highly expressed genes to use trna pools poised to translate gc-rich genes. this selective pressure would be reflected in a preferred usage of gc-rich codons within highly expressed genes. patience does experience codon selection, which is shown by the robust positive correlation between codon selection (adapative codon enrichment [ace z ] [ ] ) (see materials and methods) and level of gene expression, using total numbers of peptides as identified by mass spectrometry under stringent conditions as a surrogate for expression (fig. b) . codon selection is measured using ␦ values, or the ratio of codon frequencies in genes experiencing codon selection normalized to their frequencies in genes lacking codon selection. preferred codons are those with ␦ values greater than , indicating more frequent use in genes experiencing codon selection, frequently those expressed to greater levels. patience favors the use of gcrich codons in genes experiencing codon selection (table ) , with of preferred codons bearing gc base pairs in their third positions, whereas of nonpreferred codons end with at base pairs. moreover, the patterns of codon preference in patience (which codons are preferred and which are not) are strongly correlated with that of m. smegmatis (fig. c) , suggesting that this host imposes codon selection congruent with that currently being experienced by patience. in contrast, patience codon selection is not congruent with that imposed by hosts with more moderate gc content, such as escherichia coli (fig. c, inset) , although such surveys can never be conclusive. patience represents an intriguing example of a virus that has successfully entered the mycobacterial genetic neighborhood in its relatively recent evolutionary history. its overall gc content and codon usage profiles are distinctly different from those of its mycobacterial host, suggesting that it primarily evolved in a moderate-gc (~ %) environment. growth in high-gc bacteria may have required multiple events, including acquisition of part of a tail gene (gene ) by lateral gene transfer. nonetheless, the mismatch between viral and host genomic profiles does not appear to have been a substantial impediment to host range expan-sion, although the highly expressed viral genes are under codon selection for more efficient translation by the host apparatus. interestingly, although patience conceivably could have responded by acquisition of a trna repertoire to facilitate phage gene expression, this has not occurred; the only phage-carried trna is trna-gln (uug), and caa is not a rare codon in the patience genome (see fig. s in the supplemental material). patience is the first phage to our knowledge for which the proteomic profile in infected cells has been examined by mass spectrometry. the approach is highly informative, providing strong evidence that many of the annotated reading frames are expressed-including that are shorter than codons-and providing support for many of the translational start sites, as well as revisions of start sites of several genes. moreover, a previously unannotated gene was identified (gene ) containing only codons, and the revision of start codons indicates that two pairs of genes have significant overlaps (Ͼ bp). phage genome annotation is generally more error-prone than that of other genomes because of the abundance of small open reading frames and relatively small gene size (average mycobacteriophage gene length is bp). hplc-ms/ms adds confidence to genome annotation, especially for phages such as patience, whose coding potential does not closely match its bacterial host. it is surprising to find that at least two reading frames embedded out of frame within annotated genes are also expressed. these orfs are generally not conserved and may not express functional products, but an intriguing possibility is that this expression is a consequence of movement into higher-gc hosts, presenting a small reservoir of new products available for selection and further adaption at little evolutionary cost. we note that ribosomal profiling of phage suggests that previously unannotated genes are expressed from its genome ( ) . the recent evolutionary history of patience supports a model in which the diversity of viruses of a given host is a function of rapid movement from one host to another coupled with a landscape of diverse but closely related hosts in which the viruses evolve ( ) . the large collection of mycobacteriophages that infect the common host m. smegmatis mc encompasses considerable diversity of sequence and gc content, and it seems likely that other groups of phages have entered the high-gc environment relatively recently, with patience representing an extreme example. we note that there are bacterial strains within the order actinomycetales, such as those of corynebacterium pseudotuberculosis, corynebacterium ulcerans, and corynebacterium diphtheriae, which have moderate gc contents ( . %, . %, and . %, respectively) and may be relatives of hosts that supported patience growth in its earlier evolutionary history. their codon usage profiles (see fig. s in the supplemental material) more closely reflect those of the lower-gc mycobacteriophages such as patience. patience may share gene content with phages of such hosts, but to date, few have been characterized, other than some prophages such as phage beta of c. diphtheriae ( , ) . deeper exploration into the phages of these hosts and other hosts is thus likely to be highly informative and provide insights into viral origins and viral evolution ( ) . dna sequencing. patience was isolated using standard methods as described previously ( , ) , and the genome was sequenced using technology at the university of pittsburgh's genomics and proteomics core laboratories; a total of~ , reads were assembled to yield an average ( ) , and phamerator ( ) . the phamerator database used for genomic comparisons was mycobacteriophage_ . phams were built using blastp and/or clustalw, with similarity cutoff e values of Ϫ and . % similarity or better as described elsewhere ( ) . codon usage and codon selection. codon usage resulting from mutational biases was estimated using the collection of all genes from a genome. the fraction (f) of each codon for a given amino acid was calculated as the ratio of the codon count to the amino acid count. relative synonymous codon usage (rscu) normalizes codon frequencies so that the sum of rscu for codons of each amino acid is equal to the number of synonymous codons for that amino acid. to measure codon selection, a second codon usage table (f o ) was tabulated, limited to genes experiencing strong codon selection. for bacterial genomes, this table was constructed from homologues of sharp's set of genes whose products participate in translation ( ) . for patience, this table was constructed in three steps. first, a codon usage table was generated from % of the genome using the genes with the most extreme value of codon usage bias as determined by (where expected codon usage is calculated from the nucleotide composition). next, adaptive codon enrichment (ace u ) values ( ) are calculated for all genes as described previously ( ) , using this table to represent codon frequencies under codon selection (f o ) and the frequencies of codons among all genes in the patience genome to represent codon frequencies expected from mutational processes alone (f n ( ) , and phamerator ( ) . electron microscopy. cscl gradient-purified patience particles were applied to glow-discharged formvar-and carbon-coated copper grids ( mesh) (ted pella). they were stained with % uranyl acetate and imaged with a morgagni transmission electron microscope fitted with a hamamatsu orca hr side-model digital camera and amt software. sds-page. patience particles were concentrated and purified via cscl gradient and ultracentrifugation. the visible phage band was dialyzed against two changes of phage buffer; l of the dialyzed cscl band was pelleted by a -min spin at , rpm in a microcentrifuge. the pellet was resuspended in l of mm dithiothreitol (dtt), and then l of . m edta and l of m mgso were added. the phage was disrupted by being heated to °c for min and then sonicated on ice six times for s to disrupt the dna. the sample was then mixed with l of ϫ sds sample buffer and heated in a boiling bath for min at °c. the sample was electrophoresed through a % polyacrylamide gel containing sds and stained with coomassie brilliant blue in methanol. hplc-ms/ms. five milliliters of exponentially growing m. smegmatis mc (optical density at nm [od ] of . ) in h -adc medium ( ) was concentrated to a -l volume via low-speed centrifugation and infected with patience at a multiplicity of infection (moi) of . phage particles were allowed to adsorb for min, and then . ml of fresh h medium was added to the culture and incubated with shaking for h at °c; the od was monitored throughout to follow cell growth and lysis. at min and min postadsorption, a -ml aliquot was removed from the culture, the cells were pelleted via centrifugation ( min, , rpm in a microcentrifuge), and the supernatant was removed. the cell pellet was frozen at Ϫ °c and then shipped overnight on wet ice to the university of california, davis proteomics core (ucdpc) (http://proteomics.ucdavis.edu). there, the cells were lysed via a magna lyser, the insoluble fraction was removed, and the soluble proteins were precipitated, digested with trypsin, and cleaned up using a macrospin column. the peptides were then separated using an easy-lc ii high-pressure liquid chromatography (hplc) system and loaded into a q exactive orbitrap mass spectrometer with a proxeon nanospray source (thermo) for tandem ms analysis. detected spectra and fragmentation profiles were matched against a database comprised of a six-frame translation of the patience genome, the annotated proteins of m. smegmatis mc , and uniprot using x! tandem. peptide matches were analyzed using scaf-fold . the "relaxed" settings (as reported in table s in the supplemental material) used a peptide false discovery rate (fdr) of % and a protein fdr of %; the "stringent" settings used a peptide fdr of . % and a protein fdr of . %. estimation of relative protein abundance was determined by normalizing the total number of spectra detected to the gene size. supplemental material for this article may be found at http://mbio.asm.org /lookup/suppl/doi: . /mbio. - /-/dcsupplemental. text s , pdf file, . mb. figure s , pdf file, . mb. figure s , pdf file, . mb. figure s , pdf file, . mb. figure s , pdf file, . mb. figure s , pdf file, . mb. figure s , pdf file, . mb. table s , pdf file, . mb. the secret lives of mycobacteriophages science education alliance phage hunters advancing genomics and evolutionary science phage hunters integrating research and education (phire) program. . complete genome sequences of mycobacteriophages kwazulu-natal research institute for tuberculosis and hiv mycobacterial genetics course students, phage hunters integrating research and education program on the nature of mycobacteriophage diversity and host preference exploring the mycobacteriophage metaproteome: phage genomics as an educational platform deciphering the biology of mycobacterium tuberculosis from the complete genome sequence origins of highly mosaic mycobacteriophage genomes cluster m mycobacteriophages bongo, pegleg, and rey with unusually large repertoires of trna isotypes functional role of mycobacteriophage transfer rnas synonymous codon usage analysis of the mycobacteriophage bxz and its plating bacteria m. smegmatis: identification of highly and lowly expressed 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bioinformatic tool for comparative bacteriophage genomics variation in the strength of selected codon usage bias among bacteria dna sequence, structure and gene expression of mycobacteriophage l : a phage system for mycobacterial genetics key: cord- -cc dzycl authors: richman, douglas d. title: antiviral drug discovery to address the covid- pandemic date: - - journal: mbio doi: . /mbio. - sha: doc_id: cord_uid: cc dzycl the magnitude of the morbidity and mortality inflicted upon the global population in less than year has driven the inescapable conclusion that the discovery and development of effective antiviral drugs for covid- are urgent and should be prioritized. the antiviral drug discovery programs that emerged for hiv and hepatitis c virus have enabled technology and expertise to accelerate this process for sars-cov- . the description of candidate lead inhibitors for the viral main protease (m(pro)) exemplifies this accelerated approach and reminds us of the needs and opportunities for addressing this pandemic. t he need for more effective antiviral drugs against sars-cov- is undeniable. even with the identification of the first effective drug for coronavirus infection, remdesivir, the need for additional drugs remains critical. regardless of whether or when a vaccine becomes available, antivirals for sars-cov- will still be needed for several reasons: the unlikelihood that a vaccine will be % effective, the incompleteness of vaccine coverage because of both vaccine hesitancy and the numerous logistical challenges to accomplishing prompt large-scale immunization of the majority of the population, the possibility of limited durability of vaccine protection, the need for additional prophylaxis for high-risk subjects and poor vaccine responders, and the future value of effective antiviral treatment for middle east respiratory syndrome (mers) and new coronaviruses that will likely emerge from zoonoses. unmet needs for new antivirals include additional and greater efficacy, oral bioavailability, utility for prophylaxis as well as treatment, and the knowledge that combination therapy can enhance efficacy and prevent the emergence of drug resistance. although drugs to modulate the late immunopathological complications are also needed, only highly active antiviral drugs will be beneficial for prophylaxis, the treatment of early disease, and perhaps the reduction of transmissibility. remdesivir targets the viral rna-dependent rna polymerase (rdrp), and additional inhibitors of that enzyme are in development, as mentioned below. as viral enzymes are critical for replication and represent the majority of targets of all approved antiviral drugs, other coronavirus enzymes represent obvious candidate targets for effective drugs. with protease inhibitors proven as effective for antivirals against hiv and hepatitis c virus (hcv), the two coronavirus proteases, the main protease (m pro ) and the papain-like protease (pl pro ), also stand out as clear candidate targets for antiviral drug discovery. hattori et al. ( ) examined two indole-chloropyridinyl-ester derivatives, grl- and grl- , which they had designed against sars-cov before the emergence of sars-cov- . the m pro s of the two viruses share significant sequence and structural similarities. to their credit, hattori et al. use multiple assays to document activity (inhibition of infectivity, replication, and cytopathic effects) in two different cell lines, and importantly, they conscientiously characterize cellular toxicity. too many publica-tions fail to carefully recognize that the reduction of replication that is being measured may be attributable to diminished host cell viability. in fact, the data in the work of hattori et al. suggest that the purported activity against sars-cov- of the two hiv protease inhibitors, lopinavir and nelfinavir, is probably attributable to cellular toxicity. moreover, the claims of activity for chloroquine and hydroxychloroquine may be attributable to cellular toxicity according to the data in the work of hattori et al. they also confirm with structural modeling and high-performance liquid chromatography and mass spectrometry (hplc/ms) that their inhibitors of m pro dock and then covalently bind in the catalytic site. this information will provide invaluable guidance for the modification of the inhibitors when designing yet more potent derivatives. in addition to the identification of compounds by hattori et al. of note, hattori et al. ( ) found no significant anti-sars-cov- activity for several compounds reportedly active against sars-cov- , including lopinavir, nelfinavir, nitazoxanide, favipiravir, and hydroxychloroquine, while the proven drug remdesivir displayed activity at nontoxic concentrations. it has been frustrating to observe the widespread rush to use these drugs both for patient management and in numerous redundant clinical trials, when the in vitro data should be sufficient to discourage this enthusiasm, especially when their in vitro potency against coronaviruses is so low compared to their potency against their proven targets. the urgency of curbing the covid- pandemic has motivated a large number of investigators globally to identify new drugs and repurpose old ones to target viral proteins, as well as some host cell targets essential for viral replication. the only antiviral yet shown to have clinical efficacy is the rdrp inhibitor remdesivir. these studies were conducted in subjects with moderate to severe disease, while efficacy with antiviral drugs generally is greatest when administered early in disease. the results of planned studies with outpatient administration to subjects at high risk for severe disease, of aerosolized administration, and of remdesivir in combination with interferon beta- a are eagerly awaited. other ribonucleoside inhibitors of rdrp, which are orally bioavailable, unlike remdesivir, have entered clinical trials. mk- (formerly eidd- ) has activity against sars-cov, mers, and sars-cov- in mice and is currently being studied in humans ( ) . mk- appears to act by inducing frequent nucleoside transitions during rna replication, resulting in increased error rates, which probably account for impaired replication. at- is a prodrug of a guanosine ribonucleotide analog, which was well tolerated with substantial activity in studies of hcv-infected subjects ( ) . it has in vitro activity against sars-cov- , justifying its investigation for covid- ( ) . early studies with parenteral interferon beta- b ( ) and inhaled interferon beta (as yet unpublished) showed encouraging results. numerous clinical trials are being conducted with specific antibodies, both from convalescent-phase plasma and as monoclonal antibody preparations, but no randomized controlled trials to document activity clinically have yet been published. repurposing already available drugs is appealing because the availability of the drug along with pharmacokinetic and toxicity profiles facilitates expeditious evaluation for covid- . as mentioned above, several such drugs have been and remain under study because of ready availability rather than compelling in vitro activity against sars-cov- . an exception is camostat mesylate, which has been approved for the treatment of pancreatic cancer in japan. camostat mesylate inhibits the host serine protease tmprss , which dramatically enhances sars-cov- replication by proteolytically cleaving the spike (s) protein of the virus to activate the spike to mediate cell entry ( ) . with human safety and pharmacokinetics already characterized, camostat mesylate is in several clinical trials as a repurposed drug for covid- . the severe complications of covid- appear to be more the consequence of a misguided, pathological host immune response to ongoing viral infection ( ) . numerous studies are in progress to identify effective modulators of this dysregulated immune response with some early indications of efficacy of the corticosteroid dexa-methasone ( ) . the potential benefits of inhibitors of interleukin- and other cytokines and chemokines are being examined in multiple clinical trials, but evidence of clinical efficacy is pending. the mitsuya group has a track record of developing drugs that are effective against hiv infection. these include the reverse transcriptase inhibitors ddc (zalcitabine) and ddi (didanosine), the protease inhibitor darunavir, and the reverse transcriptase translocation inhibitor islatavir ( ) ( ) ( ) . with the need for antiviral drugs for covid- , we can all hope that not only will this group be successful in identifying effective coronavirus drugs, but that numerous other investigators are successful in finding drugs against multiple targets. we have learned with other viral diseases that to address the challenges of safety, efficacy, and drug resistance, to tackle the demands of both prophylaxis and treatment, and to manage both the viral replication and immunopathological aspects of covid- , numerous drugs will be needed. let us all applaud the many efforts and wish many successes in the battle against this pandemic. grl- , an indole chloropyridinyl ester, completely blocks sars-cov- infection structurebased design of antiviral drug candidates targeting the sars-cov- main protease an orally bioavailable broad-spectrum antiviral inhibits sars-cov- in human airway epithelial cell cultures and multiple coronaviruses in mice safety, pharmacokinetics and antiviral activity of at- , a novel purine nucleotide prodrug, in hcv-infected subjects with and without cirrhosis at- is a potent in vitro replication inhibitor of sars-cov- , the virus responsible for the covid- pandemic triple combination of interferon beta- b, lopinavir-ritonavir, and ribavirin in the treatment of patients admitted to hospital with covid- : an open-label, randomised, phase trial sars-cov- cell entry depends on ace and tm-prss and is blocked by a clinically proven protease inhibitor the innate immune system: fighting on the front lines or fanning the flames of covid- ? dexamethasone in hospitalized patients with covid- -preliminary report inhibition of the in vitro infectivity and cytopathic effect of human t-lymphotrophic virus type iii/lymphadenopathyassociated virus (htlv-iii/lav) by ', '-dideoxynucleosides novel bis-tetrahydrofuranylurethanecontaining nonpeptidic protease inhibitor (pi) uic- (tmc ) with potent activity against multi-pi-resistant human immunodeficiency virus in vitro '-deoxy- '-c-ethynyl- -fluoroadenosine: a nucleoside reverse transcriptase inhibitor with highly potent activity against all hiv- strains, favorable toxic profiles and stability in plasma